Sequence Motifs in MADS Transcription Factors Responsible for Specificity and Diversification of Protein-Protein Interaction
[['Our ability to derive structural information from primary protein sequences has matured [1]–[4] .', 'In contrast , the way in which the primary protein sequence defines protein-protein interaction specificity is still largely unknown .', 'Nevertheless , several approaches to answer this question have recently been described [5]–[17] .', 'These methods focus mostly on protein-peptide interactions , which are more amenable to computational and experimental analysis than interactions between full length proteins .', 'In addition , protein structures or quantitative data about interaction energy are used , which is information that is in general not available .', 'Though , current experimental proteomic techniques allow unraveling protein interaction networks at unprecedented scale [18]–[21] and this opens the door towards computational and experimental approaches for studying protein-protein interaction specificity at the sequence level .', 'Most proteins are member of protein families , which are groups of evolutionarily related proteins that are characterized by the presence of specific domains .', 'When proteins from a given family interact with each other these interactions are often highly specific [22] .', 'In this particular context it is even more difficult to analyze how interaction specificity is defined than in the general case , as the protein sequences show a high degree of overall similarity with various small local differences .', 'A particular example of such a protein family is given by the plant MIKC MADS domain transcription factor family [23] , [24] .', 'The proteins from this family contain a MADS ( M ) , Intervening ( I ) , K-box and C-terminal domain , and have the potential to form various homo- and heterodimers [25] , [26] .', 'The MADS and K-domain are known to be involved in mediating these protein-protein interactions [27] , and the I-domain is thought to be important for determination of interaction specificity [28] , [29] .', 'MADS domain protein-protein interaction specificity is tightly linked to specific functions , as exemplified by the ABC ( DE ) model of floral organ formation , which ascribes roles to specific combinations of MADS proteins in the development of particular floral organs [30]–[32] .', 'The quartet model describes how floral organ identity is specified at the molecular level by the activity of four different tetrameric MADS protein complexes [33] .', 'A second process in which specific MADS domain transcription factor complexes are involved is the timing of flowering [34] , [35] .', 'The ability to form various homo- and heterodimers is in particular relevant for eukaryotic transcription factors such as the MADS domain transcription factors [36]–[38] .', 'Aspects of dimerization that are of functional importance include differential gene regulation by formation of dimers with distinct properties , as well as the addition of the monomer-to-dimer transition as an extra layer of regulation .', 'For the MADS domain transcription factor family as well as for other protein families , duplications occurred during evolution [39] which are often lineage- or species-specific .', 'After a duplication , one of the copies experiences relaxed selection pressure and hence can evolve a specialized or new function .', 'It is currently unclear to what extent changes in protein-protein interactions contribute to this process of sub- or neo-functionalization .', 'Moreover , in several cases there is apparently no change in function between the two copies , leading to functional redundancy .', 'This should be reflected in similar protein interaction patterns , even if the amino acid sequences of the proteins have diverged .', 'Examples of redundancy in interaction patterns related to functioning have been described for members of plant MADS domain protein families [40] , [41] .', 'Similarly , examples of functional divergence related to changes in protein-protein interaction specificity have been described [42] , [43] .', 'Insight into sequence level determinants of MADS domain protein-protein interaction specificity is thus of broad biological significance .', 'Here we present a combined computational and experimental approach towards understanding how protein-protein interaction specificity is encoded in the MADS domain protein sequences .', 'We started with our recently developed interaction prediction method [44] , which provided a set of predicted interaction motifs .', 'The importance of these short amino acid sequences was tested experimentally by introducing mutations and comparing the observed interaction pattern for mutated proteins with the predicted interaction pattern .', 'We analyzed conservation and variability of those motifs , which gives insight into their role in shaping MADS domain protein functioning and the evolution of the plant MADS domain transcription factor protein-protein interaction network .', 'At a general level , we show that protein interaction data sets can be interrogated