G PROTEIN MECHANISMS: Insights from Structural Analysis

Abstract

This review is concerned with the structures and mechanisms of a superfamily of regulatory GTP hydrolases (G proteins). G proteins include Ras and its close homologs, translation elongation factors, and heterotrimeric G proteins. These proteins share a common structural core, exemplified by that of p21 ras (Ras), and significant sequence identity, suggesting a common evolutionary origin. Threedimensional structures of members of the G protein superfamily are considered in light of other biochemical findings about the function of these proteins. Relationships among G protein structures are discussed, and factors contributing to their low intrinsic rate of GTP hydrolysis are considered. Comparison of GTP-and GDP-bound conformations of G proteins reveals how specific contacts between the γ-phosphate of GTP and the switch II region stabilize potential effectorbinding sites and how GTP hydrolysis results in collapse (or reordering) of these surfaces. A GTPase-activating protein probably binds to and stabilizes the conformation of its cognate G protein that recognizes the transition state for hydrolysis, and may insert a catalytic residue into the G protein active site. Inhibitors of nucleotide release, such as the βγ subunit of a heterotrimeric G protein, bind selectively to and stabilize the GDP-bound state. Release factors, such as the translation elongation factor, Ts, also recognize the switch regions and destabilize the Mg 2+-binding site, thereby promoting GDP release. G protein-coupled receptors are expected to operate by a somewhat different mechanism, given that the GDP-bound form of many G protein α subunits does not contain bound Mg 2+ .

Kinesin motors as molecular machines

“…The mutants alter highly conserved or invariant residues of switch I and switch II, (48) elements of the kinesin motors that are structurally homologous to G protein regions that move or change in conformation upon nucleotide binding or exchange. (49) The mutants show basal ATPase activity, but activation of the motor ATPase by microtubules, which is essential for movement of the motors along microtubules, is completely blocked. (50) The two mutated residues interact with one another in wild-type motors, forming a salt bridge between the switch I and switch II regions of the motor.…”

Inactivation of Gi proteins induces an antidepressant-like effect in the mouse forced-swimming test

“…As a matter of fact, even if no clear alteration at protein or mRNA Gi protein level emerged, an hyperfunctionality of this transduction mechanism has been evidenced. Gi-proteins represent the most widespread modulatory signalling pathway in neurones (Holtz et al, 1986) and one of their principal effects is the inhibition of adenylate cyclase activity (Sprang, 1997). A significant elevation in hydrolysisresistant photoaffinity GTP analog (AAGTP) binding to Gi/o and a decrease in the ratio Gs/Gi AAGTP incorporation was seen in parietal and temporal cortices of unipolar depressive patients (Ozawa et al, 1993).…”

“…Interest in G-proteins comes from the fact that abnormalities in their function and expression have been associated with various human diseases and pathophysiological states (Spiegel, 1995). Gproteins are a ubiquitous family of proteins that play a crucial role in transducing extracellular signals to cellular targets, thus transmitting messages from cell surface receptors to cellular effectors including adenylate cyclase, phospholipase C and ion channels (Sprang, 1997). G-proteins are heterotrimeric molecules com-posed of three different subunits termed α, β and γ.…”

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…The mechanism by which ligand activated GPCRs catalyze the exchange of GDP for GTP is not clear [3]. It is however believed to involve three flexible regions designated switch I, switch II, and switch III on the α subunit that have been shown to adopt different conformations in the presence of GDP, GTPγS, and other nucleotide adducts and analogs [41,42]. Because the switch regions are at least 30Å away from the cytoplasmic peptide loops of the receptor, it has been proposed that the receptor must achieve the catalytic feat by means of "action at a distance" [43].…”

“…The suggested mechanism predicts that the ligand activated receptor effects the release of GDP by using the Gβγ-subunit to directly ("the lever arm") [43] or indirectly ("the gear shift") [36] distort the α-subunit switch regions (I and II) and allow nucleotide release and recognition [34]. Another hypothesis suggests that the activated receptor may use a "latch" on the surface of the Gα-subunit and thus prompt GDP release [37,42].…”