Data from: Residues neighboring an SH3-binding motif participate in the interaction in Vivo
Data files
Nov 08, 2024 version files 10.93 GB
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Data_after_revision.zip
5.49 GB
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Data.zip
5.44 GB
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README.md
2.87 KB
Abstract
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 bind 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.
README: Data from: Residues Neighboring an SH3-Binding Motif Participate in the Interaction *In Vivo*
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] 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
- 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
Code/Software
All scripts used to analyze this data are available at [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
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 images/
- 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.
Methods
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