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

Area of Science:

• Biochemistry
• Structural Biology
• Computational Biology

Background:

• GAF domains regulate protein activity through ligand binding, exemplified by phosphodiesterase 5 (PDE5) activation by cGMP.
• The precise residues and mechanisms governing GAF domain allostery remain largely uncharacterized.

Purpose of the Study:

• To identify and elucidate the role of specific residues in GAF domain allosteric regulation.
• To understand the mechanism by which distant residues influence GAF domain function and ligand binding.

Main Methods:

• Statistical Coupling Analysis (SCA) to identify coevolving residues.
• Molecular Dynamics (MD) simulations of wild-type and mutant PDE5 GAFa domains.
• Bioluminescence Resonance Energy Transfer (BRET)-based biosensors and full-length PDE5 assays.

Main Results:

• Two coevolving residues, L267 and F295, distant from the cGMP binding site, were identified as critical for GAF domain allostery.
• Mutations L267A and F295A significantly altered structural dynamics, cGMP interaction, and conformational changes in PDE5 GAFa domains.
• Experimental assays confirmed that these residues modulate GAF domain conformation and cGMP sensitivity.

Conclusions:

• Distant coevolving residues play a crucial role in mediating GAF domain allosteric regulation.
• A mechanism involving L267 and F295 in GAF domain allostery was proposed based on computational and experimental data.
• These findings enhance the understanding of allosteric mechanisms in proteins, particularly GAF domains.