DOI: 10.5061/dryad.sj3tx968m
title: Data from: Dissection of the role of a SH3 domain in the evolution of binding preference of paralogous proteins

description: Protein-protein interactions drive many cellular processes. Some protein
 interactions are directed by Src homology 3 (SH3) domains that bind
 proline-rich motifs on other proteins. The evolution of the binding
 specificity of SH3 domains is not completely understood, particularly
 following gene duplication. Paralogous genes accumulate mutations that can
 modify protein functions and, for SH3 domains, their binding preferences.
 Here, we examined how the binding of the SH3 domains of two paralogous
 yeast type I myosins, Myo3 and Myo5, evolved following duplication. We
 found that the paralogs have subtly different SH3-dependent interaction
 profiles. However, by swapping SH3 domains between the paralogs and
 characterizing the SH3 domains freed from their protein context, we find
 that few of the differences in interactions, if any, depend on the SH3
 domains themselves. We used ancestral sequence reconstruction to resurrect
 the pre-duplication SH3 domains and examined, moving back in time, how the
 binding preference changed. Although the closest ancestor of the two
 domains had a very similar binding preference as the extant ones, older
 ancestral domains displayed a gradual loss of interaction with the modern
 interaction partners when inserted in the extant paralogs. Molecular
 docking and experimental characterization of the free ancestral domains
 showed that their affinity with the proline motifs is likely not the cause
 for this loss of binding. Taken together, our results suggest that the SH3
 and its host protein could create intramolecular or allosteric
 interactions essential for the SH3-dependent PPIs, making domains not
 functionally equivalent even when they have the same binding specificity.
description: The data published in this dataset was collected by multiple
 methods. Among the methods used are DHFR Protein-fragment Complementation
 Assay, cytometry, ancestral sequence reconstruction with IQ-TREE and
 FastML, protein structure prediction with AlphaFold2 and AlphaFold
 Multimer, molecular docking with Haddock2.4, orthology analysis and
 coevolution predictions with EVCouplings. See the README.md file and the
 method section of the paper Dissection of the role of a SH3 domain in the
 evolution of binding preference of paralogous proteins for more
 details. File S1 :  Tables S1 - S12
 File S2 : Detailled protocols FiguresS :
 Figures S1 - S10 DataS1 : DHFR PCA results
 DataS2 : Phylogeny and sequence alignment 
 DataS3 : AlphaFold results DataS4 : Molecular
 docking input and output files DataS5: Orthology input
 and motif conservation results DataS6: EVCouplings
 output  Please refer to Lemieux et al. 2023 for details
 on the data collection and transformation.
description: All files can be opened with either R, a text editor, Excel or
 ChimeraX.
description: This README file was generated on 2023-09-19 by Pascale Lemieux. # GENERAL
 INFORMATION 1. Title of Dataset: Data from : Dissection of the role of a
 SH3 domain in the evolution of binding preference of paralogous proteins
 2. Author Information A. Principal Investigator Contact Information Name:
 Christian Landry Institution: Université Laval, Québec CA Email:
 christian.landry@bio.ulaval.ca <br> B. Associate or Co-investigator
 Contact Information Name: Pascale Lemieux Institution: Université Laval,
 Québec, CA Email: pascale.lemieux.4@ulaval.ca 3. Date of data collection
 (single date, range, approximate date): 2020-2023 4. Information about
 funding sources that supported the collection of the data: Canadian
 Institutes of Health Research (CIHR) Foundation grant 387697 and a HFSP
 grant (RGP0034/2018) to CRL SHARING/ACCESS INFORMATION 1.
 Licenses/restrictions placed on the data: CC0 1.0 Universal (CC0 1.0)
 Public Domain 2. Links to publications that cite or use the data: Lemieux,
 P., Bradley, D., Dubé, A. K., Dionne, U. & Landry, C. R.
