High-resolution structure of RGS17 suggests a role for Ca2+ in promoting the GTPase-activating protein activity by RZ subfamily members

Abstract

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein–coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gαo, Gαz, and Gαi1–3 proteins in the nervous system and thereby inhibit downstream pathways, including those involved in Ca2+-dependent signaling. In contrast to other RGS proteins, little is known about RZ subfamily structure and regulation. Herein, we present the 1.5-Å crystal structure of RGS17, the most complete and highest-resolution structure of an RZ subfamily member to date. RGS17 cocrystallized with Ca2+ bound to conserved positions on the predicted Gα-binding surface of the protein. Using NMR chemical shift perturbations, we confirmed that Ca2+ binds in solution to the same site. Furthermore, RGS17 had greater than 55-fold higher affinity for Ca2+ than for Mg2+. Finally, we found that Ca2+ promotes interactions between RGS17 and activated Gα and decreases the Km for GTP hydrolysis, potentially by altering the binding mechanism between these proteins. Taken together, these findings suggest that Ca2+ positively regulates RGS17, which may represent a general mechanism by which increased Ca2+ concentration promotes the GAP activity of the RZ subfamily, leading to RZ-mediated inhibition of Ca2+ signaling.

Fragment‐Based Nuclear Magnetic Resonance Screen against a Regulator of G Protein Signaling Identifies a Binding “Hot Spot”

“…The fragments that do not make contacts with one or more of these four residues could be grown with the intention of reaching these residues and improving their inhibition of the RGS17‐Gα protein‐protein interaction. Evidence to support our findings comes from a recent study that produced the highest resolution crystal structure of RGS17 and identified multiple Ca 2+ binding sites on RGS17 (PDB ID: 6AM3) [33] . Interestingly, one of the identified Ca 2+ binding sites consisted of Y106 and E109, which is near the binding site of fragments 5 – 7 .…”

“…Evidence to support our findings comes from a recent study that produced the highest resolution crystal structure of RGS17 and identified multiple Ca 2 + binding sites on RGS17 (PDB ID: 6AM3). [33] Interestingly, one of the identified Ca 2 + binding sites consisted of Y106 and E109, which is near the binding site of fragments 5-7. The study found that Ca 2 + promoted the RGS17-Gα interaction and improved the catalytic efficiency of GTP-hydrolysis by Gα bound to RGS17.…”

Regulator of G-protein signaling (RGS) proteins as drug targets: Progress and future potentials

“…Another way to achieve selectivity for small molecules is to elucidate RGS structural information at the atomic level. For example, a recent report by Seing et al presented a 1.5 Å crystal structure of RGS17, the highest-resolution crystal structure for an RZ family member to date, that revealed novel Ca 2ϩ -binding sites (119). Expanding upon this initial discovery, Seing et al went on to discover that Ca 2ϩ binding positively regulates RGS17 GAP activity.…”