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

Abstract

Protein-protein interactions (PPIs) drive many cellular processes. Some PPIs 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 different SH3-dependent interaction profiles. However, by swapping SH3 domains between the paralogs and by characterizing the SH3 domains freed from their protein context, we find that very 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 intramolecular interaction between a SH3 and its host protein evolves through time, making domains not functionally equivalent even when they have the same binding specificity.

Cryptic genetic variation shapes the fate of gene duplicates in a protein interaction network

“…Similarly, as protein-protein interaction depends on protein abundance and binding affinity, regulatory divergence could also strongly impact how mutations in SH3 domains translate into changes in protein complex formation. Although type-1 myosins are largely functionally redundant 60 , MYO5 has a slightly higher expression level than MYO3 in vivo 44 , which we find is at least partially caused by MYO5 having a stronger promoter than MYO3 (Fig Suppl 9B). Hence, we hypothesized that mutations introduced at the MYO5 locus would cause less functional impairment than those introduced at the MYO3 locus.…”

“…Quantifying the functional effects of mutations Myo3 and Myo5 share the same arrangement of protein domains: an N-terminal myosin motor domain, a neck containing two IQ motifs, and a C-terminal tail consisting of a TH1 domain, an SH3-domain and a CA domain. The SH3 domains are short (59 amino acids long) and dictate binding to multiple partners 28,43 , making them excellent candidates for genetic manipulation.…”

“…We used a recently inferred ML phylogeny for type-1 fungal myosins 44 with Microsporidia as an outgroup to reconstruct the ancestral sequences of the C-terminal SH3 domain pre-duplication. Most probable ancestral SH3 domain sequences were reconstructed on this ML phylogeny FAST-ML (v3.11) 71, 72 .…”

“…Recent research has demonstrated that the SH3 domains in these duplicates are structurally similar (RMSD = 0.386) and functionally equivalent as they can be interchanged between the two paralogs without detectable effects on the binding of Myo3 or Myo5 to cognate interaction partners 44 . The resurrection of the ancestral domain before the gene duplication event showed that the domain has remained functionally unchanged in either paralog since duplication despite a divergence of 10% of sites (6/59 residues) from the ancestral sequence on either paralog 44 . These diverged sites represent potential cryptic divergence that could shape the future evolution of these proteins in the protein network.…”

“…In contrast, a double deletion of the two duplicates leads to synthetic lethality 29, 42, 43 , which makes Myo3/Myo5 an excellent model system for understanding sequence space exploration for redundant paralogs and their evolutionary trajectories. Recent research has demonstrated that the SH3 domains in these duplicates are structurally similar (RMSD = 0.386) and functionally equivalent as they can be interchanged between the two paralogs without detectable effects on the binding of Myo3 or Myo5 to cognate interaction partners 44 . The resurrection of the ancestral domain before the gene duplication event showed that the domain has remained functionally unchanged in either paralog since duplication despite a divergence of 10% of sites (6/59 residues) from the ancestral sequence on either paralog 44 .…”