 Dissection of the role of a SH3 domain in the evolution of binding
 preference of paralogous proteins. bioRxiv 2023.03.09.531510 (2023)
 doi:10.1101/2023.03.09.531510 3. Links to other publicly accessible
 locations of the data: None 4. Links/relationships to ancillary data sets:
 None 5. Was data derived from another source? YES A. If yes, list
 source(s): EnsemblCompara (June 2021, June 2023);
 https://useast.ensembl.org/info/data/index.html 6. Recommended citation
 for this dataset: Lemieux, P., Bradley, D., Dubé, A. K., Landry, C.R.,
 (2023). Data from : Dissection of the role of a SH3 domain in the
 evolution of binding preference of paralogous proteins. Dryad Digital
 Repository. doi:10.5061/dryad.sj3tx968m DATA & FILE OVERVIEW This
 dataset contains the Supplementary Data for the pre-print titled
 'Dissection of the role of a SH3 domain in the evolution of binding
 preference of paralogous proteins'. 1. Folder List: A) Figures S :
 Supplementary figures and their caption(Figures S1-S10) from : Dissection
 of the role of a SH3 domain in the evolution of binding preference of
 paralogous proteins B) File S1 : Supplementary Tables (Tables S1-S12) C)
 Data S1 : Raw and processed data from 3 different DHFR PCA experiment
 which quantify PPIs strength in vivo D) Data S2 : Protein sequences,
 multiple sequence alignment, phylogenetic tree of the fungal orthologs of
 the paralogs MYO3 & MYO5. Ancestral sequence reconstruction
 probabilities of their SH3 domains are also present in this repository. E)
 Data S3 : Alphafold2 structure prediction output for the ancestral SH3
 domains, in the paralog context or alone. AlphaFold Multimer output for
 the SH3 and their surrounding regions. F) Data S4 : SH3 domains and
 peptides' structures and configuration files used for the molecular
 docking with Haddock2.4. G) Data S5 : Motif conservation analysis results
 and the orthology informations used for Figure S5A H) Data S6 :
 evCouplings output for Myo3 and Myo5 proteins 2. Relationship between
 files, if important: None 3. Additional related data collected that was
 not included in the current data package: None 4. Are there multiple
 versions of the dataset? No A. If yes, name of file(s) that was updated:
 NA i. Why was the file updated? NA ii. When was the file updated? NA #
 Description of the data and file structure ## Figures S All of the
 supplementary Figures from : Dissection of the role of a SH3 domain in the
 evolution of binding preference of paralogous proteins. Figure files are
 named according to their identifier (S1 to S10). The captions of the
 supplementary figures are available in the file Caption\_FigureS.pfd ##
 File S1 File S1 contains all the Supplementary tables (Table S1 to S12)
 from : Dissection of the role of a SH3 domain in the evolution of binding
 preference of paralogous proteins. FileS1.xlsx is divided in 12 sheets and
 each one shows one supplementary table. Table S1 to S3 contain the results
 of 3 different PCA DHFR experiment. Table S1 contains the result of the
 screen testing the Myo3 & Myo5 interactome, the SH3-dependency of
 those interactions, the influence of different SH3 domains one those
 interactions. Table S2 contains the result of the screen testing if
 deleting the Myo3 and Myo5 SH3 predicted binding motifs from the preys
 impacts the PPIs between the Bait-Prey. Table S3 contains the result of
 the screen testing the effect of the protein context on the PPIs of the
 SH3 domains using strain expressing the SH3 domains without the rest of
 the protein. The expression level of the SH3 domains is modulated by the
 amount of estradiol in the media. ### Table S1 - PCA paralog Number of
 rows : 8149 Number of variables : 17 Prey.Systematic\_name : Gene
 systematic name of the protein tagged by the DHFR[3] fragment
 Prey.Standard\_name : Gene standard name of the protein tagged by the
 DHFR[3] fragment Bait.Standard\_name : Gene standard name of the protein
 tagged by the DHFR[12] fragment sh3\_sequence : SH3 domain inserted in the
 Bait protein at the extant SH3 locus plate\_number : Media plate number
 tech\_rep : technical replicate number (2 per Bait & sh3\_sequence
 combination) row : row on the plate (1536 array) of the colony column :
 column on the plate (1536 array) of the colony n.bio\_rep : number of
