Coevolving residues distant from the ligand binding site are involved in GAF domain function.

Ligand binding to GAF domains regulates the activity of associated catalytic domains in various proteins, such as the cGMP-hydrolyzing catalytic domain of phosphodiesterase 5 (PDE5) activated by cGMP binding to GAFa domain. However, the specific residues involved and the mechanism of GAF domain function remain unclear. Here, we combine computational and experimental approaches to demonstrate that two highly coevolving residues, L267 and F295, distant from the ligand binding site, play a critical role in GAF domain allostery. Statistical Coupling Analysis (SCA) of GAF domain sequences identified these residues, and molecular dynamics (MD) simulations of both apo and holo forms of wild-type and mutant (L267A, F295A) PDE5 GAFa domains revealed significant changes in structural dynamics and cGMP interaction. Mutational incorporation into a Bioluminescence Resonance Energy Transfer (BRET)-based biosensors, which detects ligand-induced conformational changes, showed altered GAF domain conformation and increased EC(50) for cGMP-induced conformational changes. Similar effects were observed in full-length PDE5 and the GAF domain fluorescent protein, miRFP670nano3. Structural analysis of conformers observed in MD simulations suggested a mechanism by which these coevolving residues influence GAF domain allostery. Our findings provide insight into the role of distant residues in GAF domain function and may enhance understanding of allostery in proteins.