to obtain sequence level insight into protein interaction specificity .', 'Members of large protein families perform in general the same biochemical tasks , e . g . transcriptional regulation , but the exact biological function of each individual protein is influenced by its unique set of interactions .', 'The approaches developed in this study set the stage for further investigations to understand how protein-protein interactions and hence protein functioning , is encoded in primary amino acid sequences .'], ["The basic premise of our computational approach , IMSS ( Interaction Motif Search and Selection ) [44] , is that specific motifs in a protein sequence together determine the proteins' interaction pattern .", 'The algorithm first searches for pairs of motifs that are overrepresented in pairs of interacting protein sequences , followed by motif pair selection via a feature selection approach .', 'This results in a set of correlated , complementary motif pairs , that when present in pairs of protein sequences predicts whether these proteins interact .', 'In general , a single motif does not determine the interaction specificity for a given protein , but rather this is determined by a combination of several motif pairs .', 'A detailed overview of IMSS is given in the Methods section , including adaptations of the method made , relative to its original implementation .', 'We applied IMSS to all members of the Arabidopsis MIKC MADS domain transcription factor family , using the available interaction data [25] .', 'Our data consists of in total 152 interactions between 35 proteins , and 478 non-interacting combinations; there is however a large spread in the number of interactions per protein , with six proteins that have only one interaction , and four that have more than 20 .', 'Previously these proteins were clustered based on their interactions [25] and although in some cases the most closely related paralogs clustered together , meaning that they have quite similar interaction specificities ( e . g . AGAMOUS ( AG ) , SHATTERPROOF1 ( SHP1 ) , SHATTERPROOF2 ( SHP2 ) and SEEDSTICK ( STK ) ) in other cases such paralogs did not cluster together and had quite different interaction specificities ( e . g . APETALA1 ( AP1 ) , CAULIFLOWER ( CAL ) and FRUITFUL ( FUL ) or SUPPRESSOR OF OVEREXPRESSION OF CO 1 ( SOC1 ) and AGAMOUS LIKE14 ( AGL14 ) ) .', 'We applied IMSS with three different settings: ( 1 ) the Arabidopsis data as described above were used as input data with the original IMSS algorithm as presented before [44] ( “ara_original” ) ; ( 2 ) usage of “ara_original” with small modifications described in the Methods section ( “ara_new” ) ; or ( 3 ) usage of Arabidopsis data together with data from additional species as input ( “all_species” ) .', 'Leave-one-out cross-validation on the Arabidopsis proteins was used to select the best performing model We also tested leave-family-out cross-validation with similar results ( not shown ) .', 'To assess the quality of our predictions , both in the cross-validation setting discussed here and in the experimental validation described below , we use the F-score , which is the harmonic mean of precision and recall .', 'With 0 . 44+/−0 . 3 ( average +/− standard deviation ) the F-score for “ara_new” was slightly higher than the F-scores for the other two settings ( “ara_original” , 0 . 41+/−0 . 3; “all_species” , 0 . 40+/−0 . 3 ) .', 'However , these differences are clearly very small , and indeed the overlap between the predicted interaction motifs is relatively large , especially between “ara_orig” and “ara_new” .', 'Around 90% of the sequence positions are simultaneously either covered or not covered by a motif in each of these two models .', 'This number is somewhat lower between these two models and the “all_species” model ( around 75% ) .', 'By comparing with randomly generated motif-hits with the same distribution over the proteins but randomized positions , we found that the overlaps in motifs between our three IMSS settings are statistically significant with p<0 . 001 ( data not shown ) .', 'For each protein the resulting predicted protein-protein interaction motifs from “ara_new” are given in Table S1 .', 'Motifs were found in all the different domains ( MADS , I , K and C ) , but occurred most frequently at the border between the MADS and I-domain , in line with the proposed role of the I-domain in determining dimerization specificity [28] , [29] , [45] .', 'This ‘hotspot’ region is homologous to a region in the human MADS domain protein Myocyte Enhancer Factor-2 ( MEF2 ) [46] that interacts with a helix of the Cabin1 protein ( Figure 1 ) .', 'The motif that is complementary to most of the motifs in the MIKC MADS hotspot region is found in the K-box of the interacting proteins , a domain that is predicted to form α-helices [47]–[49] comparable to Cabin1 .', 'This data suggests a specific mode of interaction between the I-region of plant MADS proteins and the K-domain of their interaction partners .', 'Remarkably , no interaction motifs were predicted in the first helix in the MADS domain .', 'This helix contacts