A Simulated Intermediate State for Folding and Aggregation Provides Insights into ΔN6 β2-Microglobulin Amyloidogenic Behavior
[['β2-microglobulin ( β2m ) is a 99-residue protein with a typical immunoglobulin fold comprising seven anti-parallel β-strands stabilized by a disulfide bridge ( Fig . 1 ) [1] .', 'Upon dissociation from the MHC-I heavy chain , human β2m ( Hβ2m ) is catabolised in the kidneys .', 'In individuals undergoing long-term hemodialysis the clearance process is strongly impaired and the levels of Hβ2m in the serum can increase up to 60-fold [2] .', 'The progressive accumulation of Hβ2m in the osteoarticular system , presumably driven by its affinity for type-I collagen [3] , eventually leads to amyloid assembly and the onset of dialysis-related amyloidosis ( DRA ) , a pathological condition characterized by tissue erosion and destruction [4] .', 'The wild-type Hβ2m ( WT Hβ2m ) does not form amyloid fibrils in vitro in the absence of ex vivo amyloid seeds [5] , or additional factors such as Cu2+ [6] , [7] or TFE [8] .', 'This limitation makes the determination of the aggregation mechanism of Hβ2m in physiological conditions ( 37°C , pH 7 . 5 ) a particularly challenging conundrum .', 'A major contribution towards its solution was the identification [9] , [10] , and atomic-level structural characterization [11] , of an intermediate state ( representing from 3 . 7±1 . 4% [10] up to ∼14±8% [9] of the equilibrium population ) in the folding pathway of WT Hβ2m .', 'The intermediate was termed IT because its main structural trait is a non-native trans isomerization of Pro32 .', 'Enhanced fibrillogenesis in physiological conditions ( including the ability to elongate and/or nucleate amyloid fibril assembly ) has been observed in connection with an increase in the equilibrium concentration of IT [10] , [12] , [13] , indicating that IT is highly amyloidogenic and a key player in Hβ2m fibrilogenesis .', 'While relevance of IT for β2m fibrillogenesis is widely acknowledged [9] , [10] , [14]–[16] , alternative intermediate states , which are less native-like than IT , become relevant under different experimental conditions [8] , [17]–[19] .', 'Furthermore , a variety of environmental aspects have been found to directly affect the process of fibril formation by β2m including solubility , supersaturation and ultrasonication/agitation effects [20] .', 'Recently , the single point mutant Asp76Asn ( D76N ) , a naturally occurring variant of Hβ2m , was associated with the late onset of a fatal hereditary systemic amyloidosis characterized by extensive visceral amyloid deposits .', 'However , and contrary to what occurs in DRA , in this newly discovered disease the plasma concentration of Hβ2m is not augmented [21] , [22] .', 'In vitro studies have shown that the Asp76Asn mutant is highly amyloidogenic , displaying an abundant ( ∼25% ) equilibrium population of IT under physiological conditions [22] .', 'Another recently reported single point variant of β2m for which fibrillation occurs without seeding under physiological conditions is the Arg3Ala ( R3A ) mutant [23] .', 'The latter , however , has not yet been associated with any conformational disorder .', 'In this work we focus on ΔN6 , a truncated form of Hβ2m , lacking the first six N-terminal residues .', 'This variant is potentially relevant because it represents ∼30% of ex vivo amyloid deposits extracted from DRA patients [24] .', "Radford and co-workers proposed that ΔN6 is a structural mimic of IT because it populates a conformational state that reproduces the conformational features of IT and represents 90% of ΔN6's in vitro equilibrium population [11] .", 'While there is a broad agreement regarding the ability of ΔN6 to prime the fibrillar conversion of WT Hβ2m in vitro under physiological conditions , the mechanism by which it occurs is not consensual .', 'In particular , Eichner and Radford proposed that monomeric ΔN6 conformationally converts WT Hβ2m into an amyloidogenic state in a mechanism akin to prion conversion [11] , while Bellotti and coworkers challenged the prion-like hypothesis by reporting that the WT Hβ2m does not fibrillate with monomeric ΔN6 but rather with preassembled fibrils of ΔN6 [22] .', 'However , and independently of these controversies , it is widely accepted that ΔN6 alone is able to efficiently nucleate fibrillogenesis in physiological conditions ( tlag∼35 days , 80 mM ) [11] , [12] , [25] .', 'Furthermore , it displays an enhanced amyloidogenicity at pH 6 . 