Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands

Kazlauskas, Arūnas; Schmotz, Constanze; Kesti, Tapio; Hepojoki, Jussi; Kleino, Iivari; Kaneko, Tomonori; Li, Shawn Shun‐Cheng; Saksela, Kalle

doi:10.1074/mcp.m116.060483

Cited by 12 publications

(9 citation statements)

References 51 publications

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“…Moving forward, we will continue to adapt MotifAnalyzer to look for motif‐satisfying sequences of various SLiM‐binding domains, expand our evolution‐ and enrichment‐based analyses, and create a web‐based interface. Previous studies on other SLiM‐binding domains, for example SH2 and SH3 domains, suggests that these interactions are also more specific than previously realized . MotifAnalyzer is a tool that can be used in silico to predict endogenous targets of these domains and more, for example, tyrosine kinases, using a similar pipeline (Figure ).…”

Section: Discussionmentioning

confidence: 76%

MotifAnalyzer‐PDZ: A computational program to investigate the evolution of PDZ‐binding target specificity

et al. 2019

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Recognition of short linear motifs (SLiMs) or peptides by proteins is an important component of many cellular processes. However, due to limited and degenerate binding motifs, prediction of cellular targets is challenging. In addition, many of these interactions are transient and of relatively low affinity. Here, we focus on one of the largest families of SLiM-binding domains in the human proteome, the PDZ domain. These domains bind the extreme C-terminus of target proteins, and are involved in many signaling and trafficking pathways. To predict endogenous targets of PDZ domains, we developed MotifAnalyzer-PDZ, a program that filters and compares all motif-satisfying sequences in any publicly available proteome. This approach enables us to determine possible PDZ binding targets in humans and other organisms. Using this program, we predicted and biochemically tested novel human PDZ targets by looking for strong sequence conservation in evolution. We also identified three C-terminal sequences in choanoflagellates that bind a choanoflagellate PDZ domain, the Monsiga brevicollis SHANK1 PDZ domain (mbSHANK1), with endogenously-relevant affinities, despite a lack of conservation with the targets of a homologous human PDZ domain, SHANK1. All three are predicted to be signaling proteins, with strong sequence homology to cytosolic and receptor tyrosine kinases. Finally, we analyzed and compared the positional amino acid enrichments in PDZ motifsatisfying sequences from over a dozen organisms. Overall, MotifAnalyzer-PDZ is a versatile program to investigate potential PDZ interactions. This proof-ofconcept work is poised to enable similar types of analyses for other SLiM-binding domains (e.g., MotifAnalyzer-Kinase). MotifAnalyzer-PDZ is available at

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Section: Discussionmentioning

confidence: 76%

MotifAnalyzer‐PDZ: A computational program to investigate the evolution of PDZ‐binding target specificity

et al. 2019

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“…22,23 There are also examples in scaffolding domains, including SH2 and SH3 domains, where conserved loops interact directly with the peptide and determine the selectivity of both SH2 (the EF and BG loops) and SH3 (the RT and n-Src loops) domains. [29][30][31][32][33][34][35][36][37] Work from ourselves and others strongly indicates that Class A sortases are another protein family that exhibits functionally relevant sequence variation in specificity-determining loops. 12,38,39 In our previous work, we investigated the selectivity determinants of Streptococcus pneumoniae SrtA (spSrtA) at the P1 0 position of the CWSS.…”

Section: Introductionmentioning

confidence: 99%

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

et al. 2022

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Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative β7-β8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1 0 position of the substrate motif, underlined in LPXTG, in contrast to the strict P1 0 Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of β7-β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7-β8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

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“…The largest SH3-ligand interface known is between the p67 phox SH3 domain and a 32-residue peptide from p47 phox (Kami et al, 2002), which contains a PxxP motif followed by a helix-turn-helix structure that binds to the specificity site and is the tightest known natural linear ligand for SH3 domains. Several other SH3 domains significantly increase affinity and specificity by binding simultaneously to proline-rich peptides and to folded domains within their binding partners (Lee et al, 1996), and a recent study showed that 19 SH3 domains bind much tighter to full-length proteins than to isolated peptides alone (Kazlauskas et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide Variety of Non-canonical Specificities

et al. 2017

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SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways. The majority of SH3 domains studied thus far act by binding to proline-rich sequences in partner proteins, but a growing number of studies have revealed alternative recognition mechanisms. We have comprehensively surveyed the specificity landscape of human SH3 domains in an unbiased manner using peptide-phage display and deep sequencing. Based on ∼70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses.

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Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands

Cited by 12 publications

References 51 publications

MotifAnalyzer‐PDZ: A computational program to investigate the evolution of PDZ‐binding target specificity

MotifAnalyzer‐PDZ: A computational program to investigate the evolution of PDZ‐binding target specificity

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide Variety of Non-canonical Specificities

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