both the equivalent helix in the partner MADS domain and the DNA to which the MADS domain binds [46] ( Figure 1 ) .', 'Hence , it is involved in protein-protein contacts , but our computational analysis predicts that these contacts do not contribute to interaction specificity .', 'They might be important , however , in determining interaction affinity of MADS protein dimers in general .', 'This proposed decoupling of protein-DNA and protein-protein interaction specificity determination is in line with earlier experimental observations [50] and is an example of modularity at the protein structure level , which might be important from an evolutionary perspective as it allows independent diversification of these two functions .', 'Note however that we cannot exclude the possibility that determinants of interaction specificity are present in the first helix of the MADS domain but are missed by our approach .', 'In addition to the above-mentioned hotspot region , several motifs are found in the K-domain .', 'This region is indeed known to be involved in dimerization , and in fact for a couple of proteins experimental data is available showing that mutations in the K-domain change interaction specificity .', 'In particular , for AP3 and PI , several mutations are known that influence their partner formation [51] , [52] and although IMSS does not predict motifs for AP3 , for PI indeed two motifs are predicted in the K-domain ( and none in the MADS or I-domain ) .', 'Another example of a motif occurrence in the K-domain which can explain previous experimental observations is a motif found in CAL , which coincides with the cal-4 E131K mutation [53] .', 'Besides obtaining information on the molecular mechanisms underlying MADS domain protein-protein interactions , our IMSS method can be used to predict MADS domain interactomes for other plant species .', 'In order to test the usefulness of IMSS for this purpose , interactions were predicted for MADS domain proteins of plant species from which some experimental protein-protein interaction data are available ( “all_species” model; see Text S1 and Table S2 ) .', 'These additional data consist of 188 interactions between 98 proteins .', 'Although , the “ara_new” model obtained the best prediction performance for Arabidopsis MIKC MADS domain proteins based on leave-one-out cross-validation and performed best in the experimental validation ( see below ) , it turned out to have only a very low performance on data from non-Arabidopsis species , for which it obtained a recall of only 0 . 16 .', 'We cannot calculate precision or F-score for these additional species , because in most cases only interactions are known and no information is available about non-interactions .', 'However , based on leave-one-out cross-validation the “all_species” model obtained a recall of 0 . 60+/−0 . 44 for the data from other species .', 'The reason for this strong difference in performance is that despite a quite similar position of motif hits in Arabidopsis between the models ( see above ) , their exact definition is somewhat different .', 'Some of the motifs obtained upon training with Arabidopsis information only , appeared to be too “strict” and hence , occurring only in few cases in non-Arabidopsis sequences ( data not shown ) .', 'As mentioned above , the “all_species” model obtained a F-score of 0 . 40+/−0 . 3 for the Arabidopsis interactions .', 'Hence , the performance of this model for Arabidopsis is only slightly lower than what was obtained without adding these additional data ( “ara_new” ) , and the recall on these additional data is comparable to what is obtained for Arabidopsis .', 'The model obtained after training with this augmented dataset provided insight into interaction patterns among family members across plant species ( Tables S3 and S4 ) and demonstrates the usefulness of the method for the prediction of interactions of MADS domain proteins from species for which only sequence information is available .', 'Predicted interaction motifs point to sites in the MADS protein sequence that may be essential for determination of protein-protein interaction specificity ( for comparison with existing analyses see Tables S7 and S8 ) .', 'If this hypothesis is correct , then introducing mutations in these sites should lead to modified interaction patterns .', 'We chose several Arabidopsis MIKC MADS proteins as experimental test-cases for this hypothesis .', 'When selecting validation targets , we aimed for applying mutations to different regions in the MADS proteins and using MADS proteins with a range of different biological functions .', 'In addition , our aim was to demonstrate gain of interaction partners , and in part we aimed for obtaining swaps of interactions partners ( see Text S1 ) .', 'The reason for the latter is that loss of interaction partners is in principle simple to obtain by modification of the sequence , which probably affects also the folding of the particular domain in which the mutation is introduced .', 'However , obtaining gain of interaction demands well thought amino acid changes that result in modified characteristics of an interaction site .', 'The selected MADS domain proteins for our validation approach are involved in flowering time determination ( AGAMOUS-LIKE24 ( AGL24 ) , SHORT VEGETATIVE PHASE ( SVP ) , SOC1 ) , floral meristem or organ identity specification ( AP1 , CAL and AG ) or have an unknown function ( AGL14 ) .', 'As described below , the experimental validation results for “ara_new” were somewhat better than the results for “ara_original” or “all_species”: its F-score on all mutants is 0 . 