2 ( tlag∼15 days , 80 mM ) [11] , i . e . , in conditions compatible with the mildly-acidic character of the synovial fluid of DRA patients [26] .', 'It has been suggested that the aggregation potential of ΔN6 stems from its unique ability to populate one or more aggregation-prone intermediate states [11] .', 'Therefore , a complete picture of the aggregation mechanism of ΔN6 requires disclosing the process according to which it aggregates de novo starting from the self-association of aggregation-prone monomeric states .', 'Addressing this challenge via molecular simulation is the major goal of the present work .', 'By studying the early stage of aggregation of ΔN6 one expects to get insights into the amyloidogenicity of the full-length protein .', 'The large size of the system and the long timescales involved in the process of protein aggregation strongly restrict the use of classical molecular dynamics ( e . g . based on the AMBER or GROMOS force fields ) to explore it .', 'For this reason researchers have been developing coarse-grained approaches to study protein aggregation [27] .', 'One example is the symmetrised Gō potential used to study domain-swapping ( DS ) [28] .', 'In DS two monomers exchange identical structural elements or “domains” to form a strongly bound dimer .', 'Since the DS hypothesis is based on the association of two monomers into dimers , the mechanism of fibril formation from more than two identical proteins is still unclear .', 'On the other hand since DS is a manifestation of concomitant folding and binding it requires the use of a simulation framework where the two processes compete directly with each other via a force field that accounts for competing intra- and intermolecular interactions [28] .', 'Hβ2m fibrillogenesis has been reported to be initiated by dimerization of monomers [6] , [12] , [16] , [29]–[31] including DS [32] .', 'Here , however , we will not study the dimerization of Hβ2m that may result from DS .', 'Instead , our goal in this study is to explore the early stage of the aggregation mechanism of the truncated variant ΔN6 that may occur as a side-effect of protein folding .', 'More precisely , if aggregation-prone intermediate states ( including highly native-like species ) are populated along the folding pathway of ΔN6 , they may start interacting with each other ( e . g . via solvent-exposed hydrophobic residues ) thus triggering the amyloid cascade .', 'These aggregation-prone intermediates are a by-product of the folding process and likewise their formation is exclusively driven by intra-molecular interactions .', 'Inter-molecular interactions will only start operating once the monomers representative of the intermediate state get within interaction range , which may eventually lead to their self-association into dimers .', 'Our study seeks to explore this type of ( de novo ) aggregation route for the variant ΔN6 of Hβ2m by highlighting its topological aspects , i . e . , the predictions reported here are strictly structure-based .', 'In doing so , we use a three-stage computational protocol based on an array of tools as detailed in the Methods section .', 'In the first stage , following our previous studies [33] , [34] , replica-exchange discrete molecular dynamics ( RE-DMD ) simulations of a full atomistic Gō model [35] are used to study the folding transition and to identify intermediate states in the folding pathway of ΔN6 .', 'The adopted level of structural resolution encompasses the effect on the folding mechanism of detailed atomic native contacts and fully takes into account side-chain packing , a fundamental ingredient of the folding process .', 'Combined with RE-DMD simulations , the full atomistic Gō model enables equilibrium sampling of the conformational space , a task far beyond the possibilities of routinely used classical molecular dynamics protocols , especially for a system of the size of Hβ2m .', 'While this simulation procedure captures the fundamental features of the folding process [36] , it fails to include others .', 'In particular , it neglects the effects of the pH , an important environmental parameter .', 'Indeed , it is well known that changes in pH can induce conformational changes of varying degree , ranging from structural fluctuations to modifications in secondary structure