Dissection of the role of a SH3 domain in the evolution of binding preference of paralogous proteins

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.

Methods
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.