DOI: 10.5061/dryad.79cnp5j3z
title: Data from: Residues neighboring an SH3-binding motif participate in the interaction in vivo

description: In signaling networks, protein-protein interactions are often mediated by
 modular domains that bind short linear motifs. The motifs’ sequences
 affect many factors, among them affinity and specificity, or the ability
 to bind strongly and to the appropriate partners. Using Deep Mutational
 Scanning to create a mutant library, and protein complementation assays to
 measure protein-protein interactions, we determined the in vivo binding
 strength of a library of mutants of a binding motif on the MAP kinase
 kinase Pbs2, which binds the SH3 domain of the osmosensor protein Sho1 in
 Saccharomyces cerevisiae. These measurements were made using the
 full-length endogenous proteins, in their native cellular environment. We
 find that along with residues within the canonical motif, many mutations
 in the residues neighboring the motif also modulate binding strength.
 Interestingly, all Pbs2 mutations which increase affinity are situated
 outside of the Pbs2 region that interacts with the canonical SH3 binding
 pocket, suggesting that other surfaces on Sho1 contribute to binding. We
 use predicted structures to hypothesize a model of binding which involves
 residues neighboring the canonical Pbs2 motif binding outside of the
 canonical SH3 binding pocket. We compared this predicted structure with
 known structures of SH3 domains binding peptides through residues outside
 of the motif, and put forth possible mechanisms through which Pbs2 can
 bind specifically to Sho1. We propose that for certain SH3 domain-motif
 pairs, affinity and specificity are determined by a broader range of
 sequences than what has previously been considered, potentially allowing
 easier differentiation between otherwise similar partners.
description: Multiple methods were used for data collection:
 DHFR-Protein Complementation Assay in a pooled competition
 assay DHFR-Protein Complementation Assay in individual
 colonies Growth curves Protein
 structure predictions using AlphaFold-Multimer In
 Revised Upload: Sequence and structure alignments using
 mafft and MUSTANG respectively
description: # Data from: **Residues neighboring an SH3-binding motif participate in
 the interaction *In Vivo***
 [https://doi.org/10.5061/dryad.79cnp5j3z](https://doi.org/10.5061/dryad.79cnp5j3z) ## Description of the data and file structure All data pertaining to the manuscript "**Residues neighboring an SH3-binding motif participate in the interaction *In Vivo***" by Jordan et al.  These datasets can be used with the scripts hosted at [https://github.com/Landrylab/Jordan_et_al_2024](https://github.com/Landrylab/Jordan_et_al_2024]) to recreate the analysis used in the manuscript and to recreate the figures in the manuscript. Readme.md files in the different folders describe the datasets within, including describing the columns in tabular formatted data. This repository is designed to be used with the scripts in the accompanying github repo. The unzipped "Data" folder should be placed in the same folder as the "figures" and "scripts" folders from the github repo. The data is divided into 6 major folders (7 after update): * AlphaFold_predictions contains the input that was used to generate predicted structures, and the output from AlphaFold *  demultiplexed_sequencing_reads contains the results of all the sequencing runs * DMS contains the data for the analysis of the DHFR-PCA pooled competition assays, on both DMS libraries and on reconstructed mutants * growthcurves contains the data for the growth curves of individually reconstructed mutants (Second update) Now also contains the data for Sho1 mutant growth curves as well as Pbs2 fragments growth curves * images contains the png images used in the figures of the accompanying manuscript * Solid_PCA contains the data for the DHFR_PCA of selected Pbs2 mutants on solid media - (Updated) SH3_alignments contains files used to align the sequences and structure of yeast SH3-containing proteins, and the resulting alignments (Update 30/06/2025) The related Zenodo repository now also contains these Supplementary Files: * Supplementary File 1 contains the Supplementary Figures (Figures S1 to S19) * Supplementary File 2 contains the Supplenetary Tables (Tables S1 to S3) * Supplementary File 3 contains the Supplementary Methods ## Code/Software All scripts used to analyze this data are available at [https://github.com/Landrylab/Jordan_et_al_2024](https://github.com/Landrylab/Jordan_et_al_2024) ## Usage All files can be opened using either Excel, ChimeraX, an image viewer, or a text file reader. All supplemental files included are compatible with the CC0 license waiver ## Change log 08/11/2024 Following peer review, the following changes were made to the data: * SH3_alignments folder added. It contains files used to align the sequences and structure of yeast SH3-containing proteins, and the resulting alignments. * Interaction scores in DMS/results/ were normalized. WT values normalized to 0 and median value of nonsense mutants normalized to -1. * Figures were modified, and new figures and figure panels were added in the images folder. * The Solid_PCA/results/solid_pca_results.csv file was filtered to keep only results pertaining to wild-type Pbs2 and a control where the Pbs2 motif was replaced by a stuffer sequence. * Abstract and methods entries were updated to reflect the revised manuscript. ## Change log 30/06/2025 Following an additional round of peer review, the following changes were made to the repository: * In Data_second_revision.zip, three subfolders added to the growthcurves folder. pbs2_muts_validation_growth_curves contains the data initially in the growthcurves folder. pbs2_fragments contains the data relating to growth curves measuring the interaction of fragments of Pbs2 with SH3 domains. sho1_mutants contains the data relating to growth curves of Sho1 mutants with either Pbs2 or Ybt1. * Figures were modified, and new figures and figure panels were added to the images folder. * Abstract section updated to reflect the revised manuscript. * Added Supplementary Files 1 to 3 and transferred these to Zenodo. ## Change log 23/07/2025 The supplementary files in the associated Zenodo repository were updated. * In File S1, Figure S14 was changed to remove the normalization. * In file S2, Table S6 with the strains used was reorganized. * In File S3, the supplementary methods were rewritten to be more in line with the guidelines of the journal.