content [37]–[39] .', 'Furthermore , in the case of ΔN6 the pH turns out to be a particularly relevant parameter because – as stated before – the aggregation potential of this variant is remarkably sensitive to pH changes [11] .', 'To identify the molecular roots of this dependence , it is thus crucial to establish how the pH affects the structure of the relevant conformational states , because structural changes at the monomer level ( e . g . the reorganization of aromatic side chains , which are bulky and therefore natural players in the establishment of intermolecular interactions ) will directly affect monomer association and ultimately dictate aggregation performance .', "In the second stage of our computational protocol , we investigate how the pH modulates an intermediate state's structure .", 'Leaving aside its possible effect on large-scale conformational dynamics , we can afford to accurately capture the effect of pH by employing constant pH molecular dynamics ( CpHMD ) with explicit titration [37] , [38] , [40]–[43] .', 'In doing so one also obtains conformations representative of the intermediate state with the most accurate representation of side-chain and backbone geometries , which is a requirement of the Monte Carlo ensemble docking ( MC-ED ) [33] protocol whose predictions depend critically on the structural accuracy of the analysed structures .', 'The MC-ED is a low-resolution protocol that highlights the role of shape complementarity , a major driver of protein aggregation [44] , [45] .', 'It takes pairs of monomer conformations obtained with CpHMD to generate two ensembles of putative dimer structures ( one obtained from monomers at equilibrated pH 6 . 2 and another at pH 7 . 2 ) where the number of residue pairs within interaction distance is maximized and the number of excluded volume interactions is minimized .', 'The number of contacts thus evaluated provides a measure of the quality of the geometric matching between the two monomers .', 'Therefore the MC-ED method allows predicting the residues that are most likely to trigger dimerization in an ensemble of dimers whose interface was optimized for shape complementarity .', 'The MC-ED allows analyzing the association of an exceedingly large number of monomeric conformations while discriminating between the dimer structures that are prone to further oligomerize from those that are not .', 'This is an important point because protein conformations are not static entities .', 'Indeed , they have a dynamic nature leading to structural variability even for the native state .', 'Thus , two pairs of self-associating conformations ( representing the same conformational state ) will not form exactly the same docking interface upon dimerization .', 'By exhaustively docking thousands of equilibrated conformations collected from CpHMD simulations at pH 6 . 2 and 7 . 2 this work provides a probabilistic structurally resolved picture of the dimerization interface of the identified intermediate state , the native state of ΔN6 and also the native state of WT Hβ2m at physiological and near physiological pH . In doing so , it recapitulates and rationalizes previous experimental observations , and draws new insights into the aggregation mechanism of ΔN6 , including the prediction of aggregation hot spots .'], ['The free energy ( FE ) surfaces at the folding temperature ( Tf ) , evaluated with the WHAM method [46] , reveal a well-defined intermediate basin for ΔN6 that is not present in the FE surfaces of Hβ2m ( Fig . 2A ) .', 'The native and intermediate states populate ∼38% and 11% of the equilibrium ensemble at Tf , respectively .', 'To isolate and structurally characterize the intermediate state populated by ΔN6 , which we term ΔN6-I , we performed structural clustering over an ensemble of conformations collected from DMD simulations at fixed temperature ( ∼Tf ) .', 'The intermediate species preserves the trans-isomerization of Pro32 ( as a consequence of the native-centric character of the Gō potential ) and exhibits an unstructured/disordered strand A detached from a fairly conserved core region comprising residues 21 to 94 ( i . e . strands B–G and connecting loops ) ( Fig . 