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• W2061205908 abstract "The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains. The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains. Protein-protein interactions are often mediated by conserved modular domains that bind to short linear peptide motifs. Although the members of a single domain family generally recognize the same consensus motif in their targets, individual domains possess unique binding specificities and recognize different variations of this motif. In many cases, this intrinsic specificity of individual domains plays a crucial role in directing them to their relevant biological targets and preventing aberrant cross-reactivity with nonphysiological targets (1.Stollar E.J. Garcia B. Chong P.A. Rath A. Lin H. Forman-Kay J.D. Davidson A.R. Structural, functional, and bioinformatic studies demonstrate the crucial role of an extended peptide binding site for the SH3 domain of yeast Abp1p.J. Biol. Chem. 2009; 284: 26918-26927Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 2.Zarrinpar A. Park S.H. Lim W.A. Optimization of specificity in a cellular protein interaction network by negative selection.Nature. 2003; 426: 676-680Crossref PubMed Scopus (210) Google Scholar). Thus, to comprehend fully the contribution of protein interaction domains to the functioning of cellular pathways, insight into the principles that govern their intrinsic binding specificity is required. This knowledge will ultimately allow the accurate prediction of binding specificities, which will greatly increase our ability to map and understand the signal transduction pathways within cells. In this study, we explore the factors controlling intrinsic binding specificity by investigating the specificity determinants of two yeast SH3 2The abbreviations used are:SH3Src homology 3CICdc42-interactingPDBProtein Data Baser.m.s.d.root mean square deviation. domains that can bind to the same target peptides. Src homology 3 Cdc42-interacting Protein Data Base root mean square deviation. SH3 domains are among the most common protein interaction domains and are present in proteins with diverse cellular roles including signal transduction, cytoskeleton regulation, and membrane trafficking (3.Bhattacharyya R.P. Reményi A. Yeh B.J. Lim W.A. Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits.Annu. Rev. Biochem. 2006; 75: 655-680Crossref PubMed Scopus (369) Google Scholar). These domains are ∼60 residues long and are composed of five β-strands, which are sequentially joined by the RT, N-Src, and distal loops, and a short 310-helix. SH3 domains generally recognize peptide sequences containing either +XXPXXP (class I) or PXXPXX+ (class II) “core” motifs (where X can be variety of residues and + represents either a Lys or Arg residue). The prolines of the core motif are accommodated in conserved hydrophobic grooves on one surface of the SH3 domain, which has been referred to as Surface I (1.Stollar E.J. Garcia B. Chong P.A. Rath A. Lin H. Forman-Kay J.D. Davidson A.R. Structural, functional, and bioinformatic studies demonstrate the crucial role of an extended peptide binding site for the SH3 domain of yeast Abp1p.J. Biol. Chem. 2009; 284: 26918-26927Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 4.Kim J. Lee C.D. Rath A. Davidson A.R. Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities.J. Mol. Biol. 2008; 377: 889-901Crossref PubMed Scopus (25) Google Scholar). The positively charged residue is packed against a highly conserved tryptophan and also interacts with negatively charged residues in the RT loop. Although interactions with the core motif serve as a common anchor for binding by most SH3 domains, the specificity of individual SH3 domains is strongly influenced by interactions between target peptide residues flanking the core motif and a variable surface in the SH3 domain located between RT and N-Src loops (1.Stollar E.J. Garcia B. Chong P.A. Rath A. Lin H. Forman-Kay J.D. Davidson A.R. Structural, functional, and bioinformatic studies demonstrate the crucial role of an extended peptide binding site for the SH3 domain of yeast Abp1p.J. Biol. Chem. 2009; 284: 26918-26927Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 5.Bauer F. Schweimer K. Meiselbach H. Hoffmann S. Rösch P. Sticht H. Structural characterization of Lyn-SH3 domain in complex with a herpesviral protein reveals an extended recognition motif that enhances binding affinity.Protein Sci. 2005; 14: 2487-2498Crossref