An intramolecular binding site for the myristoylated amino‐terminus of Gαi

Abstract

GPCRs act as guanine exchange factors for G‐proteins, catalyzing nucleotide release. The amino terminus of Gαi subunits is subject to reversible palmitoylation, as well as myristoylation, which is a permanent, co‐translational modification. Previous work has demonstrated that unmyristoylated Gαi proteins exhibit a disordered amino terminus in the absence of Gβγ, while myristoylated amino terminal residues are ordered even in the absence of Gβγ [Medkova, M. (2002) Biochemistry 41, 9963–9972; Preininger, A.M. (2003) Biochemistry 42, 7931–7941], suggesting an intramolecular binding site for the amino terminus of myristoylated Gαi subunits. In order to determine the location of this binding site, we utilized a combination of biophysical, biochemical and crystallographic methods. Using site‐directed‐mutagenesis combined with quenching studies, the extreme amino terminus undergoes activation‐dependent quenching by tryptophan residues within 15 Å of the extreme amino terminus, and this quenching occurs on the same timescale as that seen for nucleotide exchange. A crystal structure of a myristoylated Gαi protein demonstrates that myristoylation alters the conformation of Trp 258 (between the α‐3 helix and the β‐5 strand in the GTPase domain of Gαi proteins), and implicates this region as a participant in the intramolecular binding of the myristoylated amino terminus of Gαi proteins.This work was supported by the National Institutes of Health (Grant 2R01 EY06062‐22A2).