2B ) .', 'The detachment of strand A from the protein core and its structureless nature are likely the result of a smaller number of native interactions involving this secondary structural element , which decreases by 27% with regard to that observed in the full length species ( Fig . S5 ) .', 'The evaluation of solvent accessible surface area ( SASA ) per residue reveals that 62% of the hydrophobic core residues become highly solvent-exposed in ΔN6-I with SASA exhibiting a 3- up to 7-fold increase in Leu7 , Val9 , Leu23 and Trp95 , all located at the termini .', 'Phe30 on the BC-loop , and Ile35 in strand C are also significantly more exposed to the solvent in the intermediate state ( Fig . 2C ) .', 'These observations are particularly relevant because the exposure of aggregation-prone hydrophobic patches has been pointed out as a hallmark of protein aggregation ( reviewed in [47] ) and suggest that the identified intermediate state has a high aggregation potential .', 'We conjecture that strand A , by being exposed to the solvent , will be a particularly important structural motif for the early aggregation stage of ΔN6 .', 'The Gō potential adopted in this work does not predict an equilibrium population of a similar full-length species , with a conserved core region and detached strand A , across the folding transition of the WT variant .', 'However , there is experimental evidence that the amyloid-transition of the full-length Hβ2m is concomitant with a detachment of the N-terminal strand A [48] triggered by an acidic pH [19] , [49]–[51] or Cu2+ binding [7] , [48] , [52] .', 'Therefore it is likely that the full-length Hβ2m may undergo a similar conformational transition .', 'The ΔN6-I intermediate state identified with the Gō model highlights the importance of native topology in determining the folding space .', 'In order to investigate how the pH modulates the structure of the intermediate state , and , in particular , the degree of solvent-exposure of the unstructured strand A , we set up a series of CpHMD simulations that used the intermediate conformation as a topological template .', 'This means that a structurally refined version of ΔN6-I was prepared by taking into account the structural information provided by the native-centric model .', 'The refined structure was then used as the starting conformation in CpHMD simulations at pH 7 . 2 and pH 6 . 2 .', 'The analysis of conformational ensembles taken from the equilibrated parts of CpHMD trajectories reveals that pH 6 . 2 has a striking effect on the region comprising strand A and the AB-loop .', 'In ΔN6-I this region deviates significantly from its original position in the native structure as indicated by the large ( mean ) RMSD ( 16 Å ) obtained after optimally superimposing each analyzed intermediate conformation over the native core region .', 'The recorded RMSD at pH 6 . 2 represents an increase of 20% from that observed at pH 7 . 2 indicating a distinctively higher degree of solvent exposure at lower pH . On the other hand , the core region is better preserved at pH 6 . 2 with the ( mean ) RMSD decreasing up to 40% relative to pH 7 . 2 ( Table S1 ) .', 'The increased solvent-exposure of the N-terminal region ( comprising residues 6 to 20 ) at pH 6 . 2 can be tentatively explained on the basis of a more favorable electrostatic contribution to the free energy of solvation at this pH . At a physiological/near-physiological pH Arg12 and Lys19 are permanently protonated , Glu16 is mostly deprotonated ( only ∼0 . 3% protonation at pH 6 . 2 ) , but the protonation state of His13 changes given the similarity between the medium pH and the average pKa of its imidazole ring ( 6 . 0 ) .', "Our data hints at the possibility of a direct connection between His13's protonation state and the SASA of the N-terminal region ( comprising both strand A and the AB-loop ) at pH 6 . 2 ( Fig . S1 ) .", "Therefore , one can argue that the higher degree of protonation of His13 at pH 6 . 