A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in G alpha(i1)

作者：Thaker Tarjani M; Sarwar Maruf; Preininger Anita M; Hamm Heidi E^*; Iverson T M

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2014, 289(16): 11331-11341.

DOI：10.1074/jbc.M113.539064

• Allosteric Regulation
• G Protein-coupled Receptors (GPCR)
• G Proteins
• Heterotrimeric G Proteins
• Rhodopsin
• Signal Transduction

Background: G proteins couple receptor binding to nucleotide release via an allosteric network. Results: Mutation of allosteric sites of G(i1) stabilizes a transient signaling conformation and may highlight an allosteric connection between receptor and nucleotide. Conclusion: The P-loop interacts with Switch I in the K345L variant of G(i1). Significance: G protein signaling is critical for numerous cellular functions, and GDP release is the rate-limiting step of the cycle.
Receptor-mediated activation of the G subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 . Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type G subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the G subunit. The observed differences in relative receptor affinity for intact G(i1) subunits versus C-terminal G(i1) peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L G(i1) variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type G(i1) subunit.

• 出版日期2014-4-18