 replicate per Bait, sh3\_sequence and Prey combination area : area of the
 colony computed by pyphe from Data S1 adjust\_area : area of the colony +
 1 log2\_area : transformation to logarithm 2 scale of the adjust\_area
 plate\_max : maximum colony size per plate plate\_min : minimum colony
 size per plate PPI\_score : Protein-protein interaction (PPI) score of
 each colony med.PPI\_score : median PPI score of biological replicates
 SH3\_dep : if the Bait-Prey PPI is dependent on the SH3 domain
 (SH3-dependent), according to Wilcoxon test corrected with
 Benjamini-Hochberg (TRUE, FALSE) optSH3\_dif : if the Bait-Prey PPI is
 affected by the use of the codon optimized sequence of the extant SH3 in
 the bait, according to Wilcoxon test corrected with Benjamini-Hochberg
 (TRUE, FALSE) ### Table S2 - PCA motifΔ prey Number of rows : 7328 Number
 of variables : 16 Same variables present in Table S1 - PCA paralog except
 : motif\_deletion : if the predicted binding motif has been replaced by
 the stuffer on each Prey ### Table S3 - PCA free SH3 Number of rows :
 11835 Number of variables : 16 Same variables present in Table S1
 & Table S2 except : sh3\_domain : SH3 domain inserted in the
 genetic locus (GAL) under the control of the estradiol concentration
 DHFR12\_tag : Position of the DHFR[12] fragment tag relative to the SH3
 domain (C , N) [estradiol] : estradiol concentration in the media in nM
 ### Table S4 - PCA liquid validation PCA DHFR validation results in liquid
 media. Table S4 contains the output metrics of the R package Growthcurver
 [12] and the Number of rows : 448 Number of variables : 15 plate\_well :
 position of the culture in the 96-well plates k : carrying capacity n0 :
 population size at the beginning of the growth curve r : growth rate of
 the population t\_mid : time at which the population density reaches 0.5k
 t\_gen : fastest possible generation time auc\_l : area under the logistic
 curve auc\_e : area under the empirical curve sigma : measure of the
 goodness of fit of the parameters of the logistic equation for the data
 note : if the curve does not fit the data, a warning is written here
 Prey.Systematic\_name : Gene systematic name of the protein tagged by the
 DHFR[3] fragment sh3\_sequence : SH3 domain inserted in the Bait protein
 at the extant SH3 locus Bait.Standard\_name : Gene standard name of the
 protein tagged by the DHFR[12] fragment strain : Combination of
 sh3\_sequence and Bait.Standard\_name PPI\_score\_gc : Normalized PPI
 score obtain in liquid culture (auc\_l - (n0\*60)) See
 https://cran.r-project.org/web/packages/growthcurver/vignettes/Growthcurver-vignette.html for further description of Growthcurver output metrics. ### Table S5 - Motif prediction Using the experimentally resolved specificity matrix of the SH3 domains from Myo3 and Myo5 [13], we scanned the SH3-dependent prey protein sequences to identify the best match to the specificity matrix. The motif identified is the predicted motif that is bound by the SH3 domain of either Myo3 or Myo5 protein. Number of rows : 54 Number of variables : 7 Prey.Systematic\_name : Gene systematic name of the protein scanned by the specificity matrix Prey.Standard\_name : Gene standard name of the protein scanned by the specificity matrix motif\_Start-End : positions of the start and end (relative to the first amino acid of the protein) of the best match motif to the specificity matrix best\_match : amino acid sequence of the best match motif to the specificity matrix Max\_MSS : Score of the best match motif to the specificity matrix p-value : Confidence indication of the motif prediction Paralog : SH3 Specificity matrix used to do the scan (Myo3 or Myo5) ### Table S6 - Cytometry Cytometry measurement of Green Fluorescent Protein (GFP) expression tagged to variants of Myo3 and Myo5. The variants contain different SH3 domain at the position of the extant SH3 domain in both paralogs. The cytometry measures if the paralog variants have different level of expression in vivo. For each replicate, we aggregate the measures of 3000 individual cells. Number of rows : 75 Number of variables : 6 GFP\_tagged\_protein : Paralog tagged by the GFP sh3\_sequence : SH3 domain inserted in the paralog, creating the paralog variant bio\_rep : identifier of the replicate (1,2,3) for each paralog variant cyto\_rawfile : name of the cytometry file containing the raw data med\_rep : median of the 3000 measurements mean\_rep : mean of the 3000 measurements ### Table S7 - Molecular docking To explain the in vivo binding of the SH3 domains, we performed molecular docking with the predicted binding motifs (Table S5) to SH3 domains of interest. Myo3 and Myo5 extant SH3 experimental structures were retrieved (Data S4) for the docking. We used the AlphaFold2 prediction of AncD SH3 as well (Data S3). Table S7 contains the processed data from Data S4. The molecular docking of one motif-SH3 pair generates 400 structures which are divided in multiple clusters. The cluster are then ranked by the algorithm of Haddock2.4(i.e. 1 is the most reliable cluster). We then selected the 10 best structures (again ranked by Haddock2.4 algorithm) Number of rows : 544 Number of variables : 7 SH3.Standard\_name : Identifier of the SH3 domain used for the docking (AncD, Myo3, Myo5) Motif.Standard\_name : Gene standard name of the protein on which the motif is predicted (Table S5) Motif.Systematic\_name : Gene systematic name of the protein on which the motif is predicted (Table S5) cluster\_rank : Rank of the cluster cluster\_median\_Interaction\_energy(kcal/mol) : median of binding energy between the SH3 and the motif for the 10 best structures of each cluster target\_SH3 : SH3 predicted to bind the motif exp\_validated : if the motif was experimentally validated by the PCA experiment motifΔ prey (TRUE and FALSE) ### Table S8 - Oligonucleotides Tables S8 and Table S9 list the synthesize DNA material used to construct the yeast strains. Number of rows : 120 Number of variables : 4 Name : Systematic name of the oligonucleotide Sequence : Nucleotide sequence Description : Detailed description of the oligonucleotide Purpose : General purpose of the oligonucleotides ### Table S9 - SH3 DNA sequences Number of rows : 8 Number of variables : 2 SH3 : Identifier of the codon optimized SH3 domains codon optimized sequence : nucleotide sequence of the codon optimized SH3 domain ### Table S10 - Baits Table S10 and S11 contain the description of the Bait and Prey yeast strain used in the paper related to this dataset Number of rows : 44 Number of variables : 5 Systematic.Bait\_name : Gene systematic name of the protein tagged by the DHFR[12] fragment Standard.Bait\_name : Gene standard name of the protein tagged by the DHFR[12] fragment Reference : Publication which produced the strains Strain background : Genetic background SH3 variant : SH3 domain introduced in the Bait protein at the position of the extant SH3 domain ### Table S11 - Preys Number of rows : 305 Number of variables : 5 Systematic.Prey\_name : Gene systematic name of the protein tagged by the DHFR[3] fragment Standard.Prey\_name : Gene standard name of the protein tagged by the DHFR[3] fragment Reference : Publication which produced the strains Strain background : Genetic background Additionnal Informations : Specification regarding the strain construction ### Table S12 - Reagent table Table containing reagents, strains, tools and databases used in this study. Reagent type species, or resource : General class of reagent, strain or resource Designation : Specific name of the reagent, strain or resource Source or reference: Reagent supplier or publication which produced the strains or resource Identifiers : Catalog number, public repository or RRID of the reagent, strain or resource Additional information : Specification regarding the detailed list of strains or DNA sequences ## File S2 File S2 contains the detailed protocol for yeast strain construction and for PCA DHFR experiment and analysis. ## Data S1 Raw data obtain with the analysis of microbial growth with pyphe[1]. Directories complete\_paralog\_RD, motif\_confirmation\_RD and free\_SH3\_RD contain one DHFR PCA experiment each. DHFR PCA uses multiple selection step to allow detection of protein-protein interactions in yeast strains. Each experiment contains raw data files for different selection steps. Selection steps present in each experiment directories are the second diploid selection (S2\_diploid directories) and the second methotrexate selection (MTX2 directories). The