PubMed Scopus (40) Google Scholar, 6.Ghose R. Shekhtman A. Goger M.J. Ji H. Cowburn D. A novel, specific interaction involving the Csk SH3 domain and its natural ligand.Nat. Struct. Biol. 2001; 8: 998-1004Crossref PubMed Scopus (123) Google Scholar, 7.Kami K. Takeya R. Sumimoto H. Kohda D. Diverse recognition of non-PXXP peptide ligands by the SH3 domains from p67(phox), Grb2 and Pex13p.EMBO J. 2002; 21: 4268-4276Crossref PubMed Scopus (147) Google Scholar, 8.Lewitzky M. Harkiolaki M. Domart M.C. Jones E.Y. Feller S.M. Mona/Gads SH3C binding to hematopoietic progenitor kinase 1 (HPK1) combines an atypical SH3 binding motif, R/KXXK, with a classical PXXP motif embedded in a polyproline type II (PPII) helix.J. Biol. Chem. 2004; 279: 28724-28732Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), which has been referred to as Surface II (1.Stollar E.J. Garcia B. Chong P.A. Rath A. Lin H. Forman-Kay J.D. Davidson A.R. Structural, functional, and bioinformatic studies demonstrate the crucial role of an extended peptide binding site for the SH3 domain of yeast Abp1p.J. Biol. Chem. 2009; 284: 26918-26927Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 4.Kim J. Lee C.D. Rath A. Davidson A.R. Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities.J. Mol. Biol. 2008; 377: 889-901Crossref PubMed Scopus (25) Google Scholar). The interaction of this “extended” region of target peptides with Surface II is complicated because this surface is relatively flat and displays little conservation across the SH3 domain family. In addition, the conformation of peptides interacting with this surface is variable (4.Kim J. Lee C.D. Rath A. Davidson A.R. Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities.J. Mol. Biol. 2008; 377: 889-901Crossref PubMed Scopus (25) Google Scholar), and different peptides binding the same domain may adopt distinct conformations when binding this surface (9.Hoelz A. Janz J.M. Lawrie S.D. Corwin B. Lee A. Sakmar T.P. Crystal structure of the SH3 domain of βPIX in complex with a high affinity peptide from PAK2.J. Mol. Biol. 2006; 358: 509-522Crossref PubMed Scopus (40) Google Scholar, 10.Janz J.M. Sakmar T.P. Min K.C. A novel interaction between atrophin-interacting protein 4 and β-p21-activated kinase-interactive exchange factor is mediated by an SH3 domain.J. Biol. Chem. 2007; 282: 28893-28903Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Due to the complex nature of Surface II and the relative dearth of studies on the interaction of SH3 domains with extended peptide sequences, our understanding of these interactions is limited. Thus, accurate prediction of the specificities of SH3 domains is also hampered. We have investigated the specificity of the yeast Nbp2p SH3 (NbpSH3) and Bem1p SH3b (BemSH3b) domains. This pair of SH3 domains is unusual because they recognize the same consensus binding motif (see Fig. 3A), even though they are present in nonhomologous proteins and have a relatively low sequence identity of 36% (the sequence identity of any two randomly chosen SH3 domains is ∼30%) (11.Larson S.M. Davidson A.R. The identification of conserved interactions within the SH3 domain by alignment of sequences and structures.Protein Sci. 2000; 9: 2170-2180Crossref PubMed Scopus (135) Google Scholar). Nbp2p is an adaptor protein that recruits Ptc1p phosphatase through interactions mediated by its SH3 domain. Binding of the NbpSH3 domain to PXXP-containing sites in the Pbs2p and Bck1p kinases is required for down-regulation of the high osmolarity glycerol and cell wall integrity MAP kinase pathways, respectively (12.Hruby A. Zapatka M. Heucke S. Rieger L. Wu Y. Nussbaumer U. Timmermann S. Dünkler A. Johnsson N. A constraint network of interactions: protein-protein interaction analysis of the yeast type II phosphatase Ptc1p and its adaptor protein Nbp2p.J. Cell Sci. 2011; 124: 35-46Crossref PubMed Scopus (24) Google Scholar, 13.Mapes J. Ota I.M. Nbp2 targets the Ptc1-type 2C Ser/Thr phosphatase to the HOG MAPK pathway.EMBO J. 2004; 23: 302-311Crossref PubMed Scopus (62) Google Scholar, 14.Du Y. Walker L. Novick P. Ferro-Novick S. Ptc1p regulates cortical ER inheritance via Slt2p.EMBO J. 2006; 25: 4413-4422Crossref PubMed Scopus (59) Google Scholar). Bem1p acts as an adaptor for multiple proteins involved in establishing cell polarity including Cdc42p GTPase and the Cdc42p-activated kinases, Ste20p and Cla4p (15.Matsui Y. Matsui R. Akada R. Toh-e A. Yeast Src homology region 3 domain-binding proteins involved in bud formation.J. Cell Biol. 1996; 133: 865-878Crossref PubMed Scopus (64) Google Scholar, 16.Butty A.C. Perrinjaquet N. Petit A. Jaquenoud M. Segall J.E. Hofmann K. Zwahlen C. Peter M. A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization.EMBO J. 2002; 21: 1565-1576Crossref PubMed Scopus (169) Google Scholar, 17.Bose I. Irazoqui J.E. Moskow J.J. Bardes E.S. Zyla T.R. Lew D.J. Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p.J. Biol. Chem. 2001; 276: 7176-7186Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 18.France Y.E. Boyd C. Coleman J. Novick P.J. The polarity-establishment component Bem1p interacts with the exocyst complex through the Sec15p subunit.J. Cell Sci. 2006; 119: 876-888Crossref PubMed Scopus (33) Google Scholar, 19.Yamaguchi Y. Ota K. Ito T. A novel Cdc42-interacting domain of the yeast polarity establishment protein Bem1: implications for modulation of mating pheromone signaling.J. Biol. Chem. 2007; 282: 29-38Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The BemSH3b domain binds to the PXXP motifs within Ste20p and Cla4p kinases and also binds Cdc42p itself in a PXXP-independent manner (16.Butty A.C. Perrinjaquet N. Petit A. Jaquenoud M. Segall J.E. Hofmann K. Zwahlen C. Peter M. A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization.EMBO J. 2002; 21: 1565-1576Crossref PubMed Scopus (169) Google Scholar, 17.Bose I. Irazoqui J.E. Moskow J.J. Bardes E.S. Zyla T.R. Lew D.J. Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p.J. Biol. Chem. 2001; 276: 7176-7186Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 19.Yamaguchi Y. Ota K. Ito T. A novel Cdc42-interacting domain of the yeast polarity establishment protein Bem1: implications for modulation of mating pheromone signaling.J. Biol. Chem. 2007; 282: 29-38Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 20.Winters M.J. Pryciak P.M. Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20.Mol. Cell. Biol. 2005; 25: 2177-2190Crossref PubMed Scopus (33) Google Scholar). The interaction between the BemSH3b domain and Cdc42p involves a unique C-terminal extension (Cdc42-interacting (CI) subdomain) in the BemSH3b domain, which is also required for the folding of the whole domain (19.Yamaguchi Y. Ota K. Ito T. A novel Cdc42-interacting domain of the yeast polarity establishment protein Bem1: implications for modulation of mating pheromone signaling.J. Biol. Chem. 2007; 282: 29-38Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21.Takaku T. Ogura K. Kumeta H. Yoshida N. Inagaki F. Solution structure of a novel Cdc42 binding module of Bem1 and its interaction with Ste20 and Cdc42.J. Biol. Chem. 2010; 285: 19346-19353Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The structure of the BemSH3b domain solved in complex with a peptide from Ste20p showed that the CI subdomain is intimately packed against the SH3 domain and is involved in the interaction with the peptide (21.Takaku T. Ogura K. Kumeta H. Yoshida N. Inagaki F. Solution structure of a novel Cdc42 binding module of Bem1 and its interaction with Ste20 and Cdc42.J. Biol. Chem. 2010; 285: 19346-19353Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The similarity in binding specificities of the NbpSH3 and BemSH3b domains was first detected when they were found to both bind the same PXXP-containing motif in Ste20p (20.Winters M.J. Pryciak P.M. Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20.Mol. Cell. Biol. 2005; 25: 2177-2190Crossref PubMed Scopus (33) Google Scholar). To analyze this cross-reactivity further, we previously tested binding of the NbpSH3 and BemSH3b domains to peptides from six different yeast proteins that are either proven or likely biologically relevant target proteins for these domains (Ste20p, Cla4p, Pbs2p, Bck1p, Skm1p, and Boi2p). These peptides share a class I consensus sequence, ΨXPXRXAPXXP (where Ψ is a hydrophobic residue), which was derived for the NbpSH3 domain using phage display (22.Tong A.H. Drees B. Nardelli G. Bader G.D. Brannetti B. Castagnoli L. Evangelista M. Ferracuti S. Nelson B. Paoluzi S. Quondam M. Zucconi A. Hogue C.W. Fields S. Boone C. Cesareni G. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules.Science. 