Cosolvent Molecular Dynamics Simulation‐Based Discovery of Potential Allosteric Sites on Regulator of G Protein Signaling 4

Abstract

Regulator of G Protein Signaling 4 (RGS4) is member of a family of intracellular proteins that are characterized by the presence of a 125 amino‐acid RGS homology domain (RH) which binds to the Ga subunit of heterotrimeric G proteins. By doing so RGS4 acts as a GTPase‐accelerating protein or GAP to expedite the conversion of the GTP‐bound active form of Gα subunit of heterotrimeric G proteins to the GDP‐bound inactive form, thereby terminating signaling downstream of G protein coupled receptors (GPCRs). RGS4 is expressed widely in both the periphery and central nervous system and has been implicated in pain, schizophrenia, and the control of cardiac contractility. Inhibitors of RGS4 have been developed, but these all bind covalently to a cysteine (Cys95) in the RH domain. Therefore, we sought to identify alternative druggable sites on this protein using cosolvent molecular dynamics simulations. This technique employs a low concentration of organic probes (pyrimidine, acetonitrile, isopropyl alcohol) to find druggable “hot spots” on a protein based on the binding preference of the probes. This methodology has the advantage of utilizing protein dynamics to find druggable pockets that would be otherwise inaccessible with a single snapshot crystal structure. The top predicted sites were located in all areas of the RH domain of RGS4. Subsequently, statistical coupling analysis, a technique that identifies co‐evolving residues from analysis of multiple sequence alignments, was used to map out a functionally‐significant sector on the surface of RGS4 which overlapped with the identified hot spots, allowing for prioritization. The first site identified was expectedly the Gα subunit binding site. The second was located in a cleft in the proximity of lysine residues thought to be the binding site for phosphatidylinositol (3,4,5)‐trisphosphate (PIP3) binding. Since PIP3 is a known inhibitor of the GAP activity of RGS4, future directions entail the investigation of PIP3‐bound conformations of RGS4 by molecular dynamics simulation to better understand the mechanism of deactivation. Of other predicted sites, one is in close proximity to the purported allosteric site that contains Cys95, while the others are putative novel sites for allosteric control of RGS4.Support or Funding InformationSupported by R01 DA‐035316.