A Transient Interaction Between the P-loop and Switch I Contributes to the Allosteric Network Between Receptor and Nucleotide in G{alpha}i1 [Protein Structure and Folding]

March 4th, 2014 by Thaker, T. M., Sarwar, M., Preininger, A. M., Hamm, H. E., Iverson, T. M.

Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor-binding and the guanine nucleotide-binding sites, which are separated by over 30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα 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 transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 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 meta II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared to wild-type, but exhibits similar stability in the GTPγS-bound state. High-resolution X-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, a new conformation for Switch I was stabilized by an ionic interaction with the P-loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the P-loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi subunit.
  • Posted in Journal of Biological Chemistry, Publications
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