54 vs . 0 . 48 for the other two settings ( we also tested whether this model was still performing best when leaving out any of the mutations , which was indeed the case for all mutations ) .', 'Because of its better performance , in the presentation of the results below , we will focus on the “ara_new” results , unless otherwise indicated .', 'Note that the slightly better performance of the experimental validation in case of “ara_new” is consistent with its better performance as judged by leave-one-out cross-validation on the Arabidopsis interaction data .', 'Mutations were designed based on the predicted interaction motifs ( Table S1 , Table S5 , and Text S1 ) and the mutant proteins tested in a matrix-based yeast-two-hybrid assay against the collection of Arabidopsis MADS domain proteins [25] .', 'The approach is illustrated in detail for the AGL14 and SOC1 proteins ( Figure 2; Table 1 ) .', 'These two MADS proteins share over 50% sequence identity and group in the same phylogenetic clade [24] , but have quite different interaction patterns .', 'In contrast to AGL14 , SOC1 contains a predicted interaction motif at the junction between the MADS domain and I-region , the position that coincides with the aforementioned ‘hotspot-region’ .', 'Swapping the amino acids in this region between SOC1 and AGL14 was sufficient to exchange a large part of their interaction specificity , as was predicted by the IMSS method .', 'In particular , for the mutated SOC1 protein we predicted a loss of 13 of its interaction partners , and for the mutated AGL14 protein a gain of 15 ( the difference of two relates to the accuracy of our method for the original interaction data ) .', 'Experimentally , mutated SOC1 lost 20 of its interaction partners , and of the six that it kept , two were indeed also found as interactions partners for AGL14 .', 'Mutated AGL14 gained seven interaction partners , all of which were indeed also interacting with SOC1 .', 'As mentioned above , to assess the quality of our predictions , we use the F-score , which is the harmonic mean of precision and recall .', 'Here , the F-score is calculated for predicting interaction patterns of mutated MADS domain proteins with the predictor that is trained with original Arabidopsis interaction data only .', 'The F-score for our predictions for these two mutated proteins is 0 . 71 ( AGL14 ) and 0 . 63 ( SOC1 ) .', 'To put these values in perspective , we used as a null model the interaction pattern obtained by the wild type proteins .', 'Using this null model would give a substantially lower F-score of 0 . 56 and 0 . 38 , respectively , which means that our predictor performs much better in this case .', 'F-scores for all the experimental test-cases are shown in Table S5 .', 'All mutations that we introduced led to changes in interactions .', 'Overall , in eight out of 15 cases ( ∼53% ) the mutations we introduced led specifically to loss of interactions , in three cases ( ∼20% ) specifically to gain of interactions , and in the remaining ∼27% of cases , both loss and gain were obtained ( Figure 3 , Table 1 , and Table S6 ) .', 'The number of gains and losses that our IMSS method ( “ara_new” model ) predicted , displayed a good correlation with the experimentally observed number of gains and losses ( Pearson correlation coefficient 0 . 76 , p-value 0 . 0005 ) .', 'When separating gains and losses , the correlation coefficient values are 0 . 63 and 0 . 67 , respectively ( p-value<0 . 006 ) .', 'The changing interaction patterns were predicted with a reasonable accuracy for most of the mutated proteins ( Figure 3B; Table 1 ) .', 'In particular , only for five out of 15 cases our predictions had a worse F-score than the F-score obtained using the original interaction pattern as predictor for the mutated proteins ( null model ) .', 'For seven out of 15 mutations our predictions had a better F-score and for an additional three cases , the obtained F-scores were similar to each other .', 'These latter cases all concerned mutations in the CAL protein , which were introduced based on results of the “ara_original” model , but that in the “ara_new” model were predicted to have no effect .', 'Surprisingly , the effect of the CAL double mutation was that no interaction remained at all .', 'Almost the same holds for the AP1 double mutation ( only one interaction remained ) .', 'One particular explanation for these results could be that introduction of double mutations has the unfortunate side-effect of disturbing the structure of the proteins to a large extent .', 'This might in particular be the case when these two mutations , which are far apart in the sequence of those proteins , coincide in the 3D structure of the protein .', 'Although no structure data is available for these proteins outside the MADS domain , we investigated this possibility by performing an intramolecular correlated mutation analysis on a large set of AP1 orthologs ( for CAL , too few orthologous sequences were available ) .', 'The analysis predicted several intramolecular interactions of residues in the K-box helix around the mutation side with residues in the I-region around the mutation side , e . g . residue E149 in the K-box with residue S74 .', 'Based on this observation , we hypothesize that double mutations in AP1 and CAL ( which is closely related to AP1 ) introduce too large structural changes leading to instability or mis-folding of the protein .', 'Hence , the fact that our predictions are not correct in this case can be rationalized .', 'In SVP we introduced two single mutations and a double mutation aiming to change its interaction pattern towards that of the closely related AGL24 proteins .', 'Alternative splicing has been reported for SVP and the protein named SVP1 , which was used in this particular experiment , is encoded by the fully spliced SVP transcript [54] .', 'Although SVP and AGL24 are phylogenetically grouped in the same clade [24] , they act opposite in flowering , being a repressor and activator of this process , respectively .', 'In this case the two mutations are in close proximity in the sequence and within one predicted interaction motif positioned at the ‘hotspot region’ between the MADS and I domain of the SVP protein .', 'Swapping of this interaction motif between AGL24 and SVP1 did not result in a complete exchange of interaction pattern ( Table 1 ) .', 'Though , for one single mutation ( SVP1 S61R ) and the double SVP1 mutant ( SVP1 C58S/S61R ) at least interaction with the floral repressor protein FLOWERING LOCUS C ( FLC ) was lost , which is supposed to be important for its floral repressor function [55] .', 'Introducing the “SVP1”-motif in AGL24 did not result in interaction with FLC , but the mutated protein ( AGL24 R61S ) gained interaction with MADS AFFECTING FLOWERING 2 ( MAF2 ) , which is closely related to FLC and acts also as floral repressor [56] .', 'Probably , mutations in various interaction motifs need to be combined in order to get an exact and complete swap of interaction pattern between AGL24 and SVP1 .', 'Indeed , mutations in predicted interaction motifs in the C-terminal region of SVP1 , which is most divergent from AGL24 , showed that this part of the protein also contributes to SVP1 interaction specificity ( Table 1 ) .', 'As indicated above , our predictor performs worse than the null model in five out of 15 cases .', 'This seemingly moderate performance can be understood as caused by side effects of the mutations for at least some of the cases ( see discussion about AP1 and CAL above ) .', 'Nevertheless , in order to further investigate the statistical significance of our predictions of interaction changes upon introducing mutations and to judge the value of our predictor , we used a second null model .', 'Here , a series of models were trained with interaction data to which increasing amounts of noise were added , from 10% to 40% ( see Methods for details ) .', 'If our predictions using the experimental data are significantly different from random expectation , one would expect that the more noise is added to the data , the worse the prediction is .', 'This is indeed what we observe , with the F-score dropping continuously from 0 . 46+/−0 . 05 at 10% noise to 0 . 24+/−0 . 1 at 40% noise ( average and standard deviation over 10 different random tests ) .', 'As an extreme version of this randomization we also performed the test using fully randomized data as input , obtained by keeping the interaction network unchanged but randomly reassigning sequences to the MADS proteins .', 'This randomization was repeated 1000 times , resulting in an average F-score of 0 . 23+/−0 . 1 .', 'None of these random tests obtained a similar or higher F-score than what was obtained for our predictor trained with the experimental data ( Figure 3B ) .', 'This indicates that with p<0 . 001 our results are significantly different from random expectation .', 'The putative effect of mutations on protein-protein interaction specificity must have played an important role in the evolution of the MADS protein interaction network .', 'Our interaction motifs provide means to investigate this .', 'Of importance here is the balance between motif conservation , which leads to a static interaction network , and variability of motifs , which could lead to interaction loss or gain and potentially to neo- or sub-functionalization of particular proteins .', 'The latter is especially relevant after gene or ( partial ) genome duplications , which has played an important role in expansion of the plant MADS domain transcription factor family [39] .', 'We expect to observe overall conservation of the interaction motifs , but also variation of motifs when comparing duplicated proteins .', 'We