complete\_paralog\_RD/DHFR3\_array/ contains selection step for the large array of prey strains. In each selection step directories, there is one CSV file per plate analysed (final timepoint of the selection step). Here is the folder structure: /complete\_paralog\_RD/ # DHFR PCA raw data from the experiment testing paralog variant baits against 296 wt preys /DHFR3\_array/ # prey array, growth verification of the Yeast Protein Interaction collection strains /MTX2/ # growth on MTX at final timepoint /S2\_diploid/ # growth verification of the diploid strains before replication on MTX medium /motif\_confirmation\_RD/ # DHFR PCA raw data from the experiment testing paralog variant baits against 296 wt preys /MTX2/ # growth on MTX at final timepoint /S2\_diploid/ # growth verification of the diploid strains before replication on MTX medium /free\_SH3\_RD/ # DHFR PCA raw data from the experiment testing paralog variant baits against 296 wt preys /MTX2/ # growth on MTX at final timepoint /S2\_diploid/ # growth verification of the diploid strains before replication on MTX medium Each csv file contains 7 variables and a number of rows corresponding to the number of colonies detected by pyphe on the selection media. Variable list as defined in pyphe documentation [1]: area : single colony area centroid : coordinates of the center of the colony mean\_intensity : overall intensity (an estimator that reflects thickness as well as area) perimeter : perimeter of the colony row : row of the colony column : column of the colony circularity : circularity of the colony CSV files are analyzed with the corresponding PCA\_analysis\_X.R script available in the Github repository cited in the paper. ## Data S2 Data S2 contains 7 files with the sequence data used to reconstruct the ancestral SH3 domains and to visualize the sequences comparison in Supplementary Figures. The fungal orthologs of MYO3 and MYO5 were retrieved by EnsemblCompara [2] and SH3 domain were defined according to the SMART database v8 [3].Each file is obtained after a step of the ancestral sequence reconstruction pipeline. 1 - Fungal orthologs protein sequences retrieved by EnsemblCompara [2] as orthologs of Myo3 and Myo5 yeast proteins ortholog\_sequences.fa 2 - Multiple Sequence Alignment generated by MAFFT L-INS-i v7.453 of the fungal orthologs. ortholog\_MSA.fa 3 - Trimmed Multiple Sequence Alignment of the fungal orthologs used for the tree construction ortholog\_MSA\_trim.fa 4 - Phylogenetic tree of the fungal orthologs generated by IQ-TREE2 [5] ortholog\_phylogeny\_newick.txt 5 - Posterior probability for the reconstruction for all position in SH3 domains at each node in the phylogenetic tree produced by FAST-ML v3.11 [4] Ancestral\_MaxMarginalProb\_Char\_Indel.txt 6 - Multiple Sequence Alignment of the orthologs present in the WGD clade ortholog\_dup\_MSA.afa 7 - Protein sequence of the ancestral and extant SH3 domains tested in vivo (Figure S1E) myo3.myo5.sh3sequence.fasta ## Data S3 We used each ancestral sequence for protein structure prediction. AlphaFold2 (AF) structure prediction output for each of the SH3 domains are present in directories AncA, AncB,AncC, AncD, extantMyo3 and extantMyo5. Except for AncB, the prediction were performed with AlphaFold Colab (Febuary 2022). The names of the folder correspond to the name of the SH3 domains described in the paper related to this repository. See the Alphafold Colab Notebook documentation [6] for the description of all the files. superimposition.cxs is the file used for visualization of the superimposed SH3 predicted strutures . It can be open with ChimeraX [7] Further protein structure predictions were performed using the full length of the protein sequence modified by replacing the extant SH3 domains with an ancestral one, creating chimeras. These results can be found in the directory paralog\_variants. There is one file per prediction for the protein sequence submited to AlphaFold2 (Anc\**Myo\*.fasta) and one file for the best-ranked structure for each AlphaFold2 prediction (ranked\_0\_AncMyo*.pdb) We then used AlphaFold Multimer to predict how the binding of SH3 domains surrounded by disordered regions could be affected by those regions. We create chimeras inserting AncD in place of extant SH3 domains and submttted AncD chimeras with