2 leads to an increased solvent-exposure of that region which is concomitant with a favorably-enhanced electrostatic contribution to its free energy of solvation ( it has been shown that protonation of the imidazole side chain produces a substantial increase of that histidine's absolute solvation free energy [53] ) .", 'Seminal studies carried out by Miranker and co-workers have emphasized the importance of dimerization in the aggregation pathway of a mutational variant of Hβ2m ( P32A ) [6] .', 'The latter is a structural mimic of an intermediate state ( M* ) , which shares with IT ( and , therefore , with ΔN6 ) important structural features .', 'Of note , a trans peptidyl-prolyl His31-Ala32 bond and the re-packing of several aromatic side chains within the hydrophobic core including Phe30 and Leu62 .', 'Furthermore , Eichner and Radford have shown that the M* intermediate elutes at a retention volume identical to that of IT [12] .', 'More recently , studies carried out by Radford and co-workers on the mutational variants P5G and ΔN6 ( for which the intermediate IT represents respectively ∼60% and 90% of the equilibrium ensemble in physiological conditions ) showed that the first assembled oligomeric state in the amyloid pathway of both mutants is a dimer of IT monomers [12] .', 'Motivated by these findings , we carried out an exhaustive study of the dimerization interface in ΔN6 via MC-ED simulations .', 'We mapped the dimerization interfaces of the intermediate state ( ΔN6-I ) and of the native state ( ΔN6-N ) at pH 7 . 2 and 6 . 2 by docking pairs of conformations obtained from CpHMD simulations under those pH conditions .', 'We also investigated the dimerization interface of the native state of WT Hβ2m ( WT-N ) as a control experiment .', 'In Figure 3 we report density histograms ( DH ) of the number of intermolecular contacts at pH 7 . 2 ( Fig . 3A ) and pH 6 . 2 ( Fig . 3B ) for the dimers of the analyzed species .', 'This property provides a quantitative measure of the quality of the geometric matching between the two monomers because each dimer conformation was optimized for maximum number of interactions and minimum number of excluded volume interactions .', 'Each dimer interface was thus optimized for shape complementarity , a property that is considered a major driver of protein-protein association [44] , [45] .', 'In this sense the density histograms provide insight regarding the dimerization potential of each species .', 'In the DHs the vertical lines indicate the mean , and the mode corresponds to the highest point of the distribution ( representing the most probable number of intermolecular interactions in the population of dimers ) .', 'In order to facilitate the comparison of these data , Figures 3C–E separately report the DHs for the two considered pH values .', 'The analysis of the DHs reveals important findings .', 'First , the high similarity between the curves obtained for the WT-N species suggests that it should conserve its dimerization propensity upon changing the pH from 7 . 2 to 6 . 2 ( Fig . 3C ) .', 'Since WT-N is the most populated state [9] , [10] , [15] of the in vitro equilibrium population in physiological conditions , our observation is consistent with the conservation of the aggregation behavior of Hβ2m across this pH range [25] .', 'Our analysis further suggests that at the molecular level this behavior may be rooted in the conformational robustness exhibited by the monomeric form of WT-N across the analyzed pHs ( Table S2 ) , and , in particular , points out the importance of the protective role played by the N-terminus in maintaining the hydrophobic balance that stabilizes the native state [25] .', 'Second , ΔN6-I forms dimers with number of intermolecular contacts given by the mode ( mean ) with a probability that is up to ∼52% ( 60% ) higher than in the WT-N at pH 6 . 2 ( for ΔN6-N this probability goes up to 51% at pH 7 . 2 ) ( Fig . 3D and Fig . 3E ) .', 'This is consistent with the higher amyloidogenicity of ΔN6 at physiological/near physiological pH . Third , when the pH decreases from 7 . 2 to 6 . 2 , the mean and the mode decrease marginally for both ΔN6 conformational states .', 'However , in the case of ΔN6-I , this mild decrease goes in tandem with a significant increase ( up to 10% ) in the probability of formation of the corresponding dimers ( Fig . 3E ) .', 'On the other hand , for ΔN6-N , the dimer conformations representative of the mean and mode are less probable at pH 6 . 2 than at pH 7 . 2 ( Fig . 3D ) .', 'Since it is likely that further oligomerization will be limited by nucleation of dimers , both measures ( i . e . mean and mode ) predict that ΔN6-I plays a major role in amyloid formation at pH 6 . 2 .', 'In order to pinpoint the regions of the protein that are most likely to start dimerization , we have constructed probability contact maps for the dimer interfaces ( Fig . S2 ) .', 'The probability of each intermolecular contact was evaluated by counting the number of times the contact is present in the ensemble of dimers that was used to determine the DH .', 'We have also analyzed several representative dimer conformations ( i . e . conformations with number of intermolecular contacts corresponding to the mode and tail of the DHs ) , to gauge their importance for further oligomerization ( Fig . 