performed several analyses on the interaction motifs to test this hypothesis , the results of which are described below .', 'First , we compared predicted motifs with available non-synonymous single nucleotide polymorphism ( SNP ) data [57] .', 'Comparison of “motif density” and “SNP density” showed that these are negatively correlated ( Figure 4A ) .', 'For the ∼1500 non-synonymous SNPs falling within MADS protein sequences , 170 cases were found where a SNP was located inside a motif occurrence ( Table S10 ) .', 'Randomly generated motif occurrences with the same number of occurrences per protein as the predicted motifs were generated in 1000 trials .', 'The average overlap of SNPs with those motif occurrences was 351+/−116 , and in 965 out of 1000 random trials the overlap was larger than 170 .', 'This indicates that the experimental overlap between IMSS motifs and SNPs is significantly smaller than the randomly expected overlap ( p<\u200a=\u200a0 . 04; see Table S10 ) .', 'In addition , the cases where SNPs overlap motifs are conservative mutations ( several non-conservative SNPs do affect the MADS proteins , but they fall outside the predicted interaction motifs ) .', 'In fact , the largest contribution to the 170 overlaps between SNPs and motifs is formed by in total 122 SNPs found at two consecutive positions in AGL14 , where an S is changed to a T and a T to an S ( Ser187 , Thr188 ) .', 'The few cases with an overlap between a potentially more important SNP and an interaction motif indicate interesting candidates for putative causes of functional differences between MADS proteins in various Arabidopsis accessions .', 'This includes for instance a Q->E SNP in ANR1 that occurs in several ecotypes ( Table S10 ) .', 'The analysis above only captures short term evolutionary dynamics and might be biased by the preference of motif existence in certain sequence regions ( e . g . the motifs are generally absent in the C-region of the proteins , which is the most variable region ) .', 'To obtain further insight into the conservation of predicted interaction motifs , we analyzed the extent to which motifs are conserved in putative orthologous protein sequences from various sequenced plant genomes ( see Methods ) .', 'This conservation was compared to that of homologous regions in MADS proteins without a motif occurrence at that particular position in the sequence .', 'This approach ensures that the results are not influenced by a bias for motifs in particular protein domains .', 'The analysis showed that on average the fraction of motif characters that were completely conserved was 0 . 65+/−0 . 09 , whereas the same fraction for non-motifs was 0 . 55+/−0 . 09 ( average +/− standard deviation ) .', 'We used a Kolmogorov-Smirnov test which indicated that with p∼10−7 the distribution of fraction conservation of characters of motifs was significantly different from that of non-motif characters .', 'Focusing on motif-characters that are non-wildcards ( i . e . not “*” ) , the difference was even somewhat higher , 0 . 73+/−0 . 09 ( motifs ) vs . 0 . 58+/−0 . 07 ( non-motifs ) ( Figure 4B ) .', 'In addition , the number of non-conservative changes was much higher in the non-motifs than in the motifs; for example , a swap from K or R to D or E occurred in only 1% of the characters of motifs versus 3% of the non-motif sequence regions .', 'Note that our interaction motifs were obtained using a limited set of sequences and interaction data , and as such their conservation across various species is an independent validation of their functional importance .', 'The observed conservation of predicted interaction motifs provides supporting evidence that the IMSS motifs are under functional constraints .', 'Such functional constraints would be relaxed after a gene duplication in one of the two copies , allowing sub- or neo-functionalization to occur via modification of interaction motifs .', 'To analyze this , we first focused on indels coinciding with IMSS motifs ( see Text S1; note that here we used motifs from the “all_species” model as these are more appropriate in this context where we use sequences from various species ) .', 'This revealed several examples of duplicated proteins where an indel overlaps with an interaction motif ( Table S11 ) .', 'Specifically , out of 81 pairs of putative paralogous proteins containing an indel , 29 pairs were found in which the indel overlaps with a predicted interaction motif ( in 20 different species ) .', 'In half of these cases both of the paralogs contain a motif ( albeit in general a different one ) at the indel position ( Figure 4C ) .', 'Those cases where both copies contain an interaction motif are the result of sequence changes after the gene duplication that modified , but did not delete the interaction motif .', 'This suggests that after such duplication one of the copies indeed acquired a novel or specialized function ( option II in Figure 4C ) .', 'Hence , this analysis highlights interesting candidates for possible sub- or neo-functionalization and confirms the expected role of variability of interaction motifs in these