one extant sequence to AlphaFold Multimer. The sequences used as input are available in the directory /sh3\_regions/. The output of AlphaFold Multimer are present in the subdirectories AncDmyo3\_extantMyo5, AncDmyo5\_extantMyo3 and extantMyo3\_extantMyo5 named according to the sequences used as input. In each of those directories, you can find the AlphaFold Multimer top-5 models (ranked\_[0-4].pdb) and a ChimeraX file used for visualization of the predictions(\*.cxs). AncB structure was predicted with a later version of AlphaFold Colab Notebook (December 2022) which explains the different format of the output. It contains only the first ranked model (selected\_prediction.pdb) and the predicted aligned error of this model (predicted\_aligned\_error.json). Here is the folder structure: /AncA/ /AncB/ /prediction/ predicted\_aligned\_error.json selected\_prediction.pdb /AncC/ /AncD/ /extantMyo3/ /extantMyo5/ /paralog\_variants/ /sh3\_regions/ /AncDmyo3\_extantMyo5/ /AncDmyo5\_extantMyo3/ /extantMyo3\_extantMyo5/ \*.fasta # sequence files superimposition.cxs ## Data S4 All input and output data of the molecular docking of the SH3 domains with multiple peptides. First, the peptide structures were generated by AlphaFold2[11] and the output files are present in the AF2\_motif\_structure directory. There is one subdirectory per peptide used for the structure predictions. The names of the subdirectory are the systematic name of the gene (Y\*) encoding the protein on which the peptide is a subsequence. The gene systematic name is followed by the paralog predicted to bind the peptide (both, MYO3 or MYO5). The binding motifs are listed in Table S5. Each subdirectory contains the AlphaFold2 output files of the peptide structure prediction. See AlphaFold documentation for more detailed description of the output files[6, 11]. Then, the SH3 domain structures were prepared for the docking. We used AncD AlphaFold prediction and the experimental structures of Myo3 (PDB:1RUW) and Myo5 (PDB:1YP5) SH3s. We followed the best practices guide of Haddock2.4[8](https://www.bonvinlab.org/software/bpg/structures/) to prepare the SH3 domain structures for the docking. The docking ready structures are present in the directory structures\_sh3 named according to the SH3 domain. We used Ambiguous Interaction Restraints(AIRS)[8] to identify key positions on the SH3 domains that we expect the peptides to bind. The choice of these position is based on previous experimental work on the PDB structure 2P4R identifying which positions of a SH3 domain bind to a proline peptide. The files defining the AIRS for each SH3 domain are present in the directory AIRS\_files. Variables : resid : amino acid residu identifier relative to the first position of a protein segment segid : protein segment identifier (A or B, for docking with 2 segment) Ex. The structure of the \*\_AIRS file To indicate that position 35 on segment A should bind to residues 2,3 or 4 of segment B is the following : assign ( resid 35 and segid A) ( ( resid 2 and segid B) or ( resid 3 and segid B) or ( resid 4 and segid B) ) Lastly, to run the molecular docking with Haddock2.4 [8], we modified the configuration files with the optimal parameter for peptide docking (https://www.bonvinlab.org/software/bpg/peptides/). There is one configuration file per SH3 domain present in the directory run.csn\_files. Each file is named according to the SH3 used in the specific molecular docking. For more detailed information of the run.cns files, please refer to the Haddock2.4 documentation (https://www.bonvinlab.org/software/haddock2.4/manual/). 400 structures were generated per molecular docking, they were divided in clusters according to Root Mean Square Deviation computed by Haddock2.4 algorithm. We then used FoldX v4 [9] on the 10 best structures of each cluster for energy minimization ('RepairPDB' [9]) 10 times and to compute the interaction energy between the docked peptide and the SH3 domain ('AnalyseComplexe' [9]). The file Docking\_RD.csv contains the raw data obtain with the consecutive the molecular docking, energy minimization by FoldX v4 [9] and the interaction energy computation by FoldX v4. Docking\_RD.csv Number of rows : 4110 Variable list : Pdb : name of the pdb file containing the SH3-peptide structure