2002; 295: 321-324Crossref PubMed Scopus (582) Google Scholar). We found that although the NbpSH3 and BemSH3b domains bound to all of these sites, some sites were bound with equally strong affinities, whereas other sites were bound with remarkably different affinities by the two domains (23.Gorelik M. Stanger K. Davidson A.R. A conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function.J. Biol. Chem. 2011; 286: 19470-19477Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). For example, both domains bound to the Ste20 peptide with submicromolar affinities (see Fig. 3A), yet the NbpSH3 domain bound the Bck1 peptide with a Kd of 0.8 μm, whereas the Kd of the BemSH3b domain for this peptide was only 26 μm (see Fig. 3B). These data demonstrated that even though the NbpSH3 and BemSH3b domains share a consensus binding sequence and interact with equally high affinity with some peptides, each domain possesses its own unique binding specificity. We also identified a residue within the BemSH3b domain (Lys14), which serves to prevent high affinity binding to certain sites (e.g. Bck1) and plays a general role in maintaining the BemSH3b domain specificity required for its optimal function (23.Gorelik M. Stanger K. Davidson A.R. A conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function.J. Biol. Chem. 2011; 286: 19470-19477Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The ability of the NbpSH3 and BemSH3b domains to bind the same extended peptides with high affinity while strongly discriminating between other peptides provides a unique model system to examine the determinants of SH3 domain specificity. In this study, we address two main questions regarding the binding behavior of these domains. First, how do these two domains with very different sequences still recognize the same consensus motif and bind some peptides with almost identical affinities? Second, how do these domains also exhibit distinctive specificities toward other peptides? To answer these questions, we have solved the structure of the NbpSH3 domain in complex with the Ste20 peptide and compared it with the previously determined structure of the BemSH3b-Ste20 complex (21.Takaku T. Ogura K. Kumeta H. Yoshida N. Inagaki F. Solution structure of a novel Cdc42 binding module of Bem1 and its interaction with Ste20 and Cdc42.J. Biol. Chem. 2010; 285: 19346-19353Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Using a combination of structural analysis and in vitro assays involving mutant domains and peptides, we have dissected the binding mechanisms of these two SH3 domains. Based on our results, we describe a new general mechanism that explains the unique binding specificities of the NbpSH3 and BemSH3b domains. Our analysis also allowed us to identify previously unrecognized sites in yeast proteins that are highly selective for the BemSH3b domain. Yeast NbpSH3 (Nbp2p residues 110–172) and BemSH3b (Bem1p residues 155–252) domains were expressed from pET21d (Novagen) vector with a C-terminal His6 tag. Ste20(468–483), Bck1(800–815), Cla4(15–25), Cla4(451–461), and Boi1(391–401) peptidesSample B contained 0.5 used in the binding assays were expressed as C-terminal fusions to bacteriophage λ cI repressor carrying a C-terminal His6 tag, as described previously (24.Maxwell K.L. Davidson A.R. Mutagenesis of a buried polar interaction in an SH3 domain: sequence conservation provides the best prediction of stability effects.Biochemistry. 1998; 37: 16172-16182Crossref PubMed Scopus (85) Google Scholar). All proteins were purified using Ni-affinity chromatography and dialyzed against 50 mm sodium phosphate, pH 6.8, 100 mm NaCl buffer. A peptide corresponding to Ste20p residues 468–484 (GKFIPSRAPKPPSSA) was chemically synthesized (CanPeptide). For NMR experiments, two samples were prepared. Sample A contained 0.7 mm 15N13C-labeled NbpSH3 domain and 1.5 mm unlabeled Ste20p peptide in 50 mm phosphate, pH 6.8, 100 mm NaCl, 0.05% NaN3. Sample B contained 0.5 mm 15N13C-labeled NbpSH3 and 0.5 mm unlabeled Ste20p peptide in 50 mm phosphate buffer, pH 6.8, 100 mm NaCl, and 0.05% NaN3. Backbone and side chain chemical shifts for the NbpSH3 domain were assigned using standard triple-resonance experiments (25.Kay L.E. Pulsed field gradient multidimensional NMR methods for the study of protein structure and dynamics in solution.Prog. Biophys. Mol. Biol. 1995; 63: 277-299Crossref PubMed Scopus (137) Google Scholar) (HNCO, HN(CO)CA, CBCA(CO)NH, HNCACB, C(CO)NH, HCCH-TOCSY, and HCCH-COSY) with sample A. Interproton distances for the NbpSH3 domain were obtained with 15N-edited and 13C-edited NOESY spectra (sample A). Chemical shift assignments and structural restraints for the Ste20p peptide were derived from double-half-filtered two-dimensional 1H-1H TOCSY, COSY, and NOESY experiments (26.Gemmecker G.O.E.T. Fesik S.W. An improved method for selectively observing protons attached to 12C in the presence of 1H 13C spin pairs.J. Magn. Reson. 