4 and Fig . S3 ) .', 'We have chosen to analyze the structure of ‘mode’ dimers for consistency reasons , i . e . , because they exhibit the most likely number of intermolecular contacts ( and a minimal number of excluded volume interactions ) in the dimer interface , a property that quantifies the degree of geometric matching and shape complementarity of the interfaces in the ensemble of MC-ED generated dimers .', 'On the other hand , the analysis of ‘tail’ dimers is particularly pertinent for the WT-N species because a unique feature of its DH is a rather extended tail indicating the formation of dimers with the strongest geometric matching ( Fig . 3C ) .', 'Since shape complementarity is a major driver of protein aggregation and is maximized for ‘tail’ dimers it is important to establish if/how the existence ‘tail’ dimers may affect the aggregation performance of Hβ2m .', 'We find that at both pH values dimerization of WT-N is majorly driven by the DE-loop ( especially residues 56–60 ) ( Fig . S2A ) .', 'The analysis of several dimer conformations representative of the mode of the DH reveals that the most likely dimerization interface involves the DE-loop of one monomer that associates with the second monomer in several possible spots ( Fig . S3A ) .', 'On the other hand , the DE-loop directed interfaces of the strongly packed dimers are more specific , being based on loop-loop interactions involving the BC and DE aromatic-rich regions ( Fig . S3B ) .', 'Since the latter become unavailable for subsequent interaction , further oligomerization ( via addition of another monomer ) appears to be restricted to the potentially adhesive residues located on the EF-loop ( e . g . Phe70 and Tyr78 ) and in the C-terminus ( Trp95 ) .', "Our analysis therefore predicts that the recruitment of the aromatic-rich regions in the WT-N best geometrically matched dimers' interfaces renders these dimeric entities particularly soluble thus lowering their aggregation potential ( soluble dimerization was recently found to be a possible dead-end for aggregation in Ref . [54] ) .", 'In order to gauge the importance of residue 76 ( located in the EF-loop ) for the dimerization of Hβ2m we have selected a representative mode dimer of WT Hβ2m and used the program SCAP ( included in the Jackal package [55] ) to replace ( in each monomer ) the original amino acid Asp by an Asn thus mimicking the mutation that occurs in the systemic amyloidosis characterized by extensive visceral amyloid deposits .', 'We then computed the electrostatic potential at the interface of both dimers ( i . e . with and without the mutation ) ( Fig . S4 ) .', 'Our results are consistent with the enhanced amyloidogenicity observed in vitro for Asp76Asn with regard to Hβ2m [21] , [22] because they indicate that the mutation contributes to stabilize the dimer by decreasing the amount of repulsive electrostatic interactions between the EF-loop of one monomer with the DE-loop of the second monomer .', 'In the case of ΔN6-N the pH has a modulating effect on the dimerization interface .', 'First , the DE-loop is no longer the major player in dimerization , as both the AB- and BC-loops gain significant importance ( Fig . S2B; Fig . 4C and Fig . 4E ) .', 'The most important structural element for dimerization in ΔN6-I at pH 6 . 2 is the unstructured and detached strand A together with the adjoining AB-loop ( Fig . S2C ) .', 'Furthermore , inspection of several dimer conformations with number of intermolecular contacts equal to the mode ( Fig . 4B and Fig . 4D; Fig . S3C and Fig . S3D ) reveals that strand A facilitates fibril growth by imposing a rather straightforward oligomerization pattern .', "Indeed , strand A acts as a sticky ‘hook’ that recruits another monomer by interacting with its DE- , EF- or FG-loops ( Fig . 4B and Fig . 4D; Fig . S3C and Fig . S3D ) thereby leaving the second monomer's strand-A available for further growth .", 'These ‘sticky hook’ interactions driven by strand A clearly drive a preferred oligomerization direction that could coincide with that of the amyloid fibril axis ( Fig . 