# WEAK INTERACTIONS IN MOLECULAR MACHINERY

EDITED BY : Irene Díaz-Moreno and Rivka Isaacson PUBLISHED IN : Frontiers in Molecular Biosciences

#### Frontiers Copyright Statement

© Copyright 2007-2019 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.

The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.

Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.

Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.

As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.

All copyright, and all rights therein, are protected by national and international copyright laws.

The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use. ISSN 1664-8714 ISBN 978-2-88945-787-8 DOI 10.3389/978-2-88945-787-8

#### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

#### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

#### Dedication to Quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

#### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# WEAK INTERACTIONS IN MOLECULAR MACHINERY

Topic Editors: Irene Díaz-Moreno, University of Seville, Spain Rivka Isaacson, King's College London, United Kingdom

Diverse cellular processes depend on weak interactions between biological components. In this volume, we bring together a wealth of recent information on this topic, combining original research articles with up-to-date reviews, organized under four separate themes. In our first section on nucleic acid regulation, we include a study of the PII-NAGK-PipX-NtcA regulatory axis of cyanobacteria and discover much about the role of RNA binding protein regulation and cross-talk in the control of AU-rich mRNA. Peptide-mediated weak interactions are our second theme in which we review weak molecular interactions in clathrin-mediated endocytosis, investigate the selectivity of the G7-18NATE inhibitor peptide for the Grb7-SH2 domain target and present new structure and interactions of the TPR domain of Sgt2 with yeast chaperones and Ybr137wp. Our third part focuses on carbohydrates and includes a thorough review of how to use NMR to study transient carbohydrate–protein binding and a structural and functional study of lysostaphin–substrate interaction. In our final section, we look at functional sensors driving weak interactions by presenting the molecular basis of the dual regulation of bacterial iron sulfur cluster biogenesis by CyaY and IscX alongside a review of intramolecular fuzzy interactions involving intrinsically disordered domains. Taken together, our eBook chapters offer some recent insight into this area of scientific understanding which is still expanding exponentially.

Citation: Díaz-Moreno, I., Isaacson, R., eds. (2019). Weak Interactions in Molecular Machinery. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-787-8

# Table of Contents

*05 Editorial: Weak Interactions in Molecular Machinery* Rivka L. Isaacson and Irene Díaz-Moreno

#### SECTION 1

#### DNA AND RNA REGULATION

Sofía M. García-Mauriño, Francisco Rivero-Rodríguez, Alejandro Velázquez-Cruz, Marian Hernández-Vellisca, Antonio Díaz-Quintana, Miguel A. De la Rosa and Irene Díaz-Moreno

#### SECTION 2

### PEPTIDE-MEDIATED WEAK INTERACTIONS

*34 Insight Into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target*

Gabrielle M. Watson, William A. H. Lucas, Menachem J. Gunzburg and Jacqueline A. Wilce

*42 Structure and Interactions of the TPR Domain of Sgt2 With Yeast Chaperones and Ybr137wp*

Ewelina M. Krysztofinska, Nicola J. Evans, Arjun Thapaliya, James W. Murray, Rhodri M. L. Morgan, Santiago Martinez-Lumbreras and Rivka L. Isaacson

*53 Weak Molecular Interactions in Clathrin-Mediated Endocytosis* Sarah M. Smith, Michael Baker, Mary Halebian and Corinne J. Smith

#### SECTION 3

#### TARGETING CARBOHYDRATES AND GLYCANS

### SECTION 4

#### FUNCTIONAL SENSORS DRIVING WEAK INTERACTIONS

#### *85 The Molecular Bases of the Dual Regulation of Bacterial Iron Sulfur Cluster Biogenesis by CyaY and IscX*

Salvatore Adinolfi, Rita Puglisi, Jason C. Crack, Clara Iannuzzi, Fabrizio Dal Piaz, Petr V. Konarev, Dmitri I. Svergun, Stephen Martin, Nick E. Le Brun and Annalisa Pastore

*97 Intramolecular Fuzzy Interactions Involving Intrinsically Disordered Domains*

Miguel Arbesú, Guillermo Iruela, Héctor Fuentes, João M. C. Teixeira and Miquel Pons

# Editorial: Weak Interactions in Molecular Machinery

#### Rivka L. Isaacson1 and Irene Díaz-Moreno2

1 Department of Chemistry, King's College London, London, United Kingdom, 2 cicCartuja, Institute for Chemical Research (IIQ), University of Seville - CSIC, Seville, Spain

\*

Keywords: transient interactions, molecular machineries, structural biology, biophysics, weak contacts

#### **Editorial on the Research Topic**

#### **Weak Interactions in Molecular Machinery**

Individuals are often sustained by intense relationships with others, although few would deny the importance of those more transient interactions with service providers, colleagues, and casual acquaintances in facilitating our continued existence. These latter associations are necessarily and conveniently weaker. Nobody has time for a deep conversation with everyone they encounter during a day, and society, like so many complex systems, runs on a hierarchy of interaction strengths. Similarly, inside each crowded cell of our bodies, functions arise when communication is established through biomolecules that physically and specifically contact each other in a concerted manner to transmit messages efficiently.

"Molecular sociology" within cells involves binding events between macromolecules, such as protein–protein, protein–nucleic acid, protein–carbohydrate, and protein–membrane interactions, that are acutely orchestrated to sustain the life–death balance. These interactions between biomolecules occur on a wide range of timescales. Stable complexes, with lifetimes ranging from minutes to days, involve high affinity and high specificity binding. Amongst many others, these include irreversible enzyme inhibition and the assembly of proteins supporting the cell's ultrastructure.

#### Weak complexes are characterized by a fine balance between specificity of binding and fast turnover rate, with the majority displaying equilibrium dissociation constants within the micromolar or even millimolar range. Perhaps counter-intuitively, these weaker molecular recognition mechanisms are not uncommon and play key roles in many biological processes, including electron transfer chains in respiration and photosynthesis and the cell signaling cascades involving kinases and phosphatases. Due to technical limitations, weak complexes remain poorly understood despite their critical role in many biological events inside the cell.

Over the last few years, specific tools have been developed to analyse more transient intermolecular interactions, including NMR paramagnetic relaxation enhancement and kinetic approaches. These methods have recently been complemented by high-throughput techniques to identify novel biomolecules that weakly bind to each other. Such advances make the analysis of the transient biointeractome (the so-called trans-biointeractome) more affordable. Overall, trans-biointeractome analysis remains highly dependent on the specific technique employed. In addition, the dynamics of the system can enhance the strength of interactions that are weak. Similarly, "one person may not be able to pull a truck from the mud but many would be able to." This may also be the reason that interactions detected under controlled conditions in vivo become extremely difficult to study in isolation. Protein compartmentalization, elevated protein levels at defined moments of the cell cycle, multivalent binding, can increase (temporarily) the affinities of transient interactions, suggesting that the so-called trans-biointeractome is inherently plastic and that it evolves during cell lifespan.

#### Edited by:

Anastasia S. Politou, University of Ioannina, Greece

#### Reviewed by:

Konstantinos Tripsianes, Masaryk University, Czechia

> \*Correspondence: Irene Díaz-Moreno<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Structural Biology, a section of the journal Frontiers in Molecular Biosciences

Received: 26 November 2018 Accepted: 22 December 2018 Published: 23 January 2019

#### Citation:

Isaacson RL and Díaz-Moreno I (2019) Editorial: Weak Interactions in Molecular Machinery. Front. Mol. Biosci. 5:117. doi: 10.3389/fmolb.2018.00117

**5**

Therefore, the study of weakly interacting systems can be reliably tackled in depth only by an integrative and holistic approach combining in vitro strategies with other methods to detect such interactions within the cell. We are delighted by the breadth of techniques and systems covered in the articles of our research topic.

Unsurprisingly, given its well-documented strengths in measuring weak binding, NMR spectroscopy features heavily as a method used in this Special Issue in Frontiers in Molecular Biosciences.

Nieto provides a comprehensive mini review on using NMR to investigate binding between carbohydrates and proteins, which is particularly useful given the challenges of isotopically labeling glycans. In their original research article, the group headed by Krysztofinska et al. use NMR alongside X-ray crystallography and isothermal titration calorimetry (ITC) to analyse the structure and binding of different heat shock chaperones, via a carboxylate clamp mechanism, to a cochaperone involved in targeting proteins to membranes. Multiple proteins competing for the same binding site, as analyzed using NMR, is a theme that continues in the research article by Adinolfi et al. who also employ small-angle X-ray scattering (SAXS), biolayer interferometry (BLI), and native mass-spectrometry (MS) to dissect the dual regulation of bacterial Iron Sulfur cluster biogenesis by two proteins, CyaY and IscX. Tossavainen et al. also use NMR and SAXS, alongside Molecular Dynamics simulations to explore the variable affinity interactions between Staphylococcus aureus cell-wall digester, lysostaphin, and its substrates.

Surface plasmon resonance (SPR) is another widely used technique to measure interactions, and along with microarray technology, is employed by Watson et al. to establish the efficacy of a peptide inhibitor in blocking an important weak interaction between an SH2 domain and its phosphotyrosine target. This is a step on the way to rational drug design for cancer.

The importance and unique attributes of intrinsically disordered proteins (IDPs) are becoming increasingly recognized, acknowledged and valued. Arbesú et al. feed into this theme by presenting a fascinating mini review on the intramolecular "fuzzy" interactions that occur within intrinsically disordered domains of proteins. This mini-review perfectly links with the Frontiers topic on Function and Flexibility: Friend or Foe?

Perhaps the most self-contained story in our collection, that truly conveys the variety of weak interactions in a well-characterized pathway, is the review from the group of Forcada-Nadal et al. on the PII-NAGK-PipX-NtcA regulatory axis of cyanobacteria, which beautifully compiles the information that led to an understanding of this network. Clathrin-mediated endocytosis is an elegant process as delineated in a review from. Smith et al. who examine the vital roles of weak interactions in this choreographed cellular system. On the other hand, it becomes more and more evident that there is a need for a transition from a static to a dynamic point of view in order to take into account the biological environment during RNA binding and RNA metabolism, as reviewed by the team of García-Mauriño et al. Actually, the understanding of the mRNAs processing in highly dynamic and often transient macromolecular complexes also remains challenging.

The subjects covered in our research topic span a wide range of biological questions and methodology. We believe this is merely the tip of the iceberg and that the future will bring vast insight into the importance of weak interactions in molecular machinery.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Isaacson and Díaz-Moreno. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## The PII-NAGK-PipX-NtcA Regulatory Axis of Cyanobacteria: A Tale of Changing Partners, Allosteric Effectors and Non-covalent Interactions

Alicia Forcada-Nadal 1,2, José Luis Llácer 1,3, Asunción Contreras 2 , Clara Marco-Marín1,3 and Vicente Rubio1,3 \*

1 Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Científicas, Valencia, Spain, 2 Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain, 3 Group 739, Centro de Investigación Biomédica en Red de Enfermedades Raras – Instituto de Salud Carlos III, Valencia, Spain

#### Edited by:

Irene Diaz-Moreno, Universidad de Sevilla, Spain

#### Reviewed by:

Karl Forchhammer, Universität Tübingen, Germany Juan A. Hermoso, Consejo Superior de Investigaciones Científicas (CSIC), Spain

> \*Correspondence: Vicente Rubio<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Structural Biology, a section of the journal Frontiers in Molecular Biosciences

Received: 26 August 2018 Accepted: 18 October 2018 Published: 13 November 2018

#### Citation:

Forcada-Nadal A, Llácer JL, Contreras A, Marco-Marín C and Rubio V (2018) The PII-NAGK-PipX-NtcA Regulatory Axis of Cyanobacteria: A Tale of Changing Partners, Allosteric Effectors and Non-covalent Interactions Front. Mol. Biosci. 5:91. doi: 10.3389/fmolb.2018.00091 PII, a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. PII sensing is mediated by the PII allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of PII function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a PII regulatory network. Studies on these organisms yielded a first structure of a PII complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how PII can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which PII can control gene expression. A small adaptor protein, PipX, is sequestered by PII when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of PII-NAGK, PII-PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally PII regulatory functions and reveal the changing shapes and interactions of the T-loops of PII depending on the partner and on the allosteric effectors bound to PII. Cyanobacterial studies have also revealed that in the PII-PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components

**7**

of this PII-PipX regulatory network and of KD values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs.

Keywords: protein structure, nitrogen regulation, gene expression regulation, signaling, PII complexes, PipX complexes, NtcA structure and complexes, PlmA

Protein PII was discovered in the late sixties of last century (Stadtman, 2001), when Escherichia coli glutamine synthetase (GS) was found to exist in feed-back inhibition susceptible or refractory forms depending on the adenylylation state of one tyrosine per GS subunit. PI and PII were the first and second peaks from a gel filtration column (Shapiro, 1969). PI is a bifunctional enzyme (ATase) that adenylylates or deadenylylates GS (Jiang et al., 2007). PII controls the activity of the ATase. We now know that PII proteins are highly conserved and very widespread sensors used to transduce energy/carbon/nitrogen abundance signals in all domains of life (Kinch and Grishin, 2002; Sant'Anna et al., 2009). They are found in archaea, bacteria (Gram+ and Gram−), unicellular algae and plants. Many organisms have two or more genes for PII proteins (reviewed in Forchhammer and Lüddecke, 2016), as E. coli, that has two paralogous genes encoding PII proteins with distinct functions, one (GlnB) involved in the control of GS, and the other one (GlnK) being involved in the regulation of ammonia entry into the cell. By binding to target proteins, including channels, enzymes, or molecules involved in gene regulation and by altering the function of these target molecules, PII proteins can regulate ammonia entry, nitrogen metabolism and gene expression (Forchhammer, 2008; Llácer et al., 2008). Cyanobacteria, and particularly among them Synechococcus elongatus PCC 7942 (hereafter S. elongatus), have been and continue to be very useful organisms for studies of PII actions, fuelling structural understanding of PII regulation. Studies on these organisms exemplify very clearly how enzyme activity and gene regulation can be controlled by PII via formation of several complexes (summarized in **Figure 1**) mediated by weak intermolecular interactions that are crucially regulated by allosteric effectors of the proteins involved in these complexes. This is the focus of the present review.

