Interdomain interactions regulate GDP release from heterotrimeric G proteins.
The role of interdomain contact sites in basal GDP release from heterotrimeric G proteins is unknown. G(alpha)(o) and G(alpha)(i1) 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) (mGTPgammaS) to chimers of G(alpha)(o) and G(alpha)(i1). Although no one region of the G protein determined the GDP dissociation rate, when the C-terminal 123 amino acids in G(alpha)(i1) were replaced with those of G(alpha)(o), the GDP release rate increased 3.3-fold compared to that of wild-type G(alpha)(i1). Within the C-terminal portion, modification of four amino acids in a coil between beta4 and the alpha3 helix resulted in GDP release kinetics identical to those of wild-type G(alpha)(o). Based on the G(alpha)(i1)-GDP crystal structure of this region, Leu(232) appeared to form a hydrophobic contact with Arg(144) of the helical domain. The role of this interaction was confirmed by G(alpha)(i1) L232Q and G(alpha)(i1) R144A which displayed 2-5-fold faster GDP release rates compared to wild-type G(alpha)(i1) (t(1/2) 4.7 and 1.5 min, respectively), suggesting that interdomain bridging contacts partially determine the basal rate of GDP release from heterotrimeric G proteins.
Remmers
A E
AE
Department of Pharmacology, The University of Michigan, Ann Arbor 48109, USA.
Engel
C
C
Liu
M
M
Neubig
R R
RR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D005456:Fluorescent Dyes / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism
D005456:Fluorescent Dyes; C111797:N-methyl-3'-O-anthranoyl guanosine 5'-O-(3-thiotriphosphate); D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D062367:ortho-Aminobenzoates; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins