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 Gα subtypes (Gαs, Gαi, Gαq, and Gα12/13) and Gβγ remain poorly understood. While conserved residues in Gα subtypes are often assumed to have similar functions, they may instead modulate coupling selectivity by altering the frequency and stability of contacts at the Gα:Gβγ 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 Gαi/o and Gαq/11 subfamilies contribute differentially to Gβγ coupling. These conserved residue "hotspots" on Gαi and Gαq produced divergent functional effects on Gβγ 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.