## THE PII SIGNALING PROTEIN

S. elongatus PII (**Figure 2**), as other PII proteins, is a homotrimer of a polypeptide chain of 112 amino acids that exhibits the ferredoxin fold (βαβ)2 followed by a beta hairpin (Xu et al., 2003). The trimer (**Figure 2A**) has a hemispheric body nucleated by three antiparallel oblique (relative to the three-fold axis) β-sheets, each one formed by the 4-stranded sheet (topology ↓β2↑β3↓β1↑β4) of a subunit (see for example subunit B in the central panel of **Figure 2A**) extended on its β4 end by the Cterminal hairpin (β5-β6) of an adjacent subunit (subunit A) and on the β2 end by the β2-β3 hairpin stem (the root of the Tloop, see below) of the other subunit of the trimer (subunit C). The three sheets become continuous on the flat face of the hemispheric body via their β2-β3 hairpins (**Figure 2A**). The subunit sheets encircle like a 3-sided pyramid the three-fold axis, filling the inner space between them with their side-chains. They are covered externally by 6 helices (two per subunit) that run parallel to the β strands, contributing to the rounded shape of the hemispheric trimer (**Figure 2A**, panel to the right) and to the outer part of its equatorial flat face. In the convex face, three crevices are formed at subunits junctions between adjacent βsheets, over the β2-β3 hairpins (**Figure 2B**). These crevices host the sites for the allosteric effectors ATP/ADP [and in some species AMP (Palanca et al., 2014)] and 2-oxoglutarate (2OG) that endow PII with its sensing roles (Kamberov et al., 1995; Zeth et al., 2014) (**Figure 2B**), the nucleotides reflecting the energy status (Fokina et al., 2011) and 2OG reflecting the abundance of carbon and, inversely, the nitrogen richness (see for example Muro-Pastor et al., 2001).

Very salient structural features of PII are the long flexible Tloops (**Figures 2A,C**) formed by the 18 residues that tip the β2-β3 hairpin of each subunit (Xu et al., 2003). These loops are key elements (although not the exclusive ones, see Rajendran et al., 2011 and Schumacher et al., 2015) for PII interaction with its targets (Conroy et al., 2007; Gruswitz et al., 2007; Llácer et al., 2007, 2010; Mizuno et al., 2007; Zhao et al., 2010b; Chellamuthu et al., 2014). By binding at the boundary between the T-loop and the PII body, at the crevice formed between adjacent subunits, the adenine nucleotides and MgATP/2OG promote the adoption by the T-loop of different conformations (**Figure 2C**) (Fokina et al.,

**Abbreviations:** PII, a homotrimeric signaling protein; GlnK, GlnK3 and GlnB, different paralogous forms of PII proteins; GlnD, the bifunctional enzyme that uridylylates and deuridylylates GlnB in E. coli; GS, glutamine synthetase; AmtB and Amt, homologous trimeric bacterial transporters of ammonia; PamA, putative channel of unknown function that is encoded by sll0985 of Synechocystis sp. PCC 6803; NtcA and CRP, homologous homodimeric transcription factors of cyanobacteria and of E. coli, respectively; the imperfectly palindromic target DNA sequences to which they bind specifically are called NtcA box and CRP box, respectively; PlmA, putative homodimeric transcription factor of the GntR family that is found in cyanobacteria; PI or ATase, bifunctional enzyme that adenylylates and deadenylylates glutamine synthetase in E. coli and other enterobacteria; NAGK, N-acetyl-L-glutamate kinase; NAG, N-acetyl-L-glutamate; PipX, a small monomeric protein of cyanobacteria that can interact with PII and with NtcA; FRET, fluorescence resonance energy transfer (also known as Förster resonance energy transfer), a phenomenon in which a fluorophore emits light of its characteristic frequency when a nearby different absorbing group is excited by

light; the FRET signal decreases with the 6th power of the distance between the absorbing and emitting groups; 2OG, 2-oxoglutarate, also called α-ketoglutarate; AcCoA carboxylase, acetyl coenzyme A carboxylase; BCCP, biotin carboxyl carrier protein, the protein subunit that hosts the covalently bound biotin in many bacterial AcCoA carboxylases; PLP, pyridoxal phosphate; PipY, product of the gene that in S. elongatus is the next downstream of pipX, forming a bicistronic operon with it; it is a PLP-containing protein.

FIGURE 1 | Summary of the PII-PipX-NtcA network of S. elongatus. The network illustrates its different elements and complexes depending on nitrogen abundance (inversely related to 2OG level) and the structures of the macromolecules and complexes formed (when known). For PlmA (dimer in darker and lighter blue hues for its dimerization and DNA-binding domains, respectively) and its complex the architectural coarse model proposed (Labella et al., 2016) is shown, with the C-terminal helices of PipX (schematized in the extended conformation) pink-colored and the two PII molecules in dark red. The DNA complexed with NtcA and with NtcA-PipX is modeled from the structure of DNA-CRP (Llácer et al., 2010), since no DNA-NtcA structure has been reported. BCCP, biotin carboxyl carrier protein of bacterial acetyl CoA carboxylase (abbreviated AcCoA carboxylase); the other two components of this enzyme, biotin carboxylase and carboxyl transferase are abbreviated BC and CT, respectively. No structural model of BCC has been shown because the structure of this component has not been determined in S. elongatus and also because the structures of this protein from other bacteria lack a disordered 77-residue N-terminal portion that could be highly relevant for interaction with PII. The yellow broken arrow highlights the possibility of further PipX interactions not mediated by NtcA or PII-PlmA resulting in changes in gene expression (Espinosa et al., 2014). The solid semi-transparent yellowish arrow emerging perpendicularly from the flat network symbolizes the possibility of functional interactions of PipX not mediated by physical contacts between the macromolecules involved in the interaction, giving as an example the functional interaction with PipY. Its position outside the network tries to express the different type of interaction (relative to the physical contacts shown in the remainder of the network) as well as to place it outside the field of 2OG concentrations.

2010a; Truan et al., 2010; Maier et al., 2011; Zeth et al., 2014) that favor or disfavor PII binding to a given PII target.

The T-loop also is the target of regulatory post-translational modification (reviewed in Merrick, 2015), first recognized in the regulatory cascade of the GS of E. coli as uridylylation of Tyr51 (see **Figure 2C**, 3rd panel from the left) mediated by a glutamineregulated bifunctional PII uridylylating-deuridylylating enzyme, GlnD (Stadtman, 2001). Thus, in the enterobacterial GS regulating cascade PII is uridylylated or deuridylylated depending on whether 2-OG is abundant and L-glutamine is low or the reverse. PII-UMP activates the GS deadenylylating activity of ATase (Jiang et al., 2007), activating GS by decreasing its susceptibility to feed-back inhibition (Stadtman, 2001). This uridylylation (or in Actinobacteria adenylylation of Tyr51) occurs at least in proteobacteria and actinobacteria (Merrick, 2015), but it might be more widespread, since it has also been reported in an archaeon (Pedro-Roig et al., 2013). Structural studies with E. coli PII (Palanca and Rubio, 2017) have excluded the stabilization of the T-loop into a fixed conformation by Tyr51 uridylylation, suggesting that the Tyr51 bound UMP physically interacts with the ATase. Although Tyr51 is conserved in cyanobacteria, it is not uridylylated. The Tloop serine 49 (**Figure 2C**, 1st, 2nd and 4th panels from the left) is phosphorylated in S. elongatus under conditions of nitrogen starvation by an unknown mechanism (Forchhammer and Tandeau de Marsac, 1994), whereas the phosphatase that dephosphorylates phosphoSer49 has been identified and proven to be 2OG-sensitive (Irmler et al., 1997).

complex with AmtB (PDB 2NUU, Conroy et al., 2007); the rightmost panels show Chlamydomonas reinhardtii PII, taken from its complex with Arabidopsis thaliana NAGK (PDB 4USJ; Chellamuthu et al., 2014). (C) Illustration of different shapes of the T-loops found in distinct complexes with allosteric effectors or with partner proteins. The T-loop is shown in cartoon representation, within a semi-transparent surface representation as if this loop were isolated from the remainder of PII and from the protein partner in the complex. In the third panel, the side chain of Arg47 of E. coli GlnK is represented in sticks, given its importance for inhibiting the AmtB channel. Taken, from left to right, from: S. elongatus PII with MgATP and 2OG bound (PDB file 2XZW; Fokina et al., 2010a); S. elongatus PII-PipX (PDB 2XG8; Llácer et al., 2010); E. coli GlnK-AmtB complex (PDB 2NUU, Conroy et al., 2007); and PII-NAGK complex (PDB 2V5H; Llácer et al., 2007).

Forcada-Nadal et al. PII-NAGK-PipX-NtcA Regulatory Network

FRET studies with engineered fluorescent S.elongatus PII used as an ADP and ATP-sensitive probe (Lüddecke and Forchhammer, 2015) have challenged the claim (Radchenko et al., 2013) that PII proteins have a very slow ATPase activity that would regulate PII similarly as the signaling GTPases with bound GTP and GDP. Although this ATPase was reported as a 2OG-triggered switch that appeared an intrinsic trait of PII proteins (Radchenko et al., 2013), the FRET experiments with S. elongatus PII (Lüddecke and Forchhammer, 2015) appear to indicate that an endogenous ATPase is not a relevant mechanism for the transition of PII into the ADP state.

## PII COMPLEXES WITH CHANNELS

The first structurally solved PII complex was the one of E. coli GlnK with the AmtB ammonia channel (Conroy et al., 2007; Gruswitz et al., 2007) (**Figure 3A**) formed under nitrogen richness conditions. This structure showed that AmtB was inhibited by GlnK because the extended T-loop fits the channel entry, with the insertion into the channel of a totally extended arginine emerging from the T-loop and blocking the channel space (**Figure 2C**, 3rd panel from the left, and **Figure 3A**, zoom). The ADP-bound and MgATP/2OG-bound structures of an Archeoglobus fulgidus GlnK protein (Maier et al., 2011) indicated that 2OG may prevent GlnK binding to the ammonia channel because of induced flexing outwards (relative to the 3-fold molecular axis) of the T-loops, preventing their topographical correspondence with the three holes of the trimer of ammonia channels (**Figure 3B**). Interestingly, the T-loops of MgATP/2OG-bound S. elongatus PII (**Figure 2C**, leftmost panel) and A. fulgidus GlnK3 (**Figure 3B**) exhibited different flexed conformations (relative to the ADP-bound extended forms), and thus 2OG-binding by itself does not determine a single T-loop conformation, at least with different PII proteins.

Yeast two hybrid approaches (Osanai et al., 2005) detected the interaction between PII and the putative channel PamA (encoded by sll0985) of Synechocystis sp. PCC 6803 (from now on Synechocystis), but molecular detail on this protein is non-existent, and, therefore, it is uncertain whether such interaction might resemble the GlnK-AmtB interaction. PamA is not conserved in many cyanobacteria, and the most closely related putative protein of S. elongatus, the product of the Synpcc7942\_0610 gene, failed to give interaction signal with S. elongatus PII in yeast two hybrid assays (Castells, M.A., PhD Dissertation, Universidad de Alicante, 2010), despite the fact that the sequence identity with PamA concentrated in the C-terminal region, where PII binds in Synechocystis (Osanai et al., 2005). In vitro studies with the recombinantly produced Synechococystis PamA and PII showed that their interaction was lost in the presence of ATP and 2OG. Thus, similarly to the GlnK-AmtB and GlnK3-Amt complexes, the PII-PamA complex is formed under conditions of nitrogen abundance. However, T-loop phosphorylation did not dissociate this complex (Osanai et al., 2005). The function of PamA is not known, but its deletion from Synechocystis changed the expression of

FIGURE 3 | PII proteins and the ammonia channel. (A) The structure (PDB file 2NUU; Conroy et al., 2007) of the E. coli complex of GlnK (a PII protein in charge of ammonia channel regulation) and the ammonia channel AmtB is shown to the right, whereas the zoom to the lower left shows only a part of the complex, to highlight the interaction of one T-loop with one channel. AmtB is in semi-transparent surface representation. GlnK is in the main figure in cartoon representation with each subunit colored differently, with the side-chain of the T-loop residue Arg47 shown in sticks representation. In the zoomed image GlnK is shown in surface representation in yellow with the T-loop residues highlighted in space-filling representation, illustrating the fact that the side-chain of Arg47 is the element getting deep into the channel and blocking it. (B) Super-imposition of the structures of Archeoglobus fulgidus GlnK3 (one of the three PII proteins of the GlnK type in this archaeon; Maier et al., 2011) with ADP bound (green; PDB file code 3TA1) or with ATP and 2OG bound (yellow; PDB 3TA2) to illustrate how 2OG binding fixes the T-loops in an outwards-flexed position (relative to the positions without 2OG) that would be inappropriate for fitting the topography of the entry chambers to the three ammonia channels in trimeric Amt (the ammonia channel in this organism).

some NtcA-dependent genes (Osanai et al., 2005) by unclarified mechanisms.

