The function of interdomain interactions in controlling nucleotide exchange rates in transducin.
The intramolecular contacts in heterotrimeric G proteins that determine the rates of basal and receptor-stimulated nucleotide exchange are not fully understood. The alpha subunit of heterotrimeric G proteins consists of two domains: a Ras-like domain with structural homology to the monomeric G protein Ras and a helical domain comprised of six alpha-helices. The bound nucleotide lies in a deep cleft between the two domains. Exchange of the bound nucleotide may involve opening of this cleft. Thus interactions between the domains may affect the rate of nucleotide exchange in G proteins. We have tested this hypothesis in the alpha subunit of the rod cell G protein transducin (Galpha(t)). Site-directed mutations were prepared in a series of residues located at the interdomain interface. The proteins were expressed in vitro in a reticulocyte lysate system. The rates of basal and rhodopsin-catalyzed nucleotide exchange were determined using a trypsin digestion assay specifically adapted for kinetic measurements. Charge-altering substitutions of two residues at the interdomain interface, Lys(273) and Lys(276), increased basal nucleotide exchange rates modestly (5-10-fold). However, we found no evidence that interactions spanning the two domains in Galpha(t) significantly affected either basal or rhodopsin-catalyzed nucleotide exchange rates. These results suggest that opening of the interdomain cleft is not an energetic barrier to nucleotide exchange in Galpha(t). Experiments with Galpha(i1) suggest by comparison that the organization and function of the interdomain region differ among various G protein subtypes.
Marin
E P
EP
Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10021, USA.
Krishna
A G
AG
Archambault
V
V
Simuni
E
E
Fu
W Y
WY
Sakmar
T P
TP
D000818:Animals; D002417:Cattle; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000378:metabolism; D015293:Transducin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
D020662:Guanine Nucleotide Exchange Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D014357:Trypsin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D015293:Transducin; D008239:Lysine