Interdomain Interactions Regulate GDP Release from Heterotrimeric G Proteins

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Abstract
The role of interdomain contact sites in basal GDP release from heterotrimeric G proteins is unknown. Gαo and Gαi1 display a 5-fold difference in the rate of GDP dissociation with half-times of 2.3 ± 0.2 and 10.4 ± 1.3 min, respectively. To identify molecular determinants of the GDP release rate, we evaluated the rate of binding of the fluorescent guanine nucleotide 2‘(3‘)-O-(N-methyl-3‘-anthraniloyl)guanosine 5‘-O-(3-thiotriphosphate) (mGTPγS) to chimers of Gαo and Gαi1. Although no one region of the G protein determined the GDP dissociation rate, when the C-terminal 123 amino acids in Gαi1 were replaced with those of Gαo, the GDP release rate increased 3.3-fold compared to that of wild-type Gαi1. Within the C-terminal portion, modification of four amino acids in a coil between β4 and the α3 helix resulted in GDP release kinetics identical to those of wild-type Gαo. Based on the Gαi1-GDP crystal structure of this region, Leu232 appeared to form a hydrophobic contact with Arg144 of the helical domain. The role of this interaction was confirmed by Gαi1 L232Q and Gαi1 R144A which displayed 2−5-fold faster GDP release rates compared to wild-type Gαi1 (t1/2 4.7 and 1.5 min, respectively), suggesting that interdomain bridging contacts partially determine the basal rate of GDP release from heterotrimeric G proteins.