### PII COMPLEXES WITH ENZYMES IN CYANOBACTERIA (AND BEYOND)

The complexes of PII with the N-acetyl-L-glutamate kinase (NAGK) enzymes from S. elongatus and Arabidopsis thaliana presented a very different architecture with respect to the structure of the GlnK-AmtB complex of E. coli (Llácer et al., 2007; Mizuno et al., 2007) (**Figures 4A,B**). The PII-NAGK complex is an activating complex in which the T-loops of PII are flexed (**Figure 2C**, rightmost panel) and integrated into a

FIGURE 4 | PII-NAGK complex and active and arginine-inhibited NAGK. (A) The PII-NAGK complex of S. elongatus (PDB 2V5H; Llácer et al., 2010). Surface representations of the complex formed by two PII trimers (yellow) capping on both ends the doughnut-like NAGK hexamer (trimer of dimers; each dimer in a different color). The three-fold axis is vertical (top) or perpendicular to the page (bottom). Figure of J.L. Llácer and V. Rubio taken from Chin (2008). Reprinted with permission from AAAS (B). Cartoon representation of the S. elongatus PII-NAGK complex after removing the back NAGK dimer for clarity. The three-fold symmetry axis is vertical. Reprinted from Current Opinion in Structural Biology, 18, Llácer et al., Arginine and nitrogen storage, 673–681, 2008, with permission from Elsevier. (C) PII subunit-NAGK subunit contacts. PII, NAGK, and NAG are shown as strings, ribbons, and spheres, respectively. The contacting parts of the T-loop, B-loop, and β1–α1 connection, including some interacting side chains (in sticks), are blue, red, and green, respectively. The surfaces provided by these elements form meshworks of the same colors. The NAGK central β-sheet is green, and other β-strands and the α-helices are brownish and grayish for N- and C-domains, respectively. Some NAGK elements and PII residues are labeled. This figure and its legend reproduce with some modifications a figure and its legend of Llácer et al. (2007). The crystal structure of the complex of PII and acetylglutamate kinase reveals how PII controls the storage of nitrogen as arginine. Copyright (2007) National Academy of Sciences. (D,E), active and inactive conformations, respectively, of hexameric arginine-inhibitable NAGK. The active form is from a crystal of the enzyme from Pseudomonas aeruginosa (PDB 2BUF) while the inactive form is from the Thermotoga maritima enzyme (PDB 2BTY) (Ramón-Maiques et al., 2006). Note that the inactive form is widened relative to the active form, and that it has arginine sitting on both sides of each interdimeric junction. In the active form the nucleotide (in this case the product ADP rather than the substrate ATP) and NAG sit one in each domain of individual subunits. The NAGK observed in the PII-NAGK complex is in the active form, being stabilized in this form by its contacts with PII.

hybrid (both proteins involved) β-sheet with NAGK, forming also a hybrid ion-pair network (**Figure 4C**; Llácer et al., 2007). Apparently this flexing from an extended conformation could occur in two steps (Fokina et al., 2010b). The initial step would be mediated by a smaller loop of PII called the B-loop (**Figures 2A**, **4C**). PII binding of 2OG also favors the flexing of the T-loop (**Figure 2C**, leftmost panel) (Fokina et al., 2010a; Truan et al., 2010) although the resulting conformation appears inappropriate for interacting with NAGK. In addition, 2OG can also promote the disassembly of the PII-NAGK complex because certain PII residues like Arg9 that are involved in the binding of 2OG are also involved in the interaction with NAGK (and also with PipX, another target of PII, see below). Therefore, 2OG, an indicator of low ammonia levels (Muro-Pastor et al., 2001), abolishes PII-NAGK complex formation (Maheswaran et al., 2004) (**Figure 1**). In S. elongatus 2OG can also promote the disassembly of the PII-NAGK complex by favoring the phosphorylation of Ser49 (Forchhammer and Tandeau de Marsac, 1994; Irmler et al., 1997), since the bound phosphate sterically prevents formation of the PII-NAGK hybrid β-sheet (Llácer et al., 2007).

Plants and cyanobacteria stockpile ammonia as arginine, the protein amino acid with the largest nitrogen content (four atoms per arginine molecule). Arginine-rich proteins are very abundant in plant seeds (VanEtten et al., 1963). Cyanobacteria make non-ribosomally an arginine-rich amino acid polymer called cyanophycin (Oppermann-Sanio and Steinbüchel, 2002; Watzer and Forchhammer, 2018). The arginine stockpiling as arginine-rich macromolecules minimizes the osmotic effect while permitting rapid nitrogen mobilization for protein-building processes such as seed germination and cell multiplication. The selection of NAGK as the regulatory target stems from the fact that in many bacteria (including cyanobacteria) and in plants NAGK controls arginine synthesis via feed-back inhibition by L-arginine (Hoare and Hoare, 1966; Cunin et al., 1986; Lohmeier-Vogel et al., 2005; Beez et al., 2009). This inhibition must be overcome if large amounts of ammonia have to be stored as arginine (Llácer et al., 2008). Indeed, the PII-NAGK complex exhibits decreased inhibition by arginine (Maheswaran et al., 2004; Llácer et al., 2008).

In arginine-sensitive NAGK (**Figures 4D,E**) the N-terminal αhelix of each subunit interacts with the same helix of an adjacent dimer, chaining three NAGK homodimers into a doughnutshaped hexameric ring with three-fold symmetry and a central large hole (Ramón-Maiques et al., 2006). The NAGK reaction (phosphorylation of the γ-COOH of N-acetyl-L-glutamate, NAG, by ATP) occurs within each NAGK subunit. NAG and ATP sit over the C-edge of the central 8-stranded largely parallel β sheet of the N-terminal and C-terminal domains, respectively (**Figure 4D**; Ramón-Maiques et al., 2002). Catalysis requires the mutual approach of both domains of each subunit to allow the contact of the ATP terminal phosphate with the attacking NAG γ-COOH (Ramón-Maiques et al., 2002; Gil-Ortiz et al., 2003). Arginine, by binding in each subunit next to the N-terminal αhelix (**Figure 4E**), expands the hexameric ring hampering the contact of the reacting groups and preventing catalysis (Ramón-Maiques et al., 2006).

In the PII-NAGK complex two PII trimers sit on the threefold axis of the complex, one on each side of the NAGK ring, making contacts with the inner circumference of this ring (**Figures 4A,B**). Each PII subunit interacts via its T and B loops with each NAGK subunit (**Figure 4C**) gluing the two domains of this last subunit (**Figure 4A**). By restricting NAGK ring expansion (**Figure 4C**) even when arginine is bound, PII renders NAGK highly active (Llácer et al., 2007; Mizuno et al., 2007). PII does not compete physically with arginine for its sites on NAGK, simply these sites are widened in the PII-NAGK complex (Llácer et al., 2007), resulting in decreased apparent affinity of NAGK for arginine (as reflected in the dependency of the NAGK activity on the arginine concentration). In addition, the hybrid PII-NAGK ion pair network (**Figure 4C**) enhances the apparent affinity for NAG (assessed as the Km or S0.5 value of NAGK for NAG) of cyanobacterial NAGK (Maheswaran et al., 2004; Llácer et al., 2007). Overall, the NAGK bound to PII exhibits decreased apparent affinity for arginine and increased activity, rendering NAGK much more active in the presence of arginine than when not bound to PII (Llácer et al., 2008), something that is crucial for nitrogen storage as arginine.

NAGK appears to be a PII target only in organisms performing oxygenic photosynthesis (cyanobacteria, algae, and plants, Burillo et al., 2004). PII proteins from plants have lost the ability to bind ADP, while still binding ATP and 2OG (Lapina et al., 2018). In addition, except in Brassicae, the Cterminal part of plant PII is extended to form two helical segments and a connecting loop (Q-loop; **Figure 2B**, rightmost panels), creating a novel glutamine site, resulting in glutaminesensitivity of the PII-NAGK interaction (Chellamuthu et al., 2014). This is not the case with cyanobacterial PII, which binds both ADP and ATP and is glutamine-insensitive (Chellamuthu et al., 2014).

PII has also been shown to interact in plants (Feria-Bourrellier et al., 2010) and bacteria, including cyanobacteria (Rodrigues et al., 2014; Gerhardt et al., 2015; Hauf et al., 2016), with the biotin carboxyl carrier protein (BCCP) of the enzyme acetyl coenzyme A carboxylase (AcCoA carboxylase) (**Figure 1**), although this complex has not been characterized structurally. BCCP is the component that hosts the covalently bound biotin that shuttles between the biotin carboxylase component and the transferase component of AcCoA carboxylase (Rubio, 1986). PII-BCCP complex formation tunes down AcCoA utilization and thus subsequent fatty acid metabolism (Feria-Bourrellier et al., 2010; Gerhardt et al., 2015; Hauf et al., 2016), promoting uses of AcCoA for different purposes than the synthesis of fatty acids, and therefore linking PII to AcCoA and fatty acid metabolism. For interaction, PII has to be in the ATP-bound and 2OGfree form (**Figure 1**) (Gerhardt et al., 2015; Hauf et al., 2016), which are conditions at which PII also binds to NAGK (Llácer et al., 2008). Therefore, there could be in vivo simultaneous activation of NAGK and inhibition of AcCoA carboxylase by PII. Mutational evidence suggests the involvement of the T-loop in this interaction with AcCoA carboxylase (Hauf et al., 2016), in principle excluding PII-NAGK-BCCP ternary complex formation and raising the possibility of competition between NAGK and BCCP for PII.

The classical example of interaction of PII with an enzymatic target was with the ATase of E. coli (see introductory section), with which uridylylated or deuridylylated GlnB (GlnB is one of the two PII proteins of E. coli) can interact (Stadtman, 2001). We will not deal with this enzyme here because the PII/ATase/GS cascade of enterobacteria does not appear to have general occurrence, for example in cyanobacteria, and also because we have only partial information on the structure of the ATase (Xu et al., 2004, 2010) and no direct information on the structure of the GlnB-ATase complex, although a model for such complex has been proposed (Palanca and Rubio, 2017).

### THE PipX ADAPTOR PROTEIN AND ITS COMPLEX WITH PII

A yeast two hybrid search for proteins interacting with PII in S. elongatus identified (Burillo et al., 2004), in addition to NAGK, a small novel protein (89 amino acids) that was named PipX (PII-interacting protein X). This protein was identified later in a search (Espinosa et al., 2006) for proteins interacting with NtcA, the global nitrogen regulator of cyanobacteria (Vega-Palas et al., 1992). PipX binding to PII occurs under conditions of ammonia abundance (**Figure 1**), the same conditions prevailing for PII-NAGK complex formation (Espinosa et al., 2006). NAGK-PipX competition for PII was revealed in NAGK assays that showed that PipX decreased PII-activation and increased arginine inhibition of NAGK (Llácer et al., 2010), excluding NAGK-PII-PipX ternary complex formation. 2OG binding to PII disassembles the PII-PipX complex (Espinosa et al., 2006; Llácer et al., 2010), leaving PipX free to interact with NtcA (**Figure 1**).

The crystal structures of PII-PipX complexes of S. elongatus (Llácer et al., 2010) and of Anabaena sp. PCC7120 (Zhao et al., 2010b) provided the first structural information on PipX (**Figure 5A**), revealing that it is formed by a compact body folded as a Tudor-like domain (a horseshoe-curved β-sheet sandwich) (Lu and Wang, 2013), followed by two C-terminal helices. In the PII-PipX complex (**Figure 5B**) the three PipX molecules are enclosed in a cage formed between the flat face of the hemispheric PII trimer and its three fully extended T-loops (see **Figure 2C**, 2nd panel from the left) emerging perpendicularly to the PII flat face at its edge. The shape and orientation of these T-loops is very different relative to the PII bound to NAGK, **Figure 5C**). In turn, the caged Tudor-like domains form a homotrimer over the PII flat surface (**Figure 5B**, bottom), with the PipX self-interaction detected in yeast three-hybrid assays using PII as bridging protein (Llácer et al., 2010). Tudor-like domains characteristically interact with RNA polymerase (Steiner et al., 2002; Deaconescu et al., 2006; Shaw et al., 2008), suggesting that PipX could have some role in gene expression that would be blunted by sequestration of these domains in the PII cage.

In the structure of the Anabaena PII-PipX complex, the two C-terminal helices of each PipX molecule lie one along the other in antiparallel orientation ("flexed"), being exposed between two adjacent T-loops in transversal orientation relative to these loops (Zhao et al., 2010b). Recent structural NMR data on isolated PipX showed that when PipX is alone (that is, not bound to a partner) the C-terminal helices are "flexed" (**Figure 5A**) (Forcada-Nadal et al., 2017). As shown below, the C-terminal helices of PipX in the NtcA-PipX complex are also flexed (Llácer et al., 2010). However, in the S. elongatus PII-PipX complex only one PipX molecule presents the "flexed" conformation, whereas in the other two PipX molecules the C-terminal helix is "extended," not contacting the previous helix and emerging centrifugally outwards from the complex, between two T-loops (**Figure 5B**) (Llácer et al., 2010). PII binding might facilitate the extension of the PipX C-terminal helix, endowing the PII-PipX complex with a novel surface and novel potentialities for interaction with other components. These novel potentialities were substantiated recently by the identification, in yeast three-hybrid searches (Labella et al., 2016), of interactions of PipX in the PII-PipX complex with the homodimeric transcription factor PlmA (see proposal for the architecture of this complex in **Figure 1** bottom left; Labella et al., 2016). Interactions were not observed in yeast two-hybrid assays between PipX or PII and PlmA. Residues involved in three-hybrid interactions, mapped by site-directed mutagenesis, are largely localized in the C-terminal helix of PipX. PlmA belongs to the GntR super-family of transcriptional regulators, but is unique to cyanobacteria (Lee et al., 2003; Hoskisson and Rigali, 2009; Labella et al., 2016). Little is known about PlmA functions other that it is involved in plasmid maintenance in Anabaena sp. strain PCC7120 (Lee et al., 2003), in photosystem stoichiometry in Synechocystis sp. PCC6803 (Fujimori et al., 2005), in regulation of the highly conserved cyanobacterial sRNA YFR2 in marine picocyanobacteria (Lambrecht et al., 2018), and that it is reduced by thioredoxin, without altering its dimeric nature in Synechocystis sp. PCC6803 (Kujirai et al., 2018). The PII-PipX-PlmA ternary complex suggests that PipX can influence gene expression regulation via PlmA, although the PlmA regulon remains to be defined.