4I ) .', 'Whenever monomer association involves strand A-strand A interactions , the resulting dimers can still grow via the BC- and DE-loops ( Fig . 4F; Fig . S3D ) .', 'In order to identify putative hot spots for aggregation , we computed the probability of intermolecular interaction per residue in the subset of the 50 most frequent intermolecular interactions .', 'The latter were identified by taking the ensemble of dimers used in the evaluation of the corresponding DH .', 'Pairs involving an aromatic amino acid and His or Lys dominate in ΔN6-I dimers at pH 6 . 2 .', "In WT-N ( Fig . 5A ) the distinctive predominance of interactions involving the DE-loop illustrates this region's importance for dimerization .", 'The relevance of the DE-loop in different experimental conditions has been acknowledged by several authors [11] , [56] , [57] , including a recent study by Eisenberg and co-workers which reported a hinge motif in dimers of Hβ2m based on DE-loop swapping at pH 8 ( in the presence of DTT ) [32] and another study by Rennella et al . which reported the formation of Hβ2m dimers with a head-to-head arrangement of monomers driven by DE-loop interactions [16] .', "The aromatic residues Phe56 , Trp60 ( located on the DE loop's tip ) , Phe62 , Tyr63 and the aliphatic Leu65 are expected dimerization spots because they assist the docking of Hβ2m onto the MHC-I heavy chain [2] .", 'Phe62 , Tyr63 and Leu65 were further shown to play an important role in fibril nucleation at acidic pH 2 . 5 [58] .', 'The importance of Phe56 and Trp60 in β2m oligomer assembly based on D-D strand association ( pH∼7 ) was reported in several studies [7] , [57] .', 'Of note , Trp60 was found to be the residue involved in the largest number of intermolecular contacts in Molecular Dynamics simulations that studied intermolecular interactions establishing between monomers of β2m [59] .', 'The results reported here recapitulate that , with the exception of Leu65 , DE-loop aromatic residues are important drivers of monomer association in Hβ2m .', 'We further find that lowering the pH reduces the importance of residues Phe56 , Lys58 and Phe62 , while Trp60 becomes a particularly promiscuous interaction hub at pH 6 . 2 ( Fig . 5A ) .', "However , the analysis of dimer's conformations whose formation is triggered by this region of the protein indicates that further oligomerization is not straightforward ( Fig . S3A and Fig . S3B ) .", 'In other words , our results suggest that while the DE-loop is certainly important for dimerization , the amyloid route that is triggered by this structural element is not the most efficient one .', 'Since the native state is the dominant conformational state in physiological ( and near physiological ) pH , this observation rationalizes the little amyloidogenic character of WT Hβ2m in those conditions .', 'Also , in line with this idea , we find that in ΔN6 , which is considerably more amyloidogenic than Hβ2m , the importance of Trp60 ( and nearby residues ) is substantially reduced , especially in ΔN6-I at pH 6 . 2 .', 'This observation is particularly relevant because at this pH the cleaved mutant is more amyloidogenic .', 'In ΔN6-N ( Fig . 5B ) , the region comprising the AB-loop ( residues 10 to 20 ) exhibits an increased probability to form intermolecular contacts at both pH values .', 'Due to increased solvent exposure of the BC-loop ( residues 28 to 34 ) ( Fig . 2C ) , there is also a significant enhancement of the participation of Phe30 and His31 ( especially at pH 6 . 2 ) .', 'Direct involvement of the BC-loop in ΔN6 oligomer assembly ( in physiological and near physiological pH ) was reported in Ref .', '[11] .', 'Furthermore , His31 was found to be a major contributor to the intermolecular contacts established in association interfaces within hexamers of H13F [7] and tetramers of DCIM50 , the E50C Hβ2m mutant disulfide-linked homodimer [57] , in physiological conditions ( in the presence of Cu2+ or 20% TFE and fibril seeds , respectively ) .', 'It was also observed to be a component of the non-covalent interface between two ΔN6 nanobody-trapped domain-swapped dimers ( pH 5 . 0 ) in the respective crystal asymmetric unit along with the aromatic residues Phe56 and Trp60 [31] .', 'The region comprising the end of strand F and the FG-loop ( residues 84 to 90 ) – which is not involved in dimerization of Hβ2m – becomes especially relevant for ΔN6-N ( pH 7 . 2 ) and ΔN6-I ( at both pHs ) ( Fig . 