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• W2010516785 abstract "Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. Analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and that binding hotspots tend to be phosphorylated in heterooligomers. Although the majority of complexes do not show significant estimated stability differences upon phosphorylation or dephosphorylation, for about one-third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are more likely to be evolutionary conserved than other interfacial residues. Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. Analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and that binding hotspots tend to be phosphorylated in heterooligomers. Although the majority of complexes do not show significant estimated stability differences upon phosphorylation or dephosphorylation, for about one-third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are more likely to be evolutionary conserved than other interfacial residues. Phosphosites tend to be located on interfaces in protein structural complexes Phosphorylation does not change binding affinity significantly for more than half cases Phosphorylation at binding hotspots is frequent and may disrupt the complex formation Phosphosites are more conserved than other interface sites Cellular regulatory mechanisms provide a sensitive, specific and robust response to external stimuli and posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability (Olsen et al., 2006Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2642) Google Scholar, Ptacek and Snyder, 2006Ptacek J. Snyder M. Charging it up: global analysis of protein phosphorylation.Trends Genet. 2006; 22: 545-554Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Schlessinger, 2000Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3350) Google Scholar). Such dynamic regulation is achieved through reversibility and fast kinetics of posttranslational modifications, such as when, for example, a phosphate group can be quickly attached and removed by kinases and phosphatases, respectively. Indeed, adding or removing a dianionic phosphate group somewhere on a protein might change its physico-chemical properties, stability, kinetics, and dynamics (Johnson, 2009Johnson L.N. The regulation of protein phosphorylation.Biochem. Soc. Trans. 2009; 37: 627-641Crossref PubMed Scopus (198) Google Scholar). Recent phosphoproteomic analyses have revealed that the majority of proteins in a mammalian cell are phosphorylated (Olsen et al., 2006Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2642) Google Scholar, Olsen et al., 2010Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. et al.Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1046) Google Scholar), so regulatory mechanisms involving phosphorylation are very widespread. Many signaling and other types of pathways involve a dense network of protein-protein interactions, and the reaction rates of these processes, among other factors, will depend on protein concentrations and association/dissociation constants of protein assemblies. Phosphorylation can be used to modulate the nature and the strength of protein-protein interactions, thereby regulating protein binding and coordinating different pathways. If phosphorylation occurs at or near a binding interface, it may directly affect the binding energy of the complex. At the same time, phosphorylation of a site outside a binding interface may cause long-range conformational changes through allosteric mechanisms and affect the binding of the partner, as observed for the classical example of glycogen phosphorylase (Jenal and Galperin, 2009Jenal U. Galperin M.Y. Single domain response regulators: molecular switches with emerging roles in cell organization and dynamics.Curr. Opin. Microbiol. 2009; 12: 152-160Crossref PubMed Scopus (66) Google Scholar, Lin et al., 1997Lin K. Hwang P.K. Fletterick R.J. Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases.Structure. 1997; 5: 1511-1523Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Another aspect of coupling between phosphorylation and binding is the recognition of the phosphates by special phospho-Ser/Thr or Tyr binding domains (such as 14-3-3, SH2, MH2, and others); such a process may release the protein from autoinhibition and result in activation and subsequent signal propagation, as in the case of Src kinases (Schlessinger, 2000Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3350) Google Scholar). Finally it has been shown that flexible regions and intrinsically disordered proteins have a tendency to be phosphorylated, and phosphorylation might induce disorder-to-order as well as order-to-disorder transitions (Antz et al., 1999Antz C. Bauer T. Kalbacher H. Frank R. Covarrubias M. Kalbitzer H.R. Ruppersberg J.P. Baukrowitz T. Fakler B. Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate.Nat. Struct. Biol. 1999; 6: 146-150Crossref PubMed Scopus (64) Google Scholar, Collins et al., 2008Collins M.O. Yu L. Campuzano I. Grant S.G. Choudhary J.S. Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder.Mol. Cell. Proteomics. 2008; 7: 1331-1348Crossref PubMed Scopus (130) Google Scholar, Gsponer et al., 2008Gsponer J. Futschik M.E. Teichmann S.A. Babu M.M. Tight regulation of unstructured proteins: from transcript synthesis to protein degradation.Science. 2008; 322: 1365-1368Crossref PubMed Scopus (335) Google Scholar, Radhakrishnan et al., 1997Radhakrishnan I. Pérez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions.Cell. 1997; 91: 741-752Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). In this article, we analyze the effect of phosphorylation on protein binding for different types of complexes from the human proteome varying by stability and the nature of the interacting subunits. We show that there exists a coupling between phosphorylation and protein-protein binding for all types of heterooligomeric and weak transient homooligomeric complexes. Computational alanine scanning experiments and analysis of the energetic effect of attaching/removing phosphate groups show that phosphorylation may modulate the strength of interactions directly on interfaces and that binding hotspots have a tendency to be phosphorylated for heterooligomers. Although for many Ser/Thr/Tyr sites we did not find significant stability differences upon attaching/removing the phosphate group, for one-third of all complexes this brings about a relatively large change in binding energy (more than 2 kcal/mol). We analyze the effect of phosphorylation on protein function and show that several pathways, especially the hemostasis pathway, are enriched with phosphoproteins and phosphosites. Finally, we show that phosphosites on interfaces are more likely to be evolutionarily conserved than other interfacial residues. Using a nonredundant set of 933 structures of phosphorylated human hetero- and homooligomeric complexes (see Experimental Procedures for detail), we observed on average two phosphorylation sites (pTyr, pSer, or pThr) per protein. Note that the majority of protein complexes do not have actual phosphate groups in the Protein Data Bank (PDB) structures (Zanzoni et al., 2011Zanzoni A. Carbajo D. Diella F. Gherardini P.F. Tramontano A. Helmer-Citterich M. Via A. Phospho3D 2.0: an enhanced database of three-dimensional structures of phosphorylation sites.Nucleic Acids Res. 2011; 39: D268-D271Crossref PubMed Scopus (33) Google Scholar). As one can see from Figure 1, the distribution of fractions of phosphosites in phosphoproteins is quite narrow with a large majority of all phosphocomplexes having about 5%-10% of all Ser, Thr, or Tyr residues phosphorylated. The distribution has a long tail, however, which is consistent with the fact that proteins with multiple phosphorylation sites occur more often than expected by chance, in agreement with previous studies for Arabidopsis thaliana (Riaño-Pachón et al., 2010Riaño-Pachón D.M. Kleessen S. Neigenfind J. Durek P. Weber E. Engelsberger W.R. Walther D. Selbig J. Schulze W.X. Kersten B. Proteome-wide survey of phosphorylation patterns affected by nuclear DNA polymorphisms in Arabidopsis thaliana.BMC Genomics. 2010; 11: 411Crossref PubMed Scopus (20) Google Scholar). Overall, we observed the relative fractions of the types of phosphosites to be ∼40% pSer, ∼25% pThr, and ∼35% pTyr in protein structural complexes, and this observation did not depend on whether the complexes represented hetero- or homooligomers. The frequencies of pSer, pThr, and pTyr observed in structural complexes were quite different from those obtained in high-throughput experiments for phosphoproteomes, which identified only a small fraction of pTyr sites (Hunter and Sefton, 1980Hunter T. Sefton B.M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine.Proc. Natl. Acad. Sci. USA. 1980; 77: 1311-1315Crossref PubMed Scopus (1529) Google Scholar, Olsen et al., 2006Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2642) Google Scholar). This discrepancy may be explained by the observation that hydrophobic Tyr is more likely to be found in structured regions, whereas Ser and Thr are frequently found in disordered and flexible regions. Indeed, it was reported recently that almost half of pTyr sites were located within conserved protein domains (Sugiyama et al., 2008Sugiyama N. Nakagami H. Mochida K. Daudi A. Tomita M. Shirasu K. Ishihama Y. Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis.Mol. Syst. Biol. 2008; 4: 193Crossref PubMed Scopus (301) Google Scholar). Moreover, tyrosine phosphorylation might occur on less abundant proteins compared to serine and threonine phosphorylation, hence the statistics for rather redundant phosphoproteomes may differ from our nonredundant set. Further, we studied the coupling between phosphorylation and protein-protein binding by examining binding interfaces and locations of phosphosites in complexes (see Table S1 available online). Overall, we found that the association between phosphorylation sites and binding interfaces is very strong for heterooligomers (Fisher's exact test, p value = 7.4e-15) and significant but not so prominent for weak transient homooligomers (p value = 0.0008) (Figure 1; Table 1; Table S2). No association was found for permanent and strong transient homooligomers. Because the stability of the complex depends on the number of subunits, we also performed a similar analysis restricted to dimers and found a similar trend (p value = 5.3e-06 for heterooligomers). The tendency of phosphosites to be involved in binding did not correlate with the estimated stability of heterooligomers, which in turn were generally less stable than all homoologomers according to our analysis (Wilcoxon rank-sum test, p value = 0.03). These results are consistent with our previous study, which showed that transient complexes that bind different protein partners using the same interface (promiscuous binding) are enriched with Tyr, Ser, and Thr (among a few other residues) on their interfaces, and their phosphorylation may provide the switch between different functional pathways (Tyagi et al., 2009Tyagi M. Shoemaker B.A. Bryant S.H. Panchenko A.R. Exploring functional roles of multibinding protein interfaces.Protein Sci. 2009; 18: 1674-1683Crossref PubMed Scopus (23) Google Scholar).Table 1Properties of Phosphorylation Sites on Protein Binding InterfacesAllAll HeterooligomersAll HomooligomersHomooligomersWeakStrongPermanentAbundance on interface1.5e-13∗7.4e-15∗0.0978.2e-04∗0.4170.054Structural properties Protomer ASAaASA of a given protomer without binding partner.2.2e-16∗2.2e-16∗0.0650.0570.3180.137 ΔASAbDifference in ASA upon complex formation.4.5e-08∗1.7e-09∗0.4820.6090.3610.050 No. of hydrogen bonds per site5.9e-05∗5.3e-04∗0.043∗0.042∗0.2720.202 No. of residue-residue contacts per site2.8e-04∗1.1e-03∗0.2170.5020.4240.452Energetic propertiescΔΔΔGala is the difference in binding energy upon Ala substitution. ΔΔΔGp is the difference in binding energy upon attaching/removing phosphate groups to phosphorylation sites on interfaces. The p values for ΔΔΔGp indicate whether the distribution is significantly shifted to positive values. See also Figure S1 and S2. ΔΔΔGala1.8e-03∗2.7e-04∗0.4940.1810.6700.390 ΔΔΔGp2.2e-16∗1.3e-12∗1.3e-08∗2.8e-05∗1.1e-04∗1.6e-05∗Evolutionary conservation of site0.018∗0.016∗0.2960.5580.1130.654All values are presented as p values. The “Abundance on interface” row presents p values calculated by Fisher's exact test showing association between being phosphorylated and location on binding interface (compared to surface). All other rows present p values calculated by Wilcoxon rank-sum test showing the difference between phosphosites and nonphosphosites on binding interfaces with respect to different properties. Significant p values (after Holm-Bonferroni correction) showing enrichment of phosphosites with a given property are denoted with an asterisk (∗). ASA, accessible surface area.a ASA of a given protomer without binding partner.b Difference in ASA upon complex formation.c ΔΔΔGala is the difference in binding energy upon Ala substitution. ΔΔΔGp is the difference in binding energy upon attaching/removing phosphate groups to phosphorylation sites on interfaces. The p values for ΔΔΔGp indicate whether the distribution is significantly shifted to positive values. See also Figure S1 and S2. Open table in a new tab All values are presented as p values. The “Abundance on interface” row presents p values calculated by Fisher's exact test showing association between being phosphorylated and location on binding interface (compared to surface). All other rows present p values calculated by Wilcoxon rank-sum test showing the difference between phosphosites and nonphosphosites on binding