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| = The human SH3 domain specificity map reveals a wide variety of novel non-canonical peptide-binding specificities = Joan Teyra 1+, Haiming Huang 1,3+, Shobhit Jain 1,2, Taehyung Kim 1,2, Xinyu Guan 4, Aiping Dong 4, Yanli Liu 4,5, Jinrong Min 4,5, Yufeng Tong 4, Philip M. Kim 1,2,3, Gary D. Bader 1,2,3 and Sachdev S. Sidhu 1,3,* |
= Comprehensive analysis of the human SH3 domain family reveals a wide variety of non-canonical specificities = Joan Teyra^1(+)^, Haiming Huang^1,2(+)^, Shobhit Jain^1,3^, Xinyu Guan^4^, Aiping Dong^4^, Yanli Liu^4^, Wolfram Tempel^4^, Jinrong Min^4,5^, Yufeng Tong^4,6^, Philip M. Kim^1,2,3^, Gary D. Bader^1,2,3^ and Sachdev S. Sidhu^1,2,(*)^ |
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| SH3 domains are small protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways, including cell growth regulation, endocytosis and cytoskeleton control. Canonically, SH3 domains are thought to act via physical binding to poly-proline-containing linear regions in proteins. Although these binding preferences have been confirmed for the majority of SH3 domains tested thus far, a growing number of studies have revealed exceptions, suggesting alternative molecular mechanisms of protein recognition are possible. To identify them, we have comprehensively surveyed the specificity landscape of human SH3 domains, for the first time in an unbiased manner using peptide-phage display combined with deep sequencing. We obtained results for 117 domains of 320 attempted and these reveal that a large fraction of SH3 domains are non-canonical and collectively recognize a wide variety of peptide motifs, most of them previously unknown (47 of 51). Structural analysis of two SH3 domains with distinct non-canonical specificities confirms novel peptide-binding modes through an extended surface at the SH3 specificity site. In sum, our results constitute a significant contribution towards a complete understanding of the mechanisms underlying SH3-mediated cellular responses. | SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways, including cell growth regulation, endocytosis and cytoskeleton control. Canonical SH3 domains act by binding to proline-rich sequences in partner proteins. Although these binding preferences have been confirmed for the majority of SH3 domains studied thus far, 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 more than 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 towards a complete understanding of the mechanisms underlying SH3-mediated cellular responses. To enable future research, we have made publicly available the peptide-binding specificity profiles. |
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| * [[attachment:HumanSH3TableS1.xlsx|S1]] - Summary of Human SH3 domains analyzed in phage display screen * [[attachment:HumanSH3TableS2.xlsx|S2]] - Summary of phage clonal ELISA results * [[attachment:HumanSH3FigureS3.docx|S3]] - Summary of ITC data * [[attachment:HumanSH3TableS4.doc|S4]] - Data collection and refinement statistics (molecular replacement) * [[attachment:HumanSH3FigureS5.docx|S5]] - Comparison of SH3 domain specificity classes between our peptide phage-based work and chip-based work from Carducci et al |
* [[attachment:HumanSH3TableS1.xlsx|Table S1]] - Sequence alignment of all SH3 domains that worked in phage display experiments * [[attachment:HumanSH3TableS2.xlsx|Table S2]] - Results summary for all human SH3 domains * [[attachment:HumanSH3TableS3.docx|Table S3]] - List of all SH3-peptide complexes in the PDB cataloged by specificity classes * [[attachment:HumanSH3TableS4.doc|Table S4]] - Phage clonal ELISA results |
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| * [[attachment:PWM.zip|PWM files]] - Position weight matrices of SH3 domains * [[attachment:PWM.zip|PWM files]] - Logos of SH3 domains |
* [[attachment:SH3_PWM.zip|PWM files]] - Position weight matrices of SH3 domains * [[attachment:SH3_Logos.zip|Logo images]] - Logos of SH3 domains Note: PWM and Logo file names are formatted as: Gene_name-Domain_position-PWM_number (e.g. PWM ABL2-1_1-1 has ABL2 gene name, 1/1 domain position, 1 PWM number (out of 2)) |
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| 1 The Donnelly Centre <<BR>> 2 Department of Computer Science, University of Toronto, Toronto, ON, Canada M5S 3E1 <<BR>> 3 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A4 <<BR>> 4 Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7 <<BR>> 5 Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada <<BR>> * To whom correspondence should be addressed. Email: sachdev.sidhu@utoronto.ca <<BR>> + JT and HH contributed equally to the study <<BR>> |
^1^The Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1 <<BR>> ^2^Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8 <<BR>> ^3^Department of Computer Science, University of Toronto, Toronto, ON, Canada M5S 3G4 <<BR>> ^4^Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7 <<BR>> ^5^Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada <<BR>> ^6^Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON Canada, M5S 1A8 <<BR>> ^(*)^To whom correspondence should be addressed. Email: sachdev.sidhu@utoronto.ca <<BR>> ^(+)^JT and HH contributed equally to the study |
Comprehensive analysis of the human SH3 domain family reveals a wide variety of non-canonical specificities
Joan Teyra1(+), Haiming Huang1,2(+), Shobhit Jain1,3, Xinyu Guan4, Aiping Dong4, Yanli Liu4, Wolfram Tempel4, Jinrong Min4,5, Yufeng Tong4,6, Philip M. Kim1,2,3, Gary D. Bader1,2,3 and Sachdev S. Sidhu1,2,(*)
Abstract
SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways, including cell growth regulation, endocytosis and cytoskeleton control. Canonical SH3 domains act by binding to proline-rich sequences in partner proteins. Although these binding preferences have been confirmed for the majority of SH3 domains studied thus far, 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 more than 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 towards a complete understanding of the mechanisms underlying SH3-mediated cellular responses. To enable future research, we have made publicly available the peptide-binding specificity profiles.
Supplementary Information
Table S1 - Sequence alignment of all SH3 domains that worked in phage display experiments
Table S2 - Results summary for all human SH3 domains
Table S3 - List of all SH3-peptide complexes in the PDB cataloged by specificity classes
Table S4 - Phage clonal ELISA results
Data
PWM files - Position weight matrices of SH3 domains
Logo images - Logos of SH3 domains
Note: PWM and Logo file names are formatted as: Gene_name-Domain_position-PWM_number (e.g. PWM ABL2-1_1-1 has ABL2 gene name, 1/1 domain position, 1 PWM number (out of 2))
Author Information
1The Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1
2Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8
3Department of Computer Science, University of Toronto, Toronto, ON, Canada M5S 3G4
4Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7
5Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada
6Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON Canada, M5S 1A8
(*)To whom correspondence should be addressed. Email: sachdev.sidhu@utoronto.ca
(+)JT and HH contributed equally to the study