5C ) .', 'Interestingly , the stretch of amino acids 83N-89Q was implicated in the nanobody-driven domain-swapping aggregation of ΔN6 [31] and was shown to fibrillate into amyloid in a highly acidic pH 2 . 0 [60] .', 'At neutral pH , both the BC- and DE-loops of ΔN6-N and ΔN6-I deviate significantly from their native positions ( Table S1 and Table S3 ) .', 'The cleaved N-terminus , which is more detached from the core in ΔN6-I , facilitates such conformational migration .', "Consequently , His84 located in the FG-loop ( adjoined to the BC-loop ) , is more solvent exposed in ΔN6-N and ΔN6-I than in WT-N thus becoming an important interaction hub in the mutant's dimers ( Fig . 5B and Fig . 5C ) .", 'In the ΔN6-N dimer interfaces , His84 preferentially interacts with Phe56 and Trp60 at pH 7 . 2 , while interaction with Tyr10 becomes relevant in ΔN6-I , especially at pH 6 . 2 ( see next section for further details ) .', 'The low amyloidogenicity of the Hβ2m mutational variant ΔN6/H84A in physiological or near-physiological pH [11] may thus reflect the absence of relevant interactions involving His84 .', 'Taken together , these findings thus point out to a direct participation of His84 in Hβ2m association , which adds up to a proposed indirect effect according to which His84 helps maintaining the trans-isomerization of Pro32 thus enhancing the population of IT [61] .', 'In ΔN6-I ( Fig . 5C ) residues Tyr10 and His13 , located in strand A and start of the AB-loop , gain importance especially at pH 6 . 2 .', 'Previous studies reported the participation of these residues in association interfaces within hexamers of H13F [7] and tetramers of DCIM50 [57] .', 'Furthermore , a single Tyr residue can act as the sole driving force triggering self-aggregation of a short polyalanine peptide ( through cation - π and π-stacking interactions ) 
['Introduction', 'Results', 'Discussion', 'Methods']
['A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis ( DRA ) is a cleaved variant of β2-microglobulin ( ΔN6 ) lacking the first six N-terminal residues .', 'Here we perform a computational study on ΔN6 , which provides clues to understand the amyloidogenicity of the full-length β2-microglobulin .', 'Contrary to the wild-type form , ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH . This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients .', 'Results reported in this work , based on molecular simulations , indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core .', 'Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly .', 'This study further predicts that the detachment of strand A from the core is maximized at pH 6 . 2 resulting into higher aggregation efficiency .', 'The structural mapping of the dimerization interface suggests that Tyr10 , His13 , Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis .']
['Dialysis-related amyloidosis ( DRA ) is a conformational disease that affects individuals undergoing long-term haemodialysis .', 'In DRA the progressive accumulation of protein human β2-microglobulin ( Hβ2m ) in the osteoarticular system , followed by its assembly into amyloid fibrils , eventually leads to tissue erosion and destruction .', 'Disclosing the aggregation mechanism of Hβ2m under physiologically relevant conditions represents a major challenge due to the inability of the protein to efficiently nucleate fibrillogenesis in vitro at physiological pH . On the other hand , ΔN6 , a truncated variant of Hβ2m , which is also a major component of ex vivo amyloid deposits extracted from DRA patients , is able to efficiently form amyloid fibrils de novo in physiological conditions .', 'This amyloidogenic behavior is dramatically enhanced in a slightly more acidic pH ( 6 . 2 ) compatible with the mild acidification that occurs in the synovial fluid of DRA patients .', 'In this work , an innovative three-stage methodological approach , relying on an array of molecular simulations , spanning different levels of resolution is used to investigate the initial stage of the de novo aggregation mechanism of ΔN6 in a physiologically relevant pH range .', 'We identify an intermediate state for folding and aggregation , whose potential to dimerize is enhanced at pH 6 . 2 .', 'Our results provide rationalizations for previous experimental observations and new insights into the molecular basis of DRA .']
['physics', 'biology and life sciences', 'physical sciences']