### THE GENE EXPRESSION REGULATOR NtcA

When ammonia becomes scarce the increasing 2OG levels should determine the disassembly of the PII-NAGK, PII-BCCP, and PII-PipX complexes (**Figure 1**). These same conditions promote the binding of PipX (see below) to the transcriptional regulator NtcA (**Figure 1**), an exclusive cyanobacterial factor of universal presence in this phylogenetic group (Vega-Palas et al., 1992; Herrero et al., 2001; Körner et al., 2003). The determination of the structures of NtcA from S. elongatus (**Figures 6A,B**) (Llácer et al., 2010) and from Anabaena sp. PCC7120 (Zhao et al., 2010a) confirmed the sequence-based inference (Vega-Palas et al., 1992) that NtcA is a homodimeric transcriptional regulators of the family of CRP (the cAMP-regulated transcriptional regulator of E. coli) (McKay and Steitz, 1981; Weber and Steitz, 1987). Similarly to CRP, NtcA has a C-terminal DNA binding domain of the helix-turn-helix type. In CRP, the DNA binding helices of its two C-terminal domains are inserted in two adjacent turns of the major groove of DNA that host the imperfectly palindromic target DNA sequence (called here the CRP box)

(McKay and Steitz, 1981; Weber and Steitz, 1987). The consensus DNA sequence to which NtcA binds (consensus NtcA box) is quite similar to the consensus CRP box (Berg and von Hippel, 1988; Luque et al., 1994; Jiang et al., 2000; Herrero et al., 2001; Omagari et al., 2008), and thus NtcA and CRP are expected to bind in similar ways to their target DNA sequences.

In vitro studies revealed that 2OG is an NtcA activator (Tanigawa et al., 2002; Vázquez-Bermúdez et al., 2002), increasing NtcA affinity for its target sequences. As in the case of cAMP for CRP, 2OG binds to the NtcA regulatory domain. This domain is responsible for the NtcA dimeric nature (**Figure 6A**) (Llácer et al., 2010; Zhao et al., 2010a). The regulatory domain of NtcA is highly similar to the corresponding domain of CRP (Llácer et al., 2010). The main differences reflect the changes in the characteristics of the site for the allosteric effector that enable the accommodation of 2OG instead of cAMP. Each 2OG molecule interacts in NtcA with the two (one per subunit) long interfacial helices that form the molecular backbone, crossing the molecule in its longer dimension, linking in each subunit both domains (**Figures 6A,C**) (Llácer et al., 2010; Zhao et al., 2010a). 2OG interactions with both interfacial helices favor a twist of one helix relative to the other, dragging the DNA binding domains and helices to apparently appropriate positions and interhelical distance for binding in two adjacent turns of the major groove of DNA where the NtcA box should be found (**Figures 6A,B**, and inset therein), although the experimental structure of

(bottom). (C) Superimposition of S. elongatus PII in the complex with PipX and in that with NAGK. The changes in the T-loops are very patent.

FIGURE 6 | NtcA structure, 2OG binding to it and associated conformational changes. (A,B), structures of "active" (A) and "inactive" (B) S. elongatus NtcA (PDB files 2XHK and 2XGX, respectively) (Llácer et al., 2010). The two subunits of each dimer are in different colors, with the DNA-binding domains in a lighter hue than the regulatory domain of the same subunit. In the cartoon representation used, helices are shown as cylinders to illustrate best the changes in position of the DNA binding helices and of the long interfacial helices (labeled) upon activation. Bound 2OG is shown in "active" NtcA (in spheres representation, with C and O atoms colored yellow and red, respectively). The DNA, in surface representation in white, has been modeled from the CRP-DNA structure (for details see Llácer et al., 2010). The inset superimposes the "active" and "inactive" forms colored as in the main figure to illustrate the magnitude of the changes. (C) Stereo view of sticks representation of the 2OG site residues of the "active" (green) and "inactive" (raspberry) forms of NtcA. The 2OG bound to the "active" form is distinguished by its yellow C atoms. Note that only two residues, both 2OG-interacting and highly polar, experience large changes in their positions between the inactive and the active forms: Arg128 from the long interfacial helix of the subunit that provides the bulk of the residues of the site, and Glu133 from the interfacial helix of the other subunit. They are believed to trigger the changes in the relations between the two interfacial helices that result in NtcA "activation".

DNA-bound NtcA should be determined to corroborate this proposals.

Although NtcA and CRP boxes are quite similar, plasmon resonance experiments (Forcada-Nadal et al., 2014) revealed that CRP exhibits complete selectivity and specificity for the CRP box, with absolute dependency on the presence of cAMP. In contrast, NtcA had less strict selectivity, since it still could bind to its promoters in the absence of 2OG, although with reduced affinity, and it could also bind to the the CRP promoter tested. Nevertheless, it is unlikely that NtcA could bind in vivo to the CRP boxes of cyanobacteria in those species where CRP is also present, given the much higher affinities for the CRP sites of cyanobacterial CRP and the relative concentrations in the cell of both transcriptional regulators (Forcada-Nadal et al., 2014).

While the structures of 2OG-bound NtcA of S. elongatus (Llácer et al., 2010) and of Anabaena (Zhao et al., 2010a) are virtually identical, the reported structures of "inactive" NtcA of Anabaena without 2OG (Zhao et al., 2010a) and of S. elongatus (Llácer et al., 2010) differed quite importantly in the positioning of the DNA binding domains (**Figure 1**, "Inactive forms" under "NtcA"), although in both cases the DNA binding helices were misplaced for properly accommodating the NtcA box of DNA, raising the question of whether these structural differences are species-specific or whether "inactive" NtcA can be in a multiplicity of conformations.

## PipX AS AN NtcA CO-ACTIVATOR

Soon after PipX was found to interact with NtcA (Espinosa et al., 2006), it was also shown to activate in vivo transcription of NtcA-dependent promoters under conditions of low nitrogen availability (Espinosa et al., 2006, 2007). Direct binding studies with the isolated molecules proved that PipX binding to NtcA requires 2OG (Espinosa et al., 2006). Nevertheless, as PipX was not totally essential for transcription of NtcA-dependent promoters (Espinosa et al., 2007; Camargo et al., 2014), it was concluded (1) that PipX was a coactivator of 2OG-activated NtcA-mediated transcription (Espinosa et al., 2006; Llácer et al., 2010); and (2) that the degree of activation by PipX depended on the specific NtcA-dependent promoter (Espinosa et al., 2007; Forcada-Nadal et al., 2014). Detailed plasmon resonance studies (Forcada-Nadal et al., 2014) using sensorchip-bound DNA confirmed for three Synechocystis promoters that PipX binding to promoter-bound NtcA has an absolute requirement for 2OG, since no PipX binding was observed when NtcA was bound to the DNA in absence of 2OG. In these studies PipX increased about one order of magnitude the apparent affinity of NtcA for 2OG. In other in vitro experiments with four NtcAdependent promoters of Anabaena sp. PCC 7120, PipX was also found to positively affect NtcA binding to its DNA sites (Camargo et al., 2014). The induction by PipX of increased NtcA affinity for 2OG and for its promoters could account for the PipX-triggered enhancement of NtcA-dependent transcription.

The crystal structure of the NtcA-PipX complex of S. elongatus (**Figure 7**) (Llácer et al., 2010) corresponded to one "active" NtcA dimer with one molecule of each 2OG and PipX bound to each subunit. PipX is inserted via its Tudor-like domain (**Figure 7A**), filling a crater-like cavity formed over each NtcA subunit (**Figure 7B**) largely over one regulatory domain, being limited between the DNA binding domain and the long interfacial helix of the same subunit, and the regulatory domain of the other subunit. PipX extensively interacts with the entire crater, with nearly 1200 Å2 of NtcA surface covered by each PipX molecule, of which 65% belongs to one subunit (40%, 15% and 10% belonging to the DNA-binding domain, the interfacial helix and the regulatory domain, respectively) and 35% belongs to the regulatory domain (including the interfacial helix) of the other subunit, gluing together the elements of half of the NtcA dimer in its active conformation, stabilizing this conformation (Llácer et al., 2010). This conformation is the one that binds 2OG and that should have high affinity for the DNA, thus explaining the requirement of 2OG for PipX binding and the increased affinities of NtcA for 2OG and DNA when PipX was bound to NtcA (Forcada-Nadal et al., 2014). Since a similar crater-like cavity exists in other transcription factors of the CRP family including CRP (see for example McKay and Steitz, 1981 or Weber and Steitz, 1987) it would be conceivable that PipX-mimicking proteins could exist for these other transcriptional regulators of the CRP family, although PipX cannot do such a role since it does not bind to CRP (Forcada-Nadal et al., 2014). Furthermore, a large set of highly specific contacts (Llácer et al., 2010) ensure the specificity of the binding of PipX to NtcA.

The elements of the Tudor-like domain that interact with NtcA are largely the same that interact with the flat surface of the hemispheric body of PII (many of them mediated by the upper layer of the Tudor-like β-sandwich, particularly strands β1 and β2), predicting total incompatibility for the simultaneous involvement of PipX in the NtcA and PII complexes (Llácer et al., 2010). While the Tudor-like domain monopolizes the contacts of PipX with NtcA, the C-terminal helices of PipX do not participate in these contacts and remain flexed, as in isolated PipX (Forcada-Nadal et al., 2017), protruding away from the

complex (**Figure 7A**). The coactivation functions of PipX for NtcA-mediated transcription could also involve these helices. However, in vitro experiments (Llácer et al., 2010; Camargo

counterpart in CRP and is involved in the interactions with PipX.

et al., 2014) and modeling (based on CRP) of DNA binding by the NtcA-PipX complex (**Figure 7A** and inset therein) (Llácer et al., 2010) did not support the idea of PipX binding to DNA.

(αCTD), to show that the C-terminal helices of PipX could reach this part of the polymerase. In this figure the C-terminal helix of NtcA is colored red because it has no

Alternatively, these helices could interact with RNA polymerase, particularly given the location, in the homologous CRP-DNA complex, of the binding site for the α-subunit of the C-terminal domain (αCTD) of RNA polymerase (**Figure 7C**; and discussed in Llácer et al., 2010). Further structures of PII-PipX bound to DNA alone or with at least some elements of the polymerase are needed to clarify these issues.

### THE PipX REGULATORY NETWORK IN QUANTITATIVE PERSPECTIVE

The gene encoding PII could not be deleted in S. elongatus unless the pipX gene was previously inactivated (Espinosa et al., 2009). Further studies led to the conclusion that decreasing the PII/PipX ratio results in lethality in S. elongatus, indicating that PipX sequestration into PII-PipX complexes is crucial for survival and implicating both proteins in the regulation of essential processes (Espinosa et al., 2010, 2018; Laichoubi et al., 2012). The ability of PII to prevent the toxicity of PipX suggests that PII acts as a PipX sink even under conditions in which the affinity for NtcA would be highest, supporting the idea that not all PipX effects are related to its role as NtcA co-activator. Mutational studies (Espinosa et al., 2009, 2010) and massive transcriptomic studies of S. elongatus mutants centered on PipX (Espinosa et al., 2014) also support the multifunctionality of PipX, stressing the need for additional studies, including the determination of PlmA functions and the search for further potential PipX-interacting proteins (**Figure 1**, broken yellow arrow).

Massive proteomic studies (Guerreiro et al., 2014) have estimated the number of chains of each protein of the PII-PipX network (**Figure 1**) in S. elongatus cells. The values obtained (**Table 1**) are corroborated by those obtained in focused western blot studies for some of these macromolecules (**Table 1**) (Labella et al., 2016, 2017). These quantitative data give an opportunity to evaluate the possible frequency of the different complexes and macromolecules of the PII-PipX-NtcA network (**Figure 1**) in one or another form (schematized in **Figure 8**). Of all the proteins mentioned here until now, PII is by far the most abundant in terms of polypeptide chains (**Table 1**). In comparison, the sum of all the chains of other known PII-binding proteins represents no more than 20% of the PII chains. Among these molecules is PipX, which only represents ∼10% of all the PII chains. This indicates that PII has the potential to sequester all the PipX that is present in the cell (**Figure 8**). In turn, PlmA could be fully trapped in the PII-PipX-PlmA complex if this complex has the 1:1:1 stoichiometry proposed for it (**Figure 1**) (Labella et al., 2016), since the number of PlmA chains only represent about 10% of the number of PipX chains. Thus, about 10 and 1% of the PII trimer could be as the respective PII-PipX and PII-PipX-PlmA complexes under nitrogen abundance conditions. In contrast, with nitrogen starvation all of the NtcA could be bound to PipX (**Figure 8**), given the ∼five-fold excess of PipX chains over NtcA chains (**Table 1**). Thus, assuming that under conditions at which 2OG and ATP reach high levels PII is totally unable to bind PipX, ∼80% of the PipX molecules could be free to interact with additional protein partners.

When given as percentages, the data are relative to the number of PII chains (given the value of 100).

aData from massive proteomic study (Guerreiro et al., 2014). Percentages within parentheses are data based on immunoquantification in Western blots (Labella et al., 2016, 2017).

bRounded to the closest integer or to the closest first decimal figure.

cGiven for reference, since there is no evidence of physical interaction with any of the other proteins.

dThe physical interaction of this putative channel with PII was found in Synechocystis sp. PCC6803, but the findings were not replicated in S. elongatus with the homologous product of gene Synpcc7942\_0610.