Interaction\_Energy : Interaction energy computed by FoldX v4[9] cluster: Cluster ID defined by Haddock2.4[8] rank : Rank of the cluster based on Haddock2.4 algorithm[8] preys : peptide ID (Gene systematic name, paralog predicted to bind to this peptide) SH3 : SH3 domain used for this specific docking Here is the folder structure: /AF2\_motif\_structure/ # peptide structures used as input /Y\*\_seq/ \# AlphaFold2 output /AIRS\_files/ # Ambiguous restraint files used as input /AncC\_AIRS.txt /myo3\_AIRS.txt /myo5\_AIRS.txt /run.csn\_files/ # Haddock2.4 configuration files used as input /run\_AncC\_test.csn /run\_myo3\_test.csn /run\_myo5\_test.csn /structures\_sh3/ # SH3 domain structures used as input /AncC\_clean.pdb /myo3\_clean.pdb /myo5\_clean.pdb Docking\_RD.csv # Molecular docking and energy minimization results for AncD, Myo3 and Myo5 SH3 domains ## Data S5 Input orthology information for the Figure S5 and motif conservation results for both paralogous SH3. We used orthology data to predict for extant interaction partners of Myo3 and Myo5 if there was also an interaction between the ancestral proteins. We retrieved orthology information for all yeast proteins from EnsemblCompara (June 2021) and compared it to the species included in the phylogeny used for the ancestral sequence reconstruction. Since EnsemblCompara modifies frequently its dataset, the dataset used is available in the file Compara.103.protein\_default.homologies.tsv. Compara.103.protein\_default.homologies.tsv Number of rows : 753493 Variables list : gene\_stable\_id : Gene systematic name protein\_stable\_id : Systematic name of the protein coding gene species : Species expressing the protein identity : Sequence identity of the protein to the homologous protein homology\_type : type of gene homology (one2one, one2many, many2many, etc.) homology\_gene\_stable\_id : Homologous Gene systematic name homology\_protein\_stable\_id : Homologous Systematic name of the protein coding gene homology\_species : Species expressing the homologous protein homology\_identity : Sequence identity homologous protein to the protein We also used a more recent EnsemblCompara version (June 2023) to perform a binding motif conservation prediction on the orthologs of 7 yeast proteins (Bzz1, Lsb3, Myo5, Osh2, Pkh2, Sla1, Ste20). We performed a sequence alignment with orthologs of the 7 proteins to map the binding motif on the orthologs according to the motif position on the yeast proteins. We also mapped the disordered region surrounding the predicted motifs and scored the motifs according to the specificity matrix of Myo3 and Myo5 SH3 domains. This analysis outputs are one file per the 7 yeast protein. There is one folder per specificity matrix the motifs were tested against (one for Myo3 and one for Myo5). Number of rows is equal to the number of orthologs retrieved for each yeast protein. Variable list : Species : Species taxonomic name Type : Orthology type (one2one, one2many, many2many, etc.) Taxonomy : Reference clade of the species Best\_match : predicted binding motif with the best match to the specificity matrix Max\_MSS : Score of the motif against the specificity matrix in Best\_match Finally, the file orthology\_myosins\_partners.csv contains the filtered version of EnsemblCompara dataset (June 2021) to summarize the orthology information for the interaction partners identified in the related paper. Orthology information is one of one2one, one2many, many2many, other\_paralog or within\_species\_paralog. Number of variable : 28 Number of rows : 177 No orthology information available : NA Variable list: label : Species name Sla1 : Orthology information for Sla1 protein in each species of label column Cdc10 : Orthology information for Cdc10 protein in each species of label column Syp1 : Orthology information for Syp1 protein in each species of label column ... Sla2 : Orthology information for Sla2 protein in each species of label column Pkh2 : Orthology information for Pkh2 protein in each species of label column Las17 : Orthology information for Las17 protein in each species of label column Here is the folder structure: /motif\_conservation\_results\_Myo3/ \*\_ortho\_mss\_myo3\_pwm.csv /motif\_conservation\_results\_Myo3/ \*\_ortho\_mss\_myo5\_pwm.csv