title: A transient interaction between the phosphate binding loop and switch I contributes to the allosteric network between receptor and nucleotide in Gαi1.
abstract: Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >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 the 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 metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but 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, 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 Gαi1 subunit.
authors.affiliation: Department of Biochemistry and Vanderbilt University Medical Center, Nashville, Tennessee 37232.
authors.forename: Tarjani M
authors.initials: TM
authors.lastname: Thaker
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
authors.forename: Maruf
authors.initials: M
authors.lastname: Sarwar
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
authors.forename: Anita M
authors.initials: AM
authors.lastname: Preininger
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232. Electronic address: Heidi.hamm@vanderbilt.edu.
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
authors.affiliation: Department of Biochemistry and Vanderbilt University Medical Center, Nashville, Tennessee 37232; Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232. Electronic address: tina.iverson@vanderbilt.edu.
authors.forename: T M
authors.initials: TM
authors.lastname: Iverson
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D051381:Rats
chemical_list: C522851:Gnai1 protein, rat; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: Allosteric Regulation; G Protein-coupled Receptors (GPCR); G Proteins; Heterotrimeric G Proteins; Rhodopsin; Signal Transduction