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

Area of Science:

• Molecular Biology
• Biochemistry
• Computational Biology

Background:

• The structural basis of G protein-coupled receptor (GPCR) interactions is known, but how distinct Gα subtypes selectively interact with Gβγ subunits is unclear.
• Conserved residues in Gα subtypes are often assumed to have equivalent functions, but this may not be true for Gα:Gβγ interface selectivity.

Purpose of the Study:

• To investigate the functional roles of conserved residues in Gα subtypes during Gβγ coupling.
• To determine if conserved residues exhibit differential contributions to Gβγ coupling selectivity between Gα_i/o and Gα_q/11 subfamilies.

Main Methods:

• Employed molecular dynamics (MD) simulations to model protein interactions.
• Utilized Bayesian Network Models (BNM) for interpretable machine learning analysis.
• Applied bioluminescence resonance energy transfer (BRET) assays to measure protein-protein proximity.

Main Results:

• Identified conserved residue "hotspots" on Gα_i1 and Gα_q that differentially affect Gβγ coupling.
• Demonstrated that conserved residues can have divergent functional outcomes on Gβγ coupling, challenging the assumption of functional equivalence.
• Showed that local microenvironment and paralog-specific allosteric coupling influence the function of conserved interface residues.

Conclusions:

• Conservation of residues does not equate to functional equivalence in Gα:Gβγ interactions.
• Subtype-specific mechanisms of Gβγ coupling are shaped by local contexts and allosteric effects.
• The developed framework offers a systematic approach for studying homologous protein families and has implications for drug discovery and disease variant annotation.