Compara.103.protein\_default.homologies.tsv orthology\_myosins\_partners.csv ## Data S6 Output of EVCoupling v0.2 using Myo3 protein sequence as input. EVCoupling[10] is a bioinformatic tool predicting coevolution between positions in a protein. The first step of EVCouplings is to create a sequence alignment with homologs of the query sequence, here with the Myo3 protein sequence (align directory). The second step is to compare the sequence level signal to the structural information and compute de couplings scores (respectively compare and couplings directories). Finally, EVCouplings predicts how mutations can affect the structure of the query protein (directories fold and mutate). The most important files of the output are briefly described but please refer to EVCouplings documentation for an exhaustive description of the output files (https://github.com/debbiemarkslab/EVcouplings/blob/v0.2/notebooks/output\_files\_tutorial.ipynb) [10] Most files are name according to the job name 'MYO3\_YEAST\_1-1272' which as as input the full lenght sequence of the protein Myo3 from position 1 to 1272. MYO3\_YEAST\_1-1272.a2m : Final alignment file usde to compute evolutionary couplings. "Lowercase columns indicate columns with too many gaps that did not meet the minimum column coverage threshold, which means they were excluded from the EC calculation. Lowercase columns have a '.' for gap character by convention. Additionally, sequences which did not fulfill the the minimum sequence coverage threshold (i.e. fragments with too many gaps) have been removed from this alignment." [10] MYO3\_YEAST\_1-1272\_statistics\_summary.csv : Contains information on properties of the final alignment [10] MYO3\_YEAST\_1-1272\_frequencies.csv : Contains the frequency of each character in each position of the alignment.[10] MYO3\_YEAST\_1-1272\_identities.csv : Contains the sequence identity of each sequence in the alignment to the target sequence.[10] MYO3\_YEAST\_1-1272\_\*\_ECs.pdf: Contact map files, Displays the evolutionary couplings together with experimental structure contacts. [10] MYO3\_YEAST\_1-1272\_ECs.txt : Contains the space-delimited raw output of the evolutionary couplings calculation from plmc. [10] MYO3\_YEAST\_1-1272\_CouplingScores.csv : Contains the evolutionary couplings sorted according to score, and the probability that a pair represents significant coupling rather than background noise. [10] MYO3\_YEAST\_1-1272\_enrichment.csv : Measures the how strongly individual residues are coupled by summing the coupling scores of pairs involving this residues, and then normalizing with the average level of coupling. [10] MYO3\_YEAST\_1-1272.model : Contains model parameters inferred by plmc [10] Here is the general folder structure : /evCouplings\_MYO3\_YEAST/ /align/ MYO3\_YEAST\_1-1272.a2m MYO3\_YEAST\_1-1272\_statistics\_summary.csv MYO3\_YEAST\_1-1272\_frequencies.csv MYO3\_YEAST\_1-1272\_identities.csv /compare/ MYO3\_YEAST\_1-1272\_\*\_ECs.pdf /couplings/ MYO3\_YEAST\_1-1272\_CouplingScores.csv MYO3\_YEAST\_1-1272\_enrichment.csv MYO3\_YEAST\_1-1272\_ECs.txt MYO3\_YEAST\_1-1272.model /fold/ /mutate/ ## Code/Software All the scripts used for analyses of raw data present in this dataset are available in the Github repository (https://github.com/Landrylab/Lemieux\_et\_al2023) cited in the paper. ## References [1]Kamrad, S., Rodri­guez-Lopez, M., Cotobal, C., Correia-Melo, C., Ralser M., Bahler J. (2020). 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UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021) [8]Dominguez C, Boelens R, Bonvin AMJJ. 2003. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125:1731–1737. [9]Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. 2005. The FoldX web server: an online force field. Nucleic Acids Res. 33:W382–W388. [10]Hopf, T. A. et al. The EVcouplings Python framework for coevolutionary sequence analysis. Bioinformatics 35, 1582–1584 (2019) [11]Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021) [12]Sprouffske, K. & Wagner, A. Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves. BMC Bioinformatics 17, 172 (2016) [13]Tonikian, R. et al. Bayesian modeling of the yeast SH3 domain interactome predicts spatiotemporal dynamics of endocytosis proteins. 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