interfaces with respect to different properties. Significant p values (after Holm-Bonferroni correction) showing enrichment of phosphosites with a given property are denoted with an asterisk (∗). ASA, accessible surface area. Although phosphorylation sites are usually located on protein surfaces, some of their structural properties are different from the other surface residues (Gnad et al., 2007Gnad F. Ren S. Cox J. Olsen J.V. Macek B. Oroshi M. Mann M. PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites.Genome Biol. 2007; 8: R250Crossref PubMed Scopus (381) Google Scholar, Jiménez et al., 2007Jiménez J.L. Hegemann B. Hutchins J.R. Peters J.M. Durbin R. A systematic comparative and structural analysis of protein phosphorylation sites based on the mtcPTM database.Genome Biol. 2007; 8: R90Crossref PubMed Scopus (48) Google Scholar, Zanzoni et al., 2011Zanzoni A. Carbajo D. Diella F. Gherardini P.F. Tramontano A. Helmer-Citterich M. Via A. Phospho3D 2.0: an enhanced database of three-dimensional structures of phosphorylation sites.Nucleic Acids Res. 2011; 39: D268-D271Crossref PubMed Scopus (33) Google Scholar). We analyzed the structural properties of phosphosites (sites that can be phosphorylated even if there is no actual phosphate present in the PDB structure) on interfaces to see if these properties are different from nonphosphorylated Ser/Thr/Tyr sites on interfaces. Phosphosites in heterooligomers seem to be more solvent accessible than nonphosphorylation sites in isolated protomers (on average by 23 Å2; p value = 2.2e-16) and tend to change solvent accessibility upon complex formation by burying more surface area (on average by 13Å2; p value = 2.2e-16, Table 1; Figure S1). This is consistent with our previous observation that phosphosites are predominantly located on binding interfaces. At interfaces, phosphorylation sites contribute to the complex stability by forming more hydrogen bonds and residue contacts than nonphosphosites (for hydrogen bond difference, p value = 0.0005 for heterooligomers and p value = 0.04 for weak homooligomers; Table 1). Additionally, Tyr residues tend to be located in the core of protein interfaces, playing a critical role for oligomerization through aromatic stacking interactions, its phosphorylation therefore might directly affect the binding affinity. The estimate of binding energy provides additional evidence for these findings, as shown in the following section. Residues that are essential for the structural integrity of proteins or protein complexes are called binding hotspots (Bogan and Thorn, 1998Bogan A.A. Thorn K.S. Anatomy of hot spots in protein interfaces.J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1557) Google Scholar, Tuncbag et al., 2009Tuncbag N. Gursoy A. Keskin O. Identification of computational hot spots in protein interfaces: combining solvent accessibility and inter-residue potentials improves the accuracy.Bioinformatics. 2009; 25: 1513-1520Crossref PubMed Scopus (195) Google Scholar). They are predominantly located on interaction interfaces, and their substitution by different amino acids (for example, Ala) causes large differences in binding energy (more than 1-2 kcal/mol), destabilizing the complex. The effect of such substitutions and therefore the contribution of a given site to the binding energy can be measured in terms of ΔΔΔGala (see Experimental Procedures). We performed substitutions of Tyr/Thr/Ser residues in phosphoproteins from our test set by Ala (computational alanine scanning experiments) and calculated ΔΔΔGala separately for phosphorylation and nonphosphorylation sites using the FoldX algorithm (see Experimental Procedures). Overall, the substitution of amino acids at both phosphorylation and nonphosphorylation sites destabilizes the complex, and the ΔΔΔGala distributions are significantly shifted to positive values for all homo- and heterooligomeric complexes (p values = 2.2e-16 for both). We did not detect any Ala substitutions that would result in increased stability of the native complex by more than 2 kcal/mol (negative values of ΔΔΔGala correspond to stabilizing substitutions). This implies that the interfaces are relatively well optimized, which is congruent with the previous studies (Brock et al., 2007Brock K. Talley K. Coley K. Kundrotas P. Alexov E. Optimization of electrostatic interactions in protein-protein complexes.Biophys. J. 2007; 93: 3340-3352Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Even though the majority of substitutions on interfaces do not change the binding energy very much, a significant fraction