1992; 96: 199-204Google Scholar, 27.Ikura M. Bax A. Isotope-filtered 2D NMR of a protein-peptide complex: study of a skeletal muscle myosin light chain kinase fragment bound to calmodulin.J. Am. Chem. Soc. 1992; 114: 2433-2440Crossref Scopus (284) Google Scholar) (sample B). Intermolecular NOEs between the NbpSH3 domain and Ste20p peptide were obtained from three-dimensional NOESY experiments (sample A) and 13C-edited 13C-filtered three-dimensional NOESY (28.Zwahlen C. Legault P. Vincent S.J.F. Greenblatt J. Konrat R. Kay L.E. Methods for measurement of intermolecular NOEs by multi nuclear NMR spectroscopy: Application to bacteriophage lambda N-peptide/Box B RNA complex.J. Am. Chem. Soc. 1997; 119: 6711-6721Crossref Scopus (537) Google Scholar) (sample B). All NMR experiments were carried out at 25 °C on a Varian INOVA 500 or 800 MHz spectrometers equipped with pulsed field gradients at the Quebec/Eastern Canada High Field NMR Facility. All data were processed using NMRPipe (29.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes.J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11548) Google Scholar) and subsequent analysis performed using SPARKY (30.Goddard, T. D., Kneller, D. G., SPARKY 3, University of California, San Francisco,Google Scholar). Structure calculations were performed using CYANA 2.1 (31.Güntert P. Automated NMR structure calculation with CYANA.Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1169) Google Scholar). The input constraints included dihedral angle restraints derived using TALOS (32.Cornilescu G. Delaglio F. Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology.J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar) and hydrogen bond upper and lower distance limits derived from examination of short range NOE data. Manually assigned NOE peaks were fixed in the first cycle of structure calculation and peak assignments. In the remaining cycles, all NOE peak assignments were made by CYANA. The 20 lowest energy structures, of 100 calculated in the final iteration, were used to derive the structure. Structural coordinates and NMR restraints have been deposited in the Protein Data Bank (PDB) with code 2LCS. SH3 domains at a concentration of 1 μm were titrated with peptide fusion proteins. Binding was monitored by measuring intrinsic tryptophan fluorescence of the SH3 domain (λexcitation = 295 nm, λemission = 326 nm), which increased significantly upon binding of the peptide. The experiments were carried out in 50 mm phosphate, pH 6.8, 100 mm NaCl at 25 °C. All titrations and fluorescence measurements were carried out on an Aviv ATF105 spectrofluorometer equipped with a Microlab 500 series automated titrator. Dissociation constants were calculated as described previously (24.Maxwell K.L. Davidson A.R. Mutagenesis of a buried polar interaction in an SH3 domain: sequence conservation provides the best prediction of stability effects.Biochemistry. 1998; 37: 16172-16182Crossref PubMed Scopus (85) Google Scholar). The bem1Δ and bem1 sh3b F54A strains constructed in the previous study (23.Gorelik M. Stanger K. Davidson A.R. A conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function.J. Biol. Chem. 2011; 286: 19470-19477Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) were used in the growth assays. The bem1 sh3b E17Q stain was constructed as described previously (23.Gorelik M. Stanger K. Davidson A.R. A conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function.J. Biol. Chem. 2011; 286: 19470-19477Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) from the same genetic background as the bem1Δ and bem1 sh3b F54A strains. Yeast peptone dextrose medium was used for growth assays. The cells were grown overnight, diluted to an A600 of 1.0, and spotted in 5-fold serial dilutions on yeast peptone dextrose plates. The cell growth was detected after 2 days at 30, 40.5, and 41.5 °C. To gain insight into the specificity determinants of the NbpSH3 domain and allow comparison to the BemSH3b domain, we determined the NMR solution structure of the NbpSH3 domain in complex with the Ste20 peptide. We chose the Ste20 peptide because both domains bind it with high affinity, and the structure of a complex of the BemSH3b domain with this peptide was determined previously (21.Takaku T. Ogura K. Kumeta H. Yoshida N. Inagaki F. Solution structure of a novel Cdc42 binding module of Bem1 and its interaction with Ste20 and Cdc42.J. Biol. Chem. 2010; 285: 19346-19353Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Standard NMR experiments were used to assign chemical shifts and NOEs to the SH3 domain and the peptide (25.Kay L.E. Pulsed field gradient multidimensional NMR methods for the study of protein structure and dynamics in solution.Prog. Biophys. Mol. Biol. 1995; 63: 277-299Crossref PubMed Scopus (137) Google Scholar). The structure was restrained using intrapeptide, intra-SH3, and intermolecular NOEs, hydrogen bond restraints, and dihedral angle restraints derived from TALOS (32.Cornilescu G. Delaglio F. Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology.J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar) (Table 1). The 20 lowest energy structures overlay with a backbone r.m.s.d. of 0.22 and 0.15 Å for well defined regions of the SH3 domain and Ste20 peptide, respectively (Table 1 and Fig. 1, A and B). The NbpSH3 domain displays a typical SH3 domain fold and overlays closely (backbone r.m.s.d. of 0.93Å over residues 1–59) with a previously determined crystal structure of the free NbpSH3 domain (PDB code 1YN8, chain B, Fig. 1C). The major differences map to the RT loop and N-Src-loop regions, which are directly involved in peptide binding.TABLE 1Structural statistics for the NbpSH3-Ste20 peptide complexConstraintsStructureInput dihedral anglesaDihedral angles restraints were derived using TALOS (32). (ϕ, ψ)44,43r.m.s.d. from mean structurebThe r.m.s.d. is indicated for well defined regions. NbpSH3 domain, residues −1 to 61, numbering according to Larson and Davidson (11); Ste20p peptide, residues −7 to 3, numbering according to Lim et al. (37). (Å)NOE upper distanceBackbone atoms (SH3, peptide)0.22, 0.15Intraresidual345Heavy atoms (SH3, peptide)0.67,0.84Medium range (1≤|i−j|<4)643r.m.s.d. from ideal geometryLong range (|i−j|>4)1,030Distance (Å)0.004Intermolecular184Angles (°)0.8Hydrogen bondcBased on the predicted hydrogen bonds that were derived from the examination of secondary structure NOEs.Ramachandran plotLower32Favored regions82.3%Upper32Additionally allowed regions17.7%Generously allowed regions0.1%Disallowed regions0.0%a Dihedral angles restraints were derived using TALOS (32.Cornilescu G. Delaglio F. Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology.J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar).b The r.m.s.d. is indicated for well defined regions. NbpSH3 domain, residues −1 to 61, numbering according to Larson and Davidson (11.Larson S.M. Davidson A.R. The identification of conserved interactions within the SH3 domain by alignment of sequences and structures.Protein Sci. 2000; 9: 2170-2180Crossref PubMed Scopus (135) Google Scholar); Ste20p peptide, residues −7 to 3, numbering according to Lim et al. (37.Lim W.A. Richards F.M. Fox R.O. Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains.Nature. 1994; 372: 375-379Crossref PubMed Scopus (450) Google Scholar).c Based on the predicted hydrogen bonds that were derived from the examination of secondary structure NOEs. Open table in a new tab Comparison of the NbpSH3-Ste20 and BemSH3b-Ste20 complexes shows that the Ste20 peptide adopts a similar conformation in the two complexes and contacts the same surfaces in both domains (Fig. 2A). The two complexes overlay with a backbone r.m.s.d. of 1.6 Å over the well defined regions in both structures. These correspond to SH3 domain residues 2–40 and 48–58, and the peptide residues Phe−7 to Pro3. The rest of the peptide backbone (total length of the peptide used in this study was 16 amino acids) is poorly defined in both structures (Fig. 1A) (21.Takaku T. Ogura K. Kumeta H. Yoshida N. Inagaki F. Solution structure of a novel Cdc42 binding module of Bem1 and its interaction with Ste20 and Cdc42.J. Biol. Chem. 2010; 285: 19346-19353Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In each complex, the core RXXPXXP motif (positions −3 to +3) of the Ste20 peptide interac" @default.
• W2061205908 created "2016-06-24" @default.
• W2061205908 creator A5007431455 @default.
• W2061205908 creator A5052222931 @default.
• W2061205908 date "2012-03-01" @default.
• W2061205908 modified "2023-09-30" @default.
• W2061205908 title "Distinct Peptide Binding Specificities of Src Homology 3 (SH3) Protein Domains Can Be Determined by Modulation of Local Energetics across the Binding Interface" @default.
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