These inferences are consistent with the KD values for the PII-NAGK (Llácer et al., 2007) and PII-PipX complexes in the absence of 2OG (Llácer et al., 2010) and for the PipX-NtcA complex at high 2OG and ATP (Forcada-Nadal et al., 2014) (∼0.08, 7 and 0.09µM, respectively). For the estimated cellular levels of the different components (**Table 1**), assuming a cell volume of 10−12 ml, virtually all the NAGK and ∼95% of the PipX could be PIIbound in the absence of 2OG, and ∼98% of the NtcA could be PipX-complexed in the presence of 2OG. However, the impacts of varying concentrations of 2OG on the disassembly or assembly of the complexes most likely differ for the various complexes. For example, a two-order of magnitude increase in the KD value for the PII-NAGK complex due to 2OG binding might have much less impact (a 7% decrease in the amount of NAGK bound to PII would be estimated from the mere total protein levels and KD value) than a two-order of magnitude increase in the KD for the PII-PipX complex (an 80% decrease in the amount of PipX bound estimated similarly). These estimations are very crude, since they do not take in consideration that in S. elongatus PII phosphorylation prevents NAGK binding (Heinrich et al., 2004), and that this phosphorylation is greatly influenced by the abundance of ammonia (Forchhammer and Tandeau de Marsac, 1994). Furthermore, we have not considered in these estimates the influence of the ATP concentrations, recently shown to decrease in vivo in S. elongatus upon nitrogen starvation (Doello et al., 2018). Therefore, the situation is much more complex than would be expected from the mere consideration of the abundances of the different proteins and of the KD values for the non-phosphorylated form of PII. Nevertheless, it appears desirable to estimate the influence of different 2OG levels on KD values as an important element to take into consideration in future attempts to model the concentrations of the different

FIGURE 8 | Protein complexes of the PII regulatory system in S. elongatus according to availability of ammonium in the cell, and their corresponding functional consequences. The frequencies of the different chains in the various forms are based on the levels of the proteins in the cell found in proteomic studies (Table 1). The PII trimer has been colored blue, PipX and its C-terminal helices are red, PlmA dimers have their DNA binding domains yellow or orange and their dimerization domains in two hues of green, NAGK is shown as a purple crown, and the regulatory and DNA binding domains of NtcA are given dark and light shades of blue, respectively.

complexes of PII and PipX under intermediate conditions of nitrogen richness.

### A NOVEL NETWORK MEMBER FUNCTIONALLY RELATED TO PipX

Recently, a novel protein has been identified as belonging to the PipX regulatory network (**Figure 1**, yellow 3D arrow projecting upwards from the plane of the network). In this case no direct protein-protein interaction with PipX has been shown (Labella et al., 2017). This protein (PipY), is the product of the downstream gene in the bicistronic pipXY operon. The regulatory influence of PipX on PipY was originally detected in functional, gene expression and mutational studies (Labella et al., 2017). More recently it has been concluded (Cantos et al., 2018) that PipX enhances pipY expression in cis, preventing operon polarity, a function that might implicate additional interactions of PipX with the transcription and translation machineries, by analogy with the action of NusG paralogues, which are proteins bearing, as PipX, Tudor-like domains. It has been proposed (Cantos et al., 2018) that the cis-acting function of PipX might be a sophisticated strategy for keeping the appropriate PipX-PipY stoichiometry.

PipY is an intriguing pyridoxal phosphate-containing protein that is folded as a modified TIM-barrel (**Figure 1**). The PipY structure (Tremiño et al., 2017) gives full structural backing to unsuccessful experimental attempts to show enzymatic activity of PipY and its orthologs (Ito et al., 2013). Because of these negative findings, and given the pleotropic effects of the inactivation of the PipY orthologs in microorganisms and humans, it has been concluded that these proteins have as yet unclarified roles in PLP homeostasis (Ito et al., 2013; Darin et al., 2016; Prunetti et al., 2016; Labella et al., 2017; Plecko et al., 2017). Interestingly, these proteins are widespread across phyla (Prunetti et al., 2016) and the deficiency of the human ortholog causes vitamin B6 dependent epilepsy (Darin et al., 2016; Plecko et al., 2017; Tremiño et al., 2018), providing an excellent example that investigations of cyanobacterial regulatory systems like the one summarized here can have far-reaching consequences spanning up to the realm of human and animal pathology. If any lesson can be inferred from all of the above, is that the investigation on PII and particularly on PipX proteins require further efforts.

### FINAL REMARKS

The rich PII regulatory network summarized in **Figure 1** of this review even for a unicellular microorganism with a single type of PII protein, attests the importance of PII and of its regulatory processes. This importance, possibly underrecognized until now, is highlighted, for example, by the very wide distribution of PII proteins among microorganisms and plants. Furthermore, in the many organisms with several genes for PII proteins the levels of complexity expected from PII regulatory networks may be much greater than the one presented here. Each one of these paralogous PII proteins may command a regulatory network, and it would be unlikely that these networks would not be interconnected into a large meshwork that will require the instruments of systems biology to be fully understood.

PipX also deserves deeper attention than received until now. Massive transcriptomics studies (Espinosa et al., 2014) have ascribed to this protein a paramount regulatory role in S. elongatus. For full understanding of this role further searches for PipX-interacting or functional partners like PipY appear desirable, with detailed investigation of the molecular mechanisms of the physical or the functional interactions. PlmA merits particular attention to try to characterize the roles of the PII-PipX-PlmA complex. PipY and its orthologs deserve similar attention, to try to define molecularly their PLP homeostatic functions, a need that is made more urgent by the role in pathology of the human ortholog of PipY. In addition to all of this, the structural evidence reviewed here makes conceivable that adaptor proteins capable of stabilizing active conformations of other transcriptional regulators of the CRP family could exist outside cyanobacteria, mimicking the PipX role. In summary, there are many important questions to be addressed arising from the field reviewed here, some within cyanobacteria, but others concerning whether the mechanisms and complexes exemplified here could have a parallel in other bacterial or even plant species. Clearly more investigations on PII and its partners in other phylogenetic groups using the approaches and experimental instruments used to uncover the cyanobacterial PII regulatory network would appear highly desirable.

## AUTHOR CONTRIBUTIONS

AF-N, JLL, AC, CM-M, and VR reviewed the literature and their own previous work and contributed to the discussions for writing this review. The main writer was VR, but all the authors contributed to the writing of the manuscript. AF-N and CM-M prepared the figures, with key inputs from VR.

### FUNDING

Supported by grants BFU2014-58229-P and BFU2017-84264-P from the Spanish Government.

### ACKNOWLEDGMENTS

We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). We are grateful to ESRF (Grenoble, France), Diamond (Oxfordshire, UK) and Alba (Barcelona, Spain) synchrotrons for access and for staff support to collect the data used for the determination by our group of most of the structures mentioned in this paper, which have been previously published as referred. CM-M holds a contract of CIBERER.

### REFERENCES

activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit. Proc. Natl. Acad. Sci. U.S.A. 107, 502–507. doi: 10.1073/pnas.0910097107

revealed by the structures of two hexameric N-acetylglutamate kinases, from Thermotoga maritima and Pseudomonas aeruginosa. J. Mol. Biol. 356, 695–713. doi: 10.1016/j.jmb.2005.11.079

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer JAH declared a shared affiliation, with no collaboration, with several of the authors, AF-N, JL, CM-M, and VR to the handling editor at time of review.

Copyright © 2018 Forcada-Nadal, Llácer, Contreras, Marco-Marín and Rubio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# RNA Binding Protein Regulation and Cross-Talk in the Control of AU-rich mRNA Fate

Sofía M. García-Mauriño, Francisco Rivero-Rodríguez, Alejandro Velázquez-Cruz, Marian Hernández-Vellisca, Antonio Díaz-Quintana, Miguel A. De la Rosa and Irene Díaz-Moreno\*

Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas, Seville, Spain

#### Edited by:

Maria Rosaria Conte, King's College London, United Kingdom

#### Reviewed by:

Graeme L. Conn, Emory University School of Medicine, United States Santiago Martinez-Lumbreras, King's College London, United Kingdom Scott A. Tenenbaum, University at Albany, Suny, United States Teresa Carlomagno, Leibniz University of Hanover, Germany

> \*Correspondence: Irene Díaz-Moreno<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Structural Biology, a section of the journal Frontiers in Molecular Biosciences

> Received: 26 July 2017 Accepted: 04 October 2017 Published: 23 October 2017

#### Citation:

García-Mauriño SM, Rivero-Rodríguez F, Velázquez-Cruz A, Hernández-Vellisca M, Díaz-Quintana A, De la Rosa MA and Díaz-Moreno I (2017) RNA Binding Protein Regulation and Cross-Talk in the Control of AU-rich mRNA Fate. Front. Mol. Biosci. 4:71. doi: 10.3389/fmolb.2017.00071 mRNA metabolism is tightly orchestrated by highly-regulated RNA Binding Proteins (RBPs) that determine mRNA fate, thereby influencing multiple cellular functions across biological contexts. Here, we review the interplay between six well-known RBPs (TTP, AUF-1, KSRP, HuR, TIA-1, and TIAR) that recognize AU-rich elements (AREs) at the 3 ′ untranslated regions of mRNAs, namely ARE-RBPs. Examples of the links between their cross-regulations and modulation of their targets are analyzed during mRNA processing, turnover, localization, and translational control. Furthermore, ARE recognition can be self-regulated by several factors that lead to the prevalence of one RBP over another. Consequently, we examine the factors that modulate the dynamics of those protein-RNA transient interactions to better understand the final consequences of the regulation mediated by ARE-RBPs. For instance, factors controlling the RBP isoforms, their conformational state or their post-translational modifications (PTMs) can strongly determine the fate of the protein-RNA complexes. Moreover, mRNA specific sequence and secondary structure or subtle environmental changes are also key determinants to take into account. To sum up, the whole understanding of such a fine tuned regulation is a challenge for future research and requires the integration of all the available structural and functional data by in vivo, in vitro and in silico approaches.

Keywords: mRNA fate, post-transcriptional regulation, RNA binding proteins, stability, translation

#### POST-TRANSCRIPTIONAL REGULATION OF GENE EXPRESSION BY ARE-RBPs

In eukaryotes, gene expression levels and protein abundance are often correlated but are subjected to a strict regulation. The control of mRNA metabolism allows cells to rapidly adapt to changing environmental conditions. Regulatory processes occurring after mRNA transcription—namely post-transcriptional control—strongly influence mRNA fate and, consequently, final protein levels (Vogel and Marcotte, 2012). Once mRNA transcription occurs in the nucleus, RNA Binding Proteins (RBPs) recognize the primary transcript or pre-mRNA to regulate its alternative splicing, polyadenylation, and capping (**Figure 1**). The generated mature mRNA is then transported to the cytoplasm by various other RBPs. Once in the cytoplasm, RBPs govern the stability, distribution to different cellular compartments and the translation of target mRNAs into their corresponding protein products (Matoulkova et al., 2012).

Within RBPs, ARE-RBPs function as trans-acting factors recognizing cis-acting elements in the 3′ -Unstranslated Regions (UTR) of eukaryotic mRNA enriched in adenylate and uridylate (AU-rich elements or AREs). AREs are present in 5–8% of human genes with diverse functions such as cell growth and differentiation, signal transduction, apoptosis, nutrient transport, and metabolism. This list is dominated by genes involved in transient processes, which therefore require strict expression control (Barreau et al., 2005). For instance, the length and specific pattern of AREs may contribute to mRNA lifetime (Khabar, 2005). However, the final mRNA fate will be determined by the variable and dynamic ARE-RBPs/mRNA interactions or by RBP competition for the same transcript. Besides, ARE-RBPs bind to AREs via a variety of domains including the so-called RNA-Recognition Motif (RRM), the CCCH tandem zinc finger and the K-Homology domain (KH) (Stoecklin and Anderson, 2006; Clery et al., 2008; Valverde et al., 2008; Daubner et al., 2013). A single protein can contain several of these motifs leading to simultaneous interactions with either multiple targets or multiple sites within a particular target (Shen and Malter, 2015). Additionally, most ARE-RBPs shuttle between nucleus and cytoplasm; and their functions are linked to their specific subcellular distribution (Gama-Carvalho and Carmo-Fonseca, 2001).

In this mini-review we focus on the post-transcriptional regulation exerted by six of the best studied ARE-RBPs whose cross-talk has biological relevance and has been widely reported in the literature. Moreover, we examine the multiple intracellular signals and factors controlling the interactions between these proteins. AU-binding Factor 1 (AUF1), also known as Heterogeneous Nuclear RiboNucleo-Protein D (hnRNPD), is included for being the first identified ARE-RBP (Brewer, 1991). AUF1 is generally considered to promote the decay of target mRNAs, although the stabilization of some other transcripts has been also reported (Xu et al., 2001; Stoecklin and Anderson, 2006). Since AUF1 discovery, 20 additional ARE-RBPs have been identified. That list includes those that primarily promote mRNA degradation, such as Tristetraprolin (TTP) and KH domainsplicing regulatory protein (KSRP) (Gherzi et al., 2004; Sanduja et al., 2011); those stabilizing mRNA, such as Human antigen R (HuR) (Brennan and Steitz, 2001); and translational control proteins, such as T-cell intracellular antigen 1 (TIA-1) and TIA-1–related protein (TIAR) (Kawai et al., 2006; Mazan-Mamczarz et al., 2006).