processes .', 'Unfortunately , in most cases we do not know the interaction patterns of the proteins so we cannot validate our predictions , but the Arabidopsis cases we found have clearly different interaction patterns .', 'For the proteins in the SEP clade for instance , some evidence is present that they are not fully functional redundant [58] , [59] .', 'Interestingly , the interaction motifs in the ‘hotspot region’ at the junction between MADS and I domain overlap an intron/exon boundary .', 'This provides a plausible evolutionary mechanism to generate protein interaction diversity by shifting these intron/exon boundaries after duplications or via alternative splicing ( Figure 5 ) .', 'Indeed the above mentioned cases where indels modify interaction motifs in duplicated proteins occur often in the MADS domain protein ‘hotspot region’ , in which interaction motifs overlap an intron/exon boundary .', 'An example of a change in interaction pattern via ‘splicing out’ of a predicted interaction motif is observed in a recently identified SVP splicing variant ( named SVP3; Accession: EU078686; Figure 5 ) .', 'SVP3 lost the interaction motif found in the hotspot region of the fully spliced SVP protein ( SVP1 ) leading to a large loss of protein interaction partners ( Figure 5; Table 1; Figure S2 ) .', 'Additional discussion is provided in the Supplementary information ( Text S1 , Figure S1 ) , where we also provide an analysis of the distance of predicted interaction motifs from intron/exon boundaries .', 'Based on these findings we hypothesize that shifting intron/exon borders plays a role in neo-functionalization of plant MADS domain transcription factors by direct changing of dimerization capacity ( Figure 5 ) .', 'Note that , at least in the above-mentioned SVP case , this mechanism seems to allow the duplicate to optimize in one specific interaction and avoid conflict with the original copy , by deleting other common interact
['Introduction', 'Results', 'Discussion', 'Methods']
['Protein sequences encompass tertiary structures and contain information about specific molecular interactions , which in turn determine biological functions of proteins .', 'Knowledge about how protein sequences define interaction specificity is largely missing , in particular for paralogous protein families with high sequence similarity , such as the plant MADS domain transcription factor family .', 'In comparison to the situation in mammalian species , this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana .', 'Here , we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family , using an integrated computational and experimental approach .', 'Plant MADS proteins have highly similar amino acid sequences , but their dimerization patterns vary substantially .', 'Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy .', 'Furthermore , we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species .', 'Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions .', 'In addition , various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity .', 'Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs , indicating their evolutionary importance .', 'We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization .', 'The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution .']
['Proteins are active in complexes and therefore it is of utmost importance to understand their protein-protein interaction patterns .', 'Over the last decade , various large-scale screening methods have been developed and implemented providing insight into interactomes for a number of model species .', 'Despite all these efforts , our knowledge about molecular mechanisms determining interaction specificity and underlying protein network formation is limited .', 'Here , we applied an integrated computational and experimental approach to identify short sequence motifs that determine interaction specificity for members of the large and important MADS domain transcription factor family .', 'We show that the method is able to predict MADS domain transcription factor interaction networks for non-model plant species with a reasonable accuracy .', 'Subsequently performed experiments revealed the possibility to rewire protein-protein interaction networks in a desired manner by generating small dedicated mutations in predicted interaction motifs .', 'Additional bioinformatics analyses led to interesting hypotheses about protein-protein interaction network evolution .', 'All together , these results provide insight in the way protein-protein interaction specificity is laid down in primary protein sequences and open the road towards a better understanding of protein complex formation and the possibility to control this important process .']
['plant biology/plant genomes and evolution', 'computational biology/sequence motif analysis', 'computational biology/transcriptional regulation', 'developmental biology/plant growth and development', 'molecular biology/bioinformatics', 'computational biology/macromolecular sequence analysis', 'biochemistry/bioinformatics', 'evolutionary biology/plant genetics and gene expression']