of them (10% for homooligomers and 13% for heterooligomers) contribute to a ΔΔΔGala of more than +2 kcal/mol (destabilizing the complex); in other words, they form binding hotspots. We considered whether phosphorylation events tend to involve binding hotspots. We found that for heterooligomers, the ΔΔΔGala values for amino acid substitutions at phosphorylation sites on binding interfaces are larger compared to other sites on interfaces (Wilcoxon rank-sum test, p value = 0.0003); namely, 7% of nonphosphorylation sites and 13% of phosphorylation sites correspondingly contribute more than 2 kcal/mol to ΔΔΔGala (20% of nonphosphorylation sites and 30% of phosphorylation sites have a ΔΔΔGala of more than 1 kcal/mol). In general, the association between phosphosites and binding hotspots is statistically significant for the entire dataset, and for heterooligomers in particular (Fisher's exact test, p value = 0.0006).This result does not hold true if only homooligomers are considered (Table 1; Figure S2). As mentioned previously, the majority of protein complexes in PDB do not have actual phosphate groups present. Therefore, to further assess the energetic effect of phosphorylation, we attached the phosphate group to those Ser/Thr/Tyr sites on binding interfaces that are known to be phosphorylated and calculated the change of binding energy upon phosphorylation as ΔΔΔGp (see Experimental Procedures). In the majority of cases, phosphorylation resulted in very moderate changes in the estimated binding energy of about +0.5-1.5 kcal/mol. Experimental studies on MAPK cascade scaffold protein showed that introducing phosphate increases the dissociation energy by about 1.5 kcal/mol (Serber and Ferrell, 2007Serber Z. Ferrell Jr., J.E. Tuning bulk electrostatics to regulate protein function.Cell. 2007; 128: 441-444Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, Strickfaden et al., 2007Strickfaden S.C. Winters M.J. Ben-Ari G. Lamson R.E. Tyers M. Pryciak P.M. A mechanism for cell-cycle regulation of MAP kinase signaling in a yeast differentiation pathway.Cell. 2007; 128: 519-531Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Nevertheless, overall, the ΔΔΔGp distribution was significantly shifted toward positive values (Figure 2; p value is 1.3e-08 for homooligomers and 1.3e-12 for heterooligomers). Namely, in 39% and 35% of the cases, the attachment of a phosphate group destabilized the complex for hetero- and homooligomers, respectively, by more than +2 kcal/mol. The phosphorylation of heterooligomers caused slightly higher destabilization compared to homooligomers. On the other hand, there were 8 and 64 cases where phosphorylation resulted in ΔΔΔGp values of less than −2 and -1 kcal/mol, respectively, leading to complex stabilization. There were 12 complexes in our test set where the actual phosphate group was resolved on protein interfaces; in these cases, we removed the phosphate group and assessed the effect, and in most cases the ΔΔΔGp was less than 2 kcal/mol. The evolutionary conservation of phosphorylation sites has been a topic of several studies; it has been found that phosphoproteins are more conserved in evolution than nonphosphorylated ones (Boekhorst et al., 2008Boekhorst J. van Breukelen B. Heck Jr., A. Snel B. Comparative phosphoproteomics reveals evolutionary and functional conservation of phosphorylation across eukaryotes.Genome Biol. 2008; 9: R144Crossref PubMed Scopus (61) Google Scholar, Macek et al., 2008Macek B. Gnad F. Soufi B. Kumar C. Olsen J.V. Mijakovic I. Mann M. Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation.Mol. Cell. Proteomics. 2008; 7: 299-307Crossref PubMed Scopus (325) Google Scholar), whereas the conservation of phosphorylation sites is limited (Levy et al., 2010Levy E.D. Landry C.R. Michnick S.W. Cell signaling. Signaling through cooperation.Science. 2010; 328: 983-984Crossref PubMed Scopus (42) Google Scholar). One of the reasons for weak conservation of phosphorylation sites is that the majority of phosphorylation events might have occurred relatively recently in evolution, especially Tyr phosphorylation (Chen et al., 2010Chen S.C. Chen F.C. Li W.H. Phosphorylated and nonphosphorylated serine and threonine residues evolve at different rates in mammals.Mol. Biol. Evol. 2010; 27: 2548-2554Crossref PubMed Scopus (29) Google Scholar, Gnad et al., 2010Gnad F. Forner F. Zielinska D.F. Birney E. Gunawardena J. Mann M. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria.Mol. Cell. Proteomics. 