Hydrogen-Bonding Dynamics between Adjacent Blades in G-Protein β-Subunit Regulates GIRK Channel Activation

Abstract

Functionally critical domains in the βγ-subunits of the G-protein (G βγ) do not undergo large structural rearrangements upon binding to other proteins. Here we show that a region containing Ser 67 and Asp 323 of G βγ is a critical determinant of G-protein-gated inwardly rectifying K + (GIRK) channel activation and undergoes only small structural changes upon mutation of these residues. Using an interactive experimental and computational approach, we show that mutants that form a hydrogen-bond between positions 67 and 323 do not activate a GIRK channel. We also show that in the absence of hydrogen-bonding between these positions, other factors, such as the displacement of the crucial G γ residues Pro 60 and Phe 61, can impair G βγ-mediated GIRK channel activation. Our results imply that the dynamic nature of the hydrogen-bonding pattern in the wild-type serves an important functional role that regulates GIRK channel activation by G βγ and that subtle changes in the flexibility of critical domains could have substantial functional consequences. Our results further strengthen the notion that the dynamic regulation of multiple interactions between G βγ and effectors provides for a complex regulatory process in cellular functions.