### INTERPLAY BETWEEN ARE-RBPs IN THE POST-TRANSCRIPTIONAL REGULATION OF mRNAs

It is well-known that the substrates of post-transcriptional control are RNA ribonucleoprotein particles or RNPs containing mRNA molecules covered with RBPs, rather than naked mRNA (Szostak and Gebauer, 2013) (**Figure 1**). However, our understanding of how ARE-RBPs interact with each other at different regulatory levels is rather limited. Noticeably, some RBPs regulate the mRNA that encodes their own gene products, as well as those of other RBP counterparts, establishing selfregulatory loops controlling mRNA metabolism (Pullmann et al., 2007).

A good example of cross-talk between RBPs is the one involving HuR, KSRP and TTP proteins. These three proteins compete with each other for binding to common recognition sequences in the AREs that they regulate.  Moreover, TTP acts as a negative regulator of its own mRNA (Tchen et al., 2004; Lin et al., 2007) as well as HuR mRNA, its direct antagonist in mRNA regulation (Al-Ahmadi et al., 2009). On the other hand, HuR acts as a positive translational regulator of both KSRP and HuR mRNAs (Pullmann et al., 2007; Yi et al., 2010); while both proteins regulate the stability of their own mRNAs (Winzen et al., 2007; Al-Ahmadi et al., 2009) (**Table 1**, dashed square). Consequently, the redundant feedback involving KSRP, TTP, and HuR may provide a bi-stable signal transduction circuit in which either all or none of their target mRNAs are stabilized and/or translated. More intriguingly is the role of AUF1 in this regulatory loop as it presents four isoforms generated by alternative splicing of a single mRNA transcript (Wagner et al., 1998) with different RNA-binding affinities and specificities for its target mRNAs—such as c-fos, c-myc, TNFα, VEGF, and COX-2 (Brewer, 1991; Loflin et al., 1999; Lasa et al., 2000; Xu et al., 2001; Fellows et al., 2012).

In addition to recognizing AU-rich sequences at the 3′ UTR of target mRNAs, some ARE-RBPs are able to activate splice 5′ sites followed by U-rich sequences. This is the case of TIA-1 and TIAR, that upregulate the translation of their own coding mRNAs (Le Guiner et al., 2001). Conversely, although consistent with their functional redundancy, their translation levels are negatively cross-regulated by each other (Le Guiner et al., 2001; Izquierdo and Valcárcel, 2007b; Pullmann et al., 2007) (**Table 1**, dotted square). Interestingly, TIA-1 and TIAR share common functions, acting as negative translational regulators of diverse mRNAs—such as c-myc, TNFα, VEGF, and COX-2—and are able to compensate for each other (Gueydan et al., 1999; Piecyk et al., 2000; Zhang et al., 2002; Cok et al., 2003; Dixon et al., 2003; Lu et al., 2009; Hamdollah Zadeh et al., 2015). In addition, it has been shown that HuR positively controls TIA-1 expression by enhancing its mRNA stability (Pullmann et al., 2007). By contrast, TIA-1 knockdown causes a marked increase in HuR levels, indicating that TIA-1 may contribute to lowering HuR levels in the cell (Kawai et al., 2006) (**Table 1**, black square). This is of great importance because both HuR and TIA-1 bind to cytochrome c (Cc) mRNA, respectively promoting or inhibiting its translation without affecting its mRNA stability. The struggle between HuR (antiapoptotic factor) and TIA-1 (proapoptotic factor) for the control of Cc mRNA translation underlies possible mechanisms to regulate both cellular respiration and programmed cell death. A direct binding between HuR and TIAR mRNA has also been reported (Pullmann et al., 2007) but, unexpectedly, TIAR does not seem to complex with Cc mRNA, despite the extensively shared homology between TIAR and TIA-1 (Kawai et al., 2006).

### FACTORS THAT MODULATE ARE-RBP/mRNA INTERACTIONS

Several examples of cross-talk between ARE-RBPs highlight that there must be an intricate network of regulatory events that lead to the prevalence of one RBP over the others when recognizing the same mRNA target. Thus, the regulatory activity of RBPs on gene expression is dynamic and adapts to cell conditions continuously. In this section, we briefly describe those factors for which there is evidence of their influence on the interaction between RBPs and their mRNA targets (Supplemental Figure 1).

#### RBP Isoforms

Alternative splicing is a highly regulated process that allows the synthesis of multiple different transcripts from the same gene, and is therefore an important source of protein diversity and complexity. The slight differences in amino acid sequence between isoforms can be determining for their function (Gallego-Páez et al., 2017). For example, TIA-1 and TIAR present two isoforms—a and b—in humans. Isoform a in TIA-1 and TIAR possesses 11 or 17 extra amino acids, respectively, that are critical for distinct functional properties. For instance, only TIAR isoform a—but not TIAR b and none of TIA-1 isoforms—has a translational silencing activity on the proteolytic enzyme Human Matrix Metalloproteinase-13 (HMMP13) in HEK293 cells. High levels of expression of HMMP13 have been documented in certain subset of cancers. Therefore, its downregulation by TIAR a may act as a tumor suppression mechanism (Yu et al., 2003).

As previously mentioned, AUF1 isoforms come from the alternative splicing of the same pre-mRNA. They differ as a function of the presence or absence of two independent domains encoded by exons 2 and 7. While p37AUF1 lacks both domains, p42AUF1 and p45AUF1 include a 49-amino acid domain encoded by exon 7 and p40AUF1 and p45AUF1 both contain a 19-amino acid domain encoded by exon 2. Inclusion of the exon 2-encoded sequence reduces the affinity of the first and second binding events of AUF1 dimers toward their mRNA substrates, but incorporation of the exon 7-encoded sequence increases the affinity of the second binding event. The isoformspecific differences provide unique biochemical characteristics that explain the diversity of AUF1 functions and complex regulation (Zucconi et al., 2010).

TABLE 1 | Matrix representation of the interaction of selected RBPs (vertical axis) with the mRNA of those RBPs (Upper table) and several ARE-containing mRNA targets (Lower table, horizontal axis).

#### ARE-containing mRNA

Blue colors show positive regulation, whereas negative regulations are colored in red. Gray color indicates interactions that have been described but the effects were not examined. n.d., non-described; S, Splicing; \*, Postulated regulations. The cross-talk between TTP, KSRP and HuR is highlighted by a dashed square; between HuR and TIA-1 by a black square; and between TIA-1 and TIAR by a dotted square.

#### RBP Post-Translational Modifications

Post-Translational Modifications (PTMs), such as phosphorylation, isomerization, methylation, NEDDylation, acetylation, and ubiquitination of RBPs have a major influence on their function and/or their affinity toward their targets, with the consequent impact on mRNA stability, turnover and translation efficiency (Lee, 2012). For instance, the phosphorylation of p40AUF1 in residues Ser83 and Ser87 influences the sequential binding of dimers to TNFα mRNA (Wilson et al., 2003). Single phosphorylation of Ser83 inhibits by 40% the initial dimer binding to mRNA substrate, whereas Ser87-single phosphorylation induces a 2-fold increase in the affinity of the second binding event. In addition, when simultaneous phosphorylation of both residues occurs, the negative effect on the binding affinity of Ser83 prevails over the positive effect of Ser87 (Wilson et al., 2003). Several phosphorylation sites have also been identified in TTP (Cao et al., 2006, 2014). Phosphorylated TTP binds with a lower affinity than the dephosphorylated TTP to target AREs (Carballo et al., 2001; Hitti et al., 2006). Phosphorylation of RBPs can also modify their activity without altering the affinity for mRNA targets. Such is the case of TIA-1 and TIAR, whose splicing control over the Fas gene sequence determines the expression of the pro-apoptotic membrane-bound form in detriment of the anti-apoptotic soluble one (Izquierdo and Valcárcel, 2007a). Moreover, HuR methylation has been proposed to increase the nuclear export of HuR, which could be important for mRNA localization (Li et al., 2002). NEDDylation of HuR increases its stability and lifetime, which, in turn, can affect the total levels of HuR target mRNAs due to its main stabilizing action (Embade et al., 2012; Fernández-Ramos and Martínez-Chantar, 2015).

#### RBP Conformational Changes

ARE-RBPs can undergo conformational changes upon binding to their targets (Ellis and Jones, 2008). These variations can be detected in the contact surface with mRNAs as well as in distant areas, meaning that ARE-RBPs can adapt both the local and global structure. An example of conformational changes that influence ARE recognition has been reported for KSRP. An interdomain re-arrangement, that orients the two central KH domains and their RNA-binding surfaces creating a two-domain unit, is crucial for its role in ARE-mediated mRNA decay (Supplemental Figure 2) (Díaz-Moreno et al., 2010). Additionally, some of the PTMs mentioned above can also influence the conformation of RBPs. Hence, the phosphorylation of Ser193 within the Nterminal KH motif (KH1) of KSRP leads to the unfolding of this structurally atypical and unstable domain, creating a binding site for 14-3-3ζ, driving the nuclear localization of KSRP and controlling its mRNA-degradation activity (Díaz-Moreno et al., 2009).

Another important regulation factor is the RBP oligomerization state upon mRNA recognition. HuR RRM1 domain and RRM1-2 di-domain (the main platform of cytoplasmic mRNA binding in HuR) form homodimers in solution (Benoit et al., 2010). This phenomenon is dependent on Cys13, which is able to form disulfide bonds. Such homodimerization may modulate HuR function upon oxidative stress. Moreover, the HuR RRM3 domain has been found to be involved in protein oligomerization and RNA recognition, both functions regulated by the same RRM but using different surfaces at opposite sides of the domain. The conserved Trp261 residue is key for dimerization, as the substitution by glutamic acid alters its dimerization dynamics and stabilizes the monomeric state (Scheiba et al., 2014; Díaz-Quintana et al., 2015).

### Cellular Conditions and Stress Response

Eukaryotic cells have evolved sophisticated strategies to overcome stress. One of them is the assembly of Stress Granules (SGs), which allows mRNA translation silencing and protection from degradation. Among RBPs with critical roles in neurodegenerative diseases, TIA-1 proteins are essential in SG formation (Mazan-Mamczarz et al., 2006; Vanderweyde et al., 2012). Hence, under hypoxic conditions, TIA-1 and TIAR block the expression of hypoxia-inducible factor (HIF)-1α through binding to its ARE-containing mRNA (Gottschald et al., 2010). Inhibition of this transcription factor is enhanced when both RBPs are organized into SGs. In addition, HuR also aggregates into SGs to halt the translation of specific housekeeping mRNAs under stress conditions (Bergalet et al., 2011). The deregulation of SGs results in cytoplasmic accumulation and subsequent pathologies such as Parkinson and Alzheimer (Vanderweyde et al., 2012).

Variations in pH values can also modulate the binding of TIA-1 to nucleic acids, acting as a pH-dependent molecular switch. The pKa values of the histidine imidazole groups of TIA-1 RRM2 and RRM3 are substantially higher in complexes with short RNA and DNA oligonucleotides than in the isolated domains. Interestingly, those pKa values are also controlled by slight environmental pH changes (Cruz-Gallardo et al., 2013, 2015). This fact provides valuable information to understand the pH effect on ARE-RBPs when shuttling among cellular compartments with different pHs (nucleus, cytoplasm, SGs, etc.).

During oxidative stress, AUF1 binding to mRNAs containing 8-oxo-7,8-dihydro-guanine could play a role in the selective elimination of oxidized mRNA by presumably driving their degradation (Ishii et al., 2015). Finally, HuR localization can also be altered upon different stress signals such us UV, actinomycin D or hydrogen peroxide, leading to the cytoplasmic accumulation of the protein. However, after a heat shock treatment, the decrease in HuR protein levels enhances cell survival. This phenomenon is linked to the ubiquitination of Lys182, promoting protein degradation, which finally interferes with the binding of HuR to its target mRNAs (Abdelmohsen et al., 2009).

#### mRNA Specific Sequence and Conformation

RBPs do not interact with the same affinity with every ARE-containing mRNA; instead, preferences exist for certain sequences. For instance, TIA-1 RRM domains display different binding constants during nucleic acid recognition. Indeed, the central domains (RRM2 and RRM3) constitute the mRNA binding platform of the protein. RRM2 drives the interaction with RNA, and shows the highest affinities for pyrimidine rich sequences. In turn, RRM3 enhances the overall TIA-1 binding affinity for RNA, preferentially interacting with C-rich motifs (Cruz-Gallardo et al., 2014; Wang et al., 2014; Waris et al., 2017). Moreover, HuR and TIAR interact with U- and AU-rich mRNAs in vitro, with greater affinity (≈10-fold) for the former ones. This higher affinity for U-rich mRNAs results from a higher association rate constant, mainly derived from the presence of a greater number of effective binding positions (Kim et al., 2011). However, in vivo analysis showed that HuR stabilized AU-rich mRNAs to a greater extent than U-rich mRNAs (Brennan and Steitz, 2001). Additionally, the KH domains of KSRP behave as independent binding modules with different affinities for AUrich mRNAs, explaining the broad range of targets recognized by the protein. While the fourth KH domain (KH4) is primarily responsible for mRNA binding and decay through an essential structural element in its β4, KH3 is also necessary to drive the recognition of AU- and G-rich sequences. On the other hand, all KH domains show a clear negative selection for C-rich sequences (García-Mayoral et al., 2007, 2008). Interestingly, many RNA targets of HuR, which acts antagonistically to KSRP, often contain isolated Gs but very rarely Cs (López De Silanes et al., 2004).

