An intramolecular contact in Galpha transducin that participates in maintaining its intrinsic GDP release rate.
Receptor mediated stimulation of the G protein-alpha subunit leads to exchange of GDP for GTP, activating the protein. Spontaneous GDP release from Galpha can also lead to the active state, if GTP in solution binds the nucleotide binding pocket. The purpose of this study is to evaluate the molecular determinants for maintaining the spontaneous GDP release rates between two Galpha subunits. Galpha(t) has a low rate of nucleotide release, compared to Galpha(i1). Galpha(t/i1) chimeras were used to explore the molecular basis for this behavior. The C-terminal alpha4-helix, the N-terminal 56 residues and the Switch I/II regions of Galpha(t) were shown to affect the low spontaneous GDP release rate in Galpha(t). A specific molecular contact between Asp26 and Asn191 was found in Galpha(t) that is not present in Galpha(i1). In two chimeras disrupting this interaction produced an increased spontaneous GDP release; restoring the contact present in Galpha(t) into these chimeras decreased the GDP release rate by half as compared to the original chimeras. Similarly, introduction of this contact in wild-type Galpha(i1) decreased the GDP release rate of Galpha(i1) by half. Differences in GDP release rates may reflect physiological roles these proteins play in living systems.
Thomas
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Institute for Neuroscience, Northwestern University, Chicago, Illinois, USA.
Bae
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Medkova
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Hamm
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D002678:Chimera / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D005453:Fluorescence; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D015293:Transducin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D006153:Guanosine Diphosphate; D015293:Transducin