2010; 9: 2642-2653Crossref PubMed Scopus (69) Google Scholar, Sridhara et al., 2011Sridhara V. Marchler-Bauer A. Bryant S.H. Geer L.Y. Automatic annotation of experimentally derived, evolutionarily conserved post-translational modifications onto multiple genomes.Database (Oxford). 2011; 2011: bar019Crossref PubMed Scopus (3) Google Scholar). In an attempt to clarify this controversy, we mapped phosphorylation sites on multiple sequence alignments of manually curated Conserved Domain Database (CDD) families at the superfamily level and calculated their sequence conservation. Overall, 539 protein complexes from our dataset were mapped to 292 CDD families. First, we found in consensus with other studies (Boekhorst et al., 2008Boekhorst J. van Breukelen B. Heck Jr., A. Snel B. Comparative phosphoproteomics reveals evolutionary and functional conservation of phosphorylation across eukaryotes.Genome Biol. 2008; 9: R144Crossref PubMed Scopus (61) Google Scholar, Gnad et al., 2007Gnad F. Ren S. Cox J. Olsen J.V. Macek B. Oroshi M. Mann M. PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites.Genome Biol. 2007; 8: R250Crossref PubMed Scopus (381) Google Scholar, Gray and Kumar, 2011Gray V.E. Kumar S. Rampant purifying selection conserves positions with posttranslational modifications in human proteins.Mol. Biol. Evol. 2011; 28: 1565-1568Crossref PubMed Scopus (24) Google Scholar, Zanzoni et al., 2011Zanzoni A. Carbajo D. Diella F. Gherardini P.F. Tramontano A. Helmer-Citterich M. Via A. Phospho3D 2.0: an enhanced database of three-dimensional structures of phosphorylation sites.Nucleic Acids Res. 2011; 39: D268-D271Crossref PubMed Scopus (33) Google Scholar) that phosphorylation sites are more conserved than the surface sites for heterooligomers (Wilcoxon rank-sum test, p value = 0.00001; Figure S3). Next, we went further and checked whether phosphorylation sites on interfaces are more conserved than other interface sites. Figure 3 shows the probability density plot of sequence conservation calculated with respect to background conservation of the overall family for both phosphosites and all other Tyr/Thr/Ser sites on interfaces. This figure shows that heterooligomers, unlike homooligomers, have a small peak in the positive range of interface conservation values, which is consistent with previous studies (Choi et al., 2009Choi Y.S. Yang J.S. Choi Y. Ryu S.H. Kim S. Evolutionary conservation in multiple faces of protein interaction.Proteins. 2009; 77: 14-25Crossref PubMed Scopus (48) Google Scholar). Moreover, the majority of nonphosphorylation sites on interfaces are less conserved than the family background (the mean value of the distribution is shifted toward negative values), which can be explained by the fact that protein core residues and active sites might be under stronger evolutionary pressure than Ser, Thr, and Tyr residues on interfaces. When we look at the conservation of phosphorylaion sites, it is evident that there are two almost equal populations of Ser/The/Tyr sites: those that are less conserved than the family background and those that are more conserved than the background. Overall, the conservation distribution for phosphosites is significantly shifted toward positive values compared to conservation of interfacial nonphosphosites for all complexes, and for heterooligomers in particular (p value = 0.018 for all; p value = 0.016 for heterooligomers). When calculated separately for homooligomers, this shift is not significant. Thus, we see that phosphosites are more conserved than nonphosphosites on interfaces in human complexes, implying that there is additional evolutionary pressure to conserve the phosphosites, which are important for binding events. This is also consistent with our previous observation that phosphosites in heterooligomers have a tendency to be located at the binding hot spots, and such hot spots are more evolutionarily conserved than the rest of the interface. It has been reported that phosphorylated proteins have specific molecular functions in a cell (Wang et al., 2011Wang Z. Ding G. Geistlinger L. Li H. Liu L. Zeng R. Tateno Y. Li Y. Evolution of protein phosphorylation for distinct functional modules in vertebrate genomes.Mol. Biol. Evol. 2011; 28: 1131-1140Crossref PubMed Scopus (17) Google Scholar). We analyzed our n" @default.
• W2010516785 created "2016-06-24" @default.
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• W2010516785 date "2011-12-01" @default.
• W2010516785 modified "2023-10-16" @default.
• W2010516785 title "Phosphorylation in Protein-Protein Binding: Effect on Stability and Function" @default.
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