David E. Coleman

The structure of the G protein heterotrimer Giα1β1γ2

The crystallographic structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma. The major alpha/beta interface covers switch II of alpha, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with alpha-GDP) explain stabilization of the inactive conformation of alpha by beta gamma. Repeated WD motifs in beta form a circularized sevenfold beta propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.

Tertiary and quaternary structural changes in Giα1 induced by GTP hydrolysis

Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein α subunit, Giα1. The switch II and switch III segments become disordered, and linker II connecting the Ras and α helical domains moves, thus altering the structures of potential effector and βγ binding regions. Contacts between the α-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and βγ binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring α subunits in the lattice, suggesting that multimers of α subunits or heterotrimers may play a role in signal transduction.

INVASION OF THE NUCLEOTIDE SNATCHERS : STRUCTURAL INSIGHTS