Conserved Residues in the Gα interface show subtype specificity in Gβγ coupling

Although the structural basis of selective G-protein coupling to G protein coupled receptors (GPCRs) is well characterized, the mechanisms underlying selective interactions between distinct G subtypes (Gs, Gi, Gq, and G12/13) and G{beta}{gamma} remain poorly understood. While conserved residues in G subtypes are often assumed to have similar functions, they may instead modulate coupling selectivity by altering the frequency and stability of contacts at the G:G{beta}{gamma} interface. Using molecular dynamics (MD) simulations combined with the interpretable machine learning method, Bayesian Network Model (BNM), and protein-protein proximity (BRET) assays, we show that conserved residues in the two closely related Gi/o and Gq/11 subfamilies contribute differentially to G{beta}{gamma} coupling. These conserved residue "hotspots" on Gi and Gq produced divergent functional effects on G{beta}{gamma} coupling, indicating that conservation does not ensure functional equivalence. These findings suggest that local microenvironment and paralog-specific allosteric coupling shape how conserved interface residues contribute to protein-protein coupling. The framework provides a systematic approach for dissecting subtype-specific mechanisms, with implications for drug design and for annotating the functional relevance of disease-associated variants. The computational methods used here are broadly applicable to other homologous protein families.