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PMID: 19222191 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Helix dipole movement and conformational variability contribute to allosteric GDP release in Galphai subunits.

Biochemistry ·Vol. 48 ·No. 12 ·2009-03-31 ·Pages 2630-42

Preininger AM, Funk MA, Oldham WM, Meier SM, Johnston CA, Adhikary S, Kimple AJ, Siderovski DP, Hamm HE, Iverson TM

Abstract

Heterotrimeric G proteins (Galphabetagamma) transmit signals from activated G protein-coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal alpha5 helix of Galpha subunits participates in Galpha-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the alpha5 helix of the Galpha subunit [Oldham, W. M., et al. (2006) Nat. Struct. Mol. Biol. 13, 772-777]. On the basis of this result, an engineered disulfide bond was designed to constrain the alpha5 helix of Galpha(i1) into its EPR-measured receptor-associated conformation through the introduction of cysteines at position 56 in the alpha1 helix and position 333 in the alpha5 helix (I56C/Q333C Galpha(i1)). A functional mimetic of the EPR-measured alpha5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino terminus of this helix, D328R Galpha(i1). Both proteins exhibit a dramatically elevated level of basal nucleotide exchange. The 2.9 A resolution crystal structure of I56C/Q333C Galpha(i1) in complex with GDP-AlF(4)(-) reveals the shift of the alpha5 helix toward the guanine nucleotide binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Galpha(i1) subunit further revealed altered positions for the switch regions and throughout the Galpha(i1) subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the alpha5 helix dipole and overall conformational variability during nucleotide release.

MeSH Terms
Allosteric Site Binding Sites Crystallography, X-Ray GTP-Binding Protein alpha Subunits, Gi-Go/chemistry,metabolism Guanosine Diphosphate/chemistry,metabolism Kinetics Models, Molecular Protein Conformation Protein Subunits Spectrometry, Fluorescence
Chemicals
Protein Subunits Guanosine Diphosphate GTP-Binding Protein alpha Subunits, Gi-Go
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Preininger Anita M
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Funk Michael A
Oldham William M
Meier Scott M
Johnston Christopher A
Adhikary Suraj
Kimple Adam J
Siderovski David P
Hamm Heidi E
Iverson Tina M
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Article Info
Journal
Biochemistry
Abbr.
Biochemistry
ISSN
1520-4995
DOI
10.1021/bi801853a
Published
2009-03-31
Pages
2630-42
Language
English
Region
United States
NLM ID
0370623
PMCID
PMC2736342
Subset
IM
Grants
NEI NIH HHS · R21 EY018435 · United States
NIMH NIH HHS · F30 MH074266 · United States
NIGMS NIH HHS · R01 GM082892 · United States
NIGMS NIH HHS · GM082892 · United States
NEI NIH HHS · EY06062 · United States
NIGMS NIH HHS · R01 GM082892-01A1 · United States
NEI NIH HHS · R01 EY006062 · United States
NEI NIH HHS · R01 EY006062-23 · United States
NIMH NIH HHS · F30 MH074266-03 · United States
Databases
PDB
3D7M
Corrections
ErratumIn
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External Links
PubMed source DOI: 10.1021/bi801853a PMC Full Text View journal details
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