Conformational changes in the ARE-mRNA structure have also the potential to regulate the binding affinity of RBPs. These changes may precede the binding of RBPs, as occurs with TNFα mRNA as a consequence of the stabilization of its folding mediated by divalent cations such as Mg2+ (Wilson et al., 2001a,b). In addition, the AU-rich motif of TNFα mRNA can also adopt a hairpin-like structure that inhibits specifically p37AUF1 binding, but hardly affects its interaction with HuR (Fialcowitz et al., 2005). On the other hand, the association of RBPs can cause local changes in the structure of their cognate mRNAs, which may affect the recruitment of new trans-acting factors or establish preferences for one RBP over another. Consequently, these changes would directly impact on the turnover rates of such ARE-containing mRNAs (Wilson et al., 2001b; Zucconi et al., 2010).

### DNA Recognition and Role of RBPs in DNA Damage Response

Some ARE-RBPs also have the ability to bind to DNA. Importantly, in the case of TIA-1 and TIAR, it occurs with a markedly higher affinity than both RBPs show for their mRNA targets (Suswam et al., 2005; Waris et al., 2017). In fact, it has been hypothesized that the formation of the RBP-mRNA complexes would require the direct displacement of the RBP from its DNAbinding site by the polymerase. This dual binding capacity of TIA-1 and TIAR could be potentially providing a link between transcription and splicing (Suswam et al., 2005; Mcalinden et al., 2007; Waris et al., 2017).

Interestingly, several RBPs are involved in DNA Damage Response (DDR), being recruited to DNA breaks in a Poly (ADP-Ribose) (PAR)-dependent manner and/or forming liquid-like compartments by phase separation (Kai, 2016). The formation of these phases requires the presence of an unstructured Prion-Related Domain (PRD) like the one that is present in TIA-1 and TIAR proteins (Gilks et al., 2004). Importantly, abnormal phase separation by mutated PRD-containing proteins leads to pathological protein aggregation and is associated with neurodegenerative and aging-associated diseases (Kai, 2016).

### CONCLUSIONS AND FUTURE PERSPECTIVES

Before being translated into proteins, mRNAs are subjected to a sequential and strict control by RBPs exerted by the

#### REFERENCES

recognition of AREs in their 3′ -UTRs. Regulation of mRNA homeostasis through ARE-RBPs allows the fine tuning of responses by controlling mRNA translation, degradation, or storage in diverse eukaryotic cell compartments (Glisovic et al., 2008; Ganguly et al., 2016). As reviewed above, many examples of ARE-RBP interactions have been reported in the literature, but it is still not well-understood how RBP domains collaborate or compete with each other for the modulation of its targets. The proper inspection of such a convoluted interplay between RBPs requires the combination of different methods in order to compensate the specific strengths and weaknesses of each technique. On the other hand, it becomes more and more evident the need of a transition from a static to a dynamic point of view to take into account the biological environment during RNA binding. Consequently, the integration of the information obtained by in vivo approaches with the structural data would be of great interest. Moreover, the understanding of the ARE-mRNAs processing in highly dynamic and often transient macromolecular complexes also remains challenging (Rissland, 2017). Finally, the key role of intrinsically disordered connecting linkers between RNA binding domains has acquired significant relevance in the latest reports (Basu and Bahadur, 2016). Altogether, the examples of mRNAprotein interactions by ARE-RBPs herein reviewed highlight the need for integrative studies to fully understand such a fine tuned regulation.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

#### ACKNOWLEDGMENTS

Financial support was provided by the Andalusian Government (P11-CVI-7216, BIO198); the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015-71017-P); the Spanish Ministry of Education, Culture and Sports (FPU013/04373, FPU016/01513) and the Ramón Areces Foundation.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmolb. 2017.00071/full#supplementary-material

translation mainly by altering Tristetraprolin expression, stability, and binding to adenine/uridine-rich element. Mol. Cell. Biol. 26, 2399–2407. doi: 10.1128/MCB.26.6.2399-2407.2006

Valverde, R., Edwards, L., and Regan, L. (2008). Structure and function of KH domains. FEBS J. 275, 2712–2726. doi: 10.1111/j.1742-4658.2008.06411.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer SM-L and handling Editor declared their shared affiliation.

Copyright © 2017 García-Mauriño, Rivero-Rodríguez, Velázquez-Cruz, Hernández-Vellisca, Díaz-Quintana, De la Rosa and Díaz-Moreno. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target

Gabrielle M. Watson, William A. H. Lucas, Menachem J. Gunzburg and Jacqueline A. Wilce\*

Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine), however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors.

#### Edited by:

Rivka Isaacson, King's College London, United Kingdom

#### Reviewed by:

Krystle J. McLaughlin, Lehigh University, United States Emeric Miclet, Université Pierre et Marie Curie, France

> \*Correspondence: Jacqueline A. Wilce<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Structural Biology, a section of the journal Frontiers in Molecular Biosciences

Received: 25 July 2017 Accepted: 13 September 2017 Published: 26 September 2017

#### Citation:

Watson GM, Lucas WAH, Gunzburg MJ and Wilce JA (2017) Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target. Front. Mol. Biosci. 4:64. doi: 10.3389/fmolb.2017.00064 Keywords: Grb7, SH2 domain, inhibitor specificity, BC loop, peptide inhibitor

### INTRODUCTION

For over 20 years, Src homology 2 (SH2) domains have been explored as targets for the development of potential therapeutics (Kraskouskaya et al., 2013; Morlacchi et al., 2014). SH2 domains mediate the formation of protein complexes by recognizing phosphorylated tyrosines (pY) on target tyrosine kinases and subsequently mediating their downstream effects. They are utilized by proteins with a range of functions, including enzymes such as the PLCγ and the Src kinase (Rebecchi and Pentyala, 2000; Roskoski, 2004), transcriptional regulators including STAT 1-6 (Darnell, 1997) and adaptor proteins including growth factor receptor bound protein 7 (Grb7) (Han et al., 2001). The reversible association of the SH2 domain/pY complex allows for the efficient regulation of signaling and is therefore frequently found in proteins mediating intracellular signaling pathways—including those regulating proliferation, migration and growth (Schlessinger and Lemmon, 2003). It is these same signaling networks that are susceptible to dysregulation, leading to the amplification and transmission of signals that drive cells into a cancerous state. Thus, targeting SH2 domains can serve as an effective point for the development of anti-cancer agents that effectively block the downstream effects of signaling.

Grb7 has been identified as a therapeutic target due to its role in the progression of a number of cancers including breast, pancreatic, ovarian and oesophageal cancers (Stein et al., 1994; Tanaka et al., 1997, 2006; Wang et al., 2010). In HER2+ breast cancer, Grb7 is co-overexpressed with HER2, leading to enhanced tumorigenesis and cell proliferation (Bai and Luoh, 2007; Chu et al., 2010; Pradip et al., 2013). While HER2 has a well-known role in breast cancer progression, overexpression of Grb7 has now also been identified as a significant predictor for reduced cancer-free periods and a worse prognosis for breast cancer patients (Ramsey et al., 2011). In pancreatic, oesophageal and triple negative breast cancers, Grb7 drives migratory and invasive events, with Grb7 knockdown inhibiting these processes when tested in vitro (Tanaka et al., 1997, 2000, 2006; Giricz et al., 2012). Furthermore, a significant relationship has been identified between Grb7 expression and tumor metastasis in pancreatic and esophageal cancers (Tanaka et al., 1997, 2006).

Grb7 has a multi-domain architecture consisting of an Nterminal proline rich domain, a Ras-associating (RA) domain, pleckstrin homology (PH) domain, a between the PH and SH2 (BPS) domain, and lastly a C-terminal SH2 domain (Shen and Guan, 2004). It is via the SH2 domain that Grb7 interacts with phosphorylated tyrosine kinases including growth factor receptors such as HER2, HER3 and EGFR, as well as cytoplasmic kinases including the focal adhesion kinase (FAK) (Stein et al., 1994; Daly et al., 1996; Han and Guan, 1999). Through these interactions, Grb7 mediates signaling networks controlling proliferation, migration and growth, making the Grb7-SH2 domain an attractive candidate for the development of targeted inhibitors (Han and Guan, 1999; Pero et al., 2003; Pradip et al., 2013).

SH2 domains are extremely prevalent in the proteome, with over 110 proteins bearing the domain. Thus, ensuring target selectivity is a critical aspect of the development of molecules targeting SH2 domains. Despite the high number, SH2 domains display exquisite selectivity for their substrates (Pawson, 2004). SH2 domains contain a well-characterized positively charged cleft for the pY to bind, but it is typically the residues C-terminal to the target pY that dictate the binding specificity for a substrate (Songyang et al., 1993). It has been determined that individual SH2 domains recognize characteristic binding motifs. In the case of Grb7 this motif has been identified as pYXN, where any residue is accommodated at the +1 position, and an asparagine is preferred at the +2 position (Pero et al., 2003). The SH2 domains of Grb2 and Grb7 have low amino acid identity (29%), however, the Grb2-SH2 also recognizes this binding motif, and both adaptor proteins bind to HER2 at pY1139 (Janes et al., 1997). Despite this binding similarity, Grb7 and Grb2 can have remarkable selectivity and an inhibitor has been developed that specifically binds and inhibits Grb7-SH2 (Pero et al., 2002).

The cyclic, non-phosphorylated peptide (named G7-18NATE (cyclo-(CH2CO-WFEGYDNTFPC)-amide) was developed via phage display and was found to specifically decrease binding between Grb7 and tyrosine phosphorylated HER family members in breast cancer cell extracts, but have no effect on the interaction between Grb2 and HER3 (Pero et al., 2002). When G7-18NATE was attached to the Penetratin cell permeability sequence, the peptide inhibited growth, migration and proliferation in breast cancer cell lines, and displayed synergistic effects on cell proliferation with the currently available chemotherapeutics Doxorubicin and Trastuzumab (Pero et al., 2007; Pradip et al., 2013). G7-18NATE-Penetratin was also shown to specifically inhibit the interaction between Grb7 and FAK, but not interfere with the Grb2/FAK or Grb2/EGFR interactions (Tanaka et al., 2006). This specificity for the Grb7-SH2 has also been demonstrated in vitro with surface plasmon resonance (SPR) experiments confirming that G7-18NATE specifically binds to Grb7-SH2 preferentially over the Grb2-SH2 domain (Gunzburg et al., 2012). G7-18NATE also displayed minimal binding to the SH2 domains of Grb10 and Grb14, proteins that share the same domain structure as Grb7 (with the three Grbs collectively termed the Grb7 family). Derivatives of G7-18NATE have also been developed that bind to the Grb7-SH2 domain with higher affinity than G7-18NATE (KD = 0.83 µM) but with the equivalent specificity (Watson et al., 2015; Gunzburg et al., 2016).

While it has been demonstrated repeatedly that G7-18NATE and its derivatives are specific for Grb7, there has been little investigation into the molecular basis of Grb7 ligand selectivity, except for analysis of the βD6 amino acid (Leu481 in Grb7). It is important to note that the nomenclature to be used to describe SH2 domain secondary structure is as derived by Eck et al. as illustrated in **Figure 1A** (Eck et al., 1993). While Grb10 and Grb14 possess a polar glutamine at the βD6 position, Grb7 has a hydrophobic leucine residue. When Leu481 in Grb7- SH2 was mutated to glutamine, binding to the HER2 receptor was completely abolished (Janes et al., 1997). Conversely, the opposite mutation in Grb14 (glutamine to leucine) imparted binding of Grb14 to the HER2 receptor. Together this established the importance of a leucine at the βD6 position for Grb7-SH2 domain interactions with its target motif. In the case of the Grb2- SH2 domain, however, the βD6 amino acid is a lysine and this would be predicted to interfere with ligand binding. Yet some binding to Grb2-SH2 domain by G7-18NATE still takes place, showing that it is not only the βD6 amino acid that dictates G7-18NATE binding (Gunzburg et al., 2012).

The structure of the Grb7-SH2 domain in complex with G7- 18NATE revealed the amino acid residues forming the binding site for the peptide ligand (Ambaye et al., 2011b). It showed the way in which G7-18NATE Y5 and F2 (note that single letter code will be used in reference to the peptide ligand amino acids for clarity) straddle the Grb7-SH2 domain Leu481 residue, helping to visualize the role of this amino acid in the specificity of the interaction. Leu481, and other Grb7-SH2 domain amino acids that form hydrogen bond interactions or contribute to the buried surface area with G7-18NATE are indicated in **Figure 1C**. For the most part, these residues are conserved between Grb7-SH2 domain and the closely related Grb10, Grb14, and Grb2 SH2 domains and, thus, do not help to explain the basis for Grb7 ligand specificity. Only the βE4 methionine and EF4 glutamine are unique to the Grb7-SH2 domain and may act alongside Leu481 to contribute to ligand specificity.

**Abbreviations:** BPS, between PH and SH2 domain; FAK, focal adhesion kinase; Grb, growth factor receptor bound protein; PH, pleckstrin homology; pY, phosphorylated tyrosine; RA, ras-associating; SPR, surface plasmon resonance; SH2, src homology 2; WT, wild-type.

Frontiers in Molecular Biosciences | www.frontiersin.org

displayed above the alignment at the corresponding position, and sequence numbering for the Grb7-SH2 displayed below the alignment. Grb7 amino acids that form hydrogen bond interactions with the G7-18NATE peptide are identified with filled circles and additional amino acids that contribute to the buried surface area are identified with empty circles. The unique Grb7-SH2 arginine at position 462 (BC2) is highlighted by an orange box.

In this study, we identified an additional amino acid in the Grb7-SH2 BC loop that is involved in binding to G7- 18NATE and potentially also contributes to Grb7-SH2 domain binding specificity. Based on another Grb7-SH2/peptide complex X-ray crystal structure, we postulated that the Grb7 BC2 arginine contributes to the Grb7-SH2/peptide interaction. In Grb2, Grb10, and Grb14 the BC2 residue is a serine. Using mutational analysis we demonstrate that this Grb7 residue does indeed contribute to the G7-18NATE interaction, and thus may dictate some of the Grb7 selectivity. We identified that mutating the BC2 residue in the Grb2-SH2 from serine to arginine is not sufficient to confer binding to G7-18NATE indicating that other Grb7 amino acids also contribute to binding strength and specificity. Lastly, using a pY micro-array we confirmed that G7- 18NATE is specific for Grb7 compared to a panel of 79 SH2 domains, providing extra verification of the specificity of this peptide. Together this study provides insight into the specificity of G7-18NATE for Grb7 which is critical for the development of effective therapeutics targeted to the Grb7-SH2 domain signaling in cancer.

### MATERIALS AND METHODS

### Protein and Peptide Preparation

Grb7-SH2 and Grb2-SH2 were prepared as GST fusion proteins and expressed and purified as previously described (Watson et al., 2015). Briefly, Grb7-SH2 (residues 415-532) and Grb2-SH2 (residues 58-160) were incorporated into the pGex2T plasmid and expressed in BL21(DE3)pLysS competent cells following IPTG induction (0.4 mM) at 25◦C. Subsequently, glutathione affinity chromatography and size exclusion chromatography were used to purify the soluble GST tagged proteins. To generate the Grb7-SH2 R462S and Grb2-SH2 S90R mutants, site-directed mutagenesis was utilized (Liu and Naismith, 2008). For this, the wild-type SH2 domain constructs were used as templates for polymerase chain reaction with the primers listed in Supplementary Material. The parent plasmid was subsequently removed by treatment with Dpn1 and the presence of the mutation verified by DNA sequencing. The mutant proteins were expressed and purified as per the wild-type proteins. GST alone was purified similarly to the GST-SH2 domain proteins with the exception that size exclusion chromatography was not necessary for purification.

G7-18NATE (cyclo-(CH2CO-WFEGYDNTFPC)-amide) was synthesized using standard Fmoc-chemistry and purchased from Purar Chemicals (Australia). The synthesis of G7-18NATE-PB ((cyclo-(CH2CO-WFEGYDNTFPC-RRMKWKKK(Biotin)) amide)) has been previously described (Ambaye et al., 2011a).The purity of both peptides was >95% as determined using LC-MS.

The final solution concentration of all proteins and peptides used in this study were determined spectroscopically at 280 nm using extinction coefficients predicted by the ProtParam server (Gasteiger et al., 2005).

### Binding Studies Using Surface Plasmon Resonance

SPR experiments were performed on a Biacore T100 using CM5 series S sensor chips. The GST tagged proteins were immobilized onto the sensor chip surface by amine coupling an anti-GST antibody to the surface of the chip. For this, firstly the chip was activated by 0.4 M 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide hydrochloride and 0.1 M Nhydroxysuccinimide. The polycloncal antibody (Abcam) was passed over the chip surface at 30 µg.mL−1 before the flow cells were blocked with 1 M ethanolamine. The running buffer used for the immobilization contained 50–150 mM NaPO4, 150 mM NaCl and 1 mM DTT (pH 7.4), with levels reaching between 3289 RU and 6324 RU. The GST fusion proteins were passed over the active chip surface at 0.7–0.9 µM to achieve final immobilization levels of 838-1283 RU. GST alone was immobilized on the reference flow cell (462-616 RU) as a negative control to allow for double referencing. Lyophilized G7-18NATE was resuspended in running buffer containing 150 mM NaPO4, 150 mM NaCl and 1 mM DTT (pH 7.4) and injected over the chip surface for 60 s at 30 µL.min−1 in triplicates. Due to machine error some injections were removed before analysis due to presence of spikes that rendered the sensorgrams unable to be interpreted. The interpretable data were analyzed using Scrubber2.0 (BioLogic Softward, Campbell, ACT, Australia) and GraphPad Prism.

### SH2 Domain pY Microarray

The microarray was conducted by the Protein Array and Analysis Core at the MD Anderson Cancer Center (University of Texas, USA) using standard protocols. Briefly, lyophilized G7-18NATE-PB (100 µg) was resuspended in PBST and incubated with the nitrocellulose slide pre-spotted with GST-fusion SH2 domains (full list provided as Supplementary Material). Each SH2 domain was spotted in duplicate.

### RESULTS

### The Unique Grb7-SH2 BC2 Amino Acid Engages E3 in the G7-B1 Peptide

We recently characterized the interaction between the Grb7-SH2 and a G7-18NATE derivative peptide, G7-B1 (B for bicyclic) (Gunzburg et al., 2016). In the course of our investigation, we solved the X-ray crystal structure of the Grb7-SH2/G7-B1 complex to 1.6 Å (PDB ID: 5EEQ). This revealed an interesting feature potentially underlying binding specificity of the Grb7 targeting peptides. We identified that an arginine at the BC2 position (Arg462) in the Grb7 BC loop reaches up and hydrogen bonds with the G7-B1 E3 backbone carbonyl and forms a salt bridge with the E3 sidechain carboxylate (**Figure 1B**; Gunzburg et al., 2016).

A sequence alignment of the SH2 domains of Grb2, Grb7, Grb10, and Grb14 revealed that an arginine at this BC2 position is unique to Grb7 with Grb10, Grb14, and Grb2 possessing a serine at the equivalent position (**Figure 1C**). Although the Arg462/E3 interaction has not been clearly observed in other Grb7-SH2/peptide crystal structures (PDB ID:3PQZ Ambaye et al., 2011b; PDB ID 4X6S Watson et al., 2015; Gunzburg et al., 2016), we postulated that this interaction might occur transiently and help to define the molecular basis of binding specificity of G7-18NATE for Grb7.

### Arg462 Contributes to the Binding Affinity between Grb7-SH2/G7-18NATE

To test this hypothesis, we mutated the Grb7-SH2 R462 (BC2) amino acid to a serine (the corresponding residue in the Grb2, Grb10. and Grb14 SH2 domains). Using SPR, we directly compared the binding of the cyclic peptide G7-18NATE to the Grb7-SH2 wild-type (WT) and the mutated Grb7-SH2 (R462S). The Grb7-SH2 proteins were prepared as GST fusions to enable efficient immobilization on the sensor chip surface via anti-GST antibodies. The G7-18NATE peptide was flowed over the sensor chip surface and the measured response at equilibrium used to construct binding curves that were fitted by a single-site binding model.

Upon injection of G7-18NATE, equilibrium was rapidly reached for both WT Grb7-SH2 (**Figure 2A**) and the mutated R462S Grb7-SH2 (**Figure 2B**), followed by rapid dissociation after the 60 s injection was complete. Excellent fits by a 1:1 interaction model were obtained with a R 2 of 0.99 for both interactions. The binding curves indicated that the R462 of the BC loop did indeed contribute to the binding affinity of the G7-18NATE/Grb7-SH2 interaction. While G7-18NATE bound to WT Grb7-SH2 with a KD of 2.43 µM, the R462S mutant had a 3.3-fold reduction in binding affinity for G7-18NATE, with a KD of 7.97 µM (**Figure 2C**; **Table 1**). This suggested that the BC2 arginine does indeed engage with the peptide ligand, and therefore may impart some of the specificity of Grb7 for its substrates.

#### The Corresponding Grb2 Mutation Is Insufficient to Confer Binding to G7-18NATE

We next postulated that the opposite mutation at the BC2 position in the Grb2-SH2 (serine to arginine) could confer binding of the Grb2-SH2 to G7-18NATE. This would indicate the degree to which Arg462 in Grb7 is responsible for binding specificity. We therefore generated the Grb2-SH2 S90R (BC2) mutant protein using site-directed mutagenesis and tested its binding to G7-18NATE using the same SPR experimental procedure described above. As shown in **Figure 3A**, there was

Grb7-SH2 (A) or mutated (R462S) Grb7-SH2 (B) with the concentrations of peptide tested displayed on the right of the sensorgram; (C) The corresponding binding curves were calculated from the responses at equilibrium and fitted by a 1:1 interaction model. All data points from the repeated experiments are displayed in the binding profiles.

TABLE 1 | SPR investigations into the specificity of G7-18NATE for the Grb7-SH2.

#Equilibrium dissociation constants were calculated from fits by a single-site binding model. Errors displayed are the standard error of the fits.

only a low binding response to the Grb2-SH2 WT following injection of G7-18NATE over the chip surface, reflecting low binding affinity as previously established for Grb2-SH2 compared to Grb7-SH2 (Gunzburg et al., 2012). The binding to G7-18NATE by Grb2-SH2 S90R, however, also gave rise to low responses, though slightly higher than seen for Grb2-SH2 WT (**Figure 3B**). Excellent fits were obtained by a single-site binding model (R 2 = 0.99), however the calculated equilibrium dissociation constant was higher than the highest peptide concentration measured (125 µM; **Figure 3C**). Therefore, we could not reliably determine differences in the KD between WT Grb2-SH2 and the mutated Grb2-SH2. This demonstrated, nevertheless, that the incorporation of the arginine at the BC2 position was not sufficient to significantly increase the binding of Grb2-SH2 to G7-18NATE.

### G7-18NATE Is Specific for the Grb7-SH2 Compared to a Panel of SH2 Domains

In the course of our analysis of G7-18NATE and its derivatives, specificity was assessed by comparing the binding of the peptides to the SH2 domains of Grb2, Grb7, and Grb10, and in some instances Grb14-SH2 (Watson et al., 2015; Gunzburg et al., 2016). While Grb2 shares the same consensus pYXN binding motif, Grb10 and Grb14 share significant sequence identity (approximately 70%) with Grb7-SH2 (Daly et al., 1996). We therefore considered that these were the most closely related SH2 domains for assessment of specificity for Grb7-SH2 using SPR experiments.

In order to determine whether there were other SH2 domains that G7-18NATE could bind to that we had not yet encountered, we utilized the pY reader protein microarray at the Protein Array and Analysis Core (MD Anderson Cancer Center). Here, a biotinylated G7-18NATE derivative (G7-18NATE-PB) was tested for binding against 79 SH2 domains (a complete list of tested SH2 domains is provided as Supplementary Material). The G7-18NATE derivative also contained a shortened Penetratin sequence (displayed as a schematic in **Figure 4A**), and the presence of this sequence has been previously demonstrated not to interfere with G7-18NATE binding to the Grb7-SH2 (Ambaye et al., 2011a; Watson et al., 2017). As shown in **Figure 4B**, it was clear that G7-18NATE-PB preferentially binds to the Grb7-SH2 over the suite of SH2 domains. Although this array was not quantitative, the level of fluorescence was found to be significantly higher for the positions containing the Grb7-SH2, indicative of peptide binding. This was not due to differences in the amount of protein blotted on the array, as similar fluorescence levels were detected when probed with an anti GST antibody (**Figure 4B**).

There was no observation of fluorescence at the positions corresponding to Grb2, Grb10, or Grb14, consistent with our previously published SPR experiments (Gunzburg et al., 2012). Fluorescence was observed, although at much lower intensity, at the positions corresponding to a previously untested SH2 domain—that of SH2D2A. A sequence alignment of the SH2 domains of Grb7 and SH2D2A revealed that SH2D2A also contains a serine at the BC2 position and, strikingly, a leucine at the βD6 position (**Figure 4C**). Although this is not the only SH2 domain in the array that posseses a leucine at the βD6 position, it is suggestive that leucine at this position is also determining feature for binding the G7- 18NATE peptide and its derivatives. Thus, as well as further confirming the specificity of the ligand G7-18NATE for the Grb7- SH2 domain, this experiment has also provided further insights into the defining features of Grb7 that affect ligand binding selectivity.

#### DISCUSSION

SH2 domains are frequently found in signaling pathways regulating critical processes that are perturbed in aggressive diseases like cancer. Due to this, and their well-characterized

binding mode, a number of SH2 domains have been targeted for the development of novel therapeutics (Kraskouskaya et al., 2013; Morlacchi et al., 2014). However, with over 110 SH2 domains in the proteome, ensuring selectivity for the target SH2 domain is a significant challenge in the development of specific and potent inhibitors. Grb7 is a signaling adaptor protein that has been specifically targeted through the development of cyclic peptide inhibitors that are able to inhibit proliferation and migration in breast cancer cell lines and reduce tumor metastasis in a mouse model of pancreatic cancer (Tanaka et al., 2006; Pradip et al., 2013). The cyclic peptide, G7-18NATE, has been demonstrated to be specific for the Grb7-SH2 domain over the closely related SH2 domains of Grb2, Grb10, and Grb14, and this specificity has, to date, been attributed to the Grb7 specific leucine at the βD6 position (Janes et al., 1997; Gunzburg et al., 2012). Here, we have investigated whether additional amino acids also contribute to the specificity of Grb7 for its ligands.

Based on the high-resolution X-ray crystal structure of the Grb7-SH2/G7-B1 complex, we identified an arginine in the Grb7-SH2 BC loop that mediates additional hydrogen bond interactions with the inhibitor peptide. We identified that this arginine is unique to Grb7 compared to the SH2 domains of Grb2, Grb10 and Grb14 (where there is a serine at the comparable position) and considered that this interaction could contribute to the specificity for Grb7 of all the G7-18NATE derived peptide ligands. Using mutagenesis we henceforth determined that Arg462 contributes to the binding affinity between Grb7- SH2 and G7-18NATE. The converse mutation in the Grb2-SH2 domain did not confer binding to G7-18NATE, suggesting other amino acids also define Grb7 specificity. We also probed a panel of 79 SH2 domains with a biotinylated version of G7-18NATE and identified that the peptide is highly specific for the Grb7- SH2 domain. The only other SH2 domain that showed detectable binding by G7-18NATE was that of SH2D2A. Interestingly, while SH2D2A does not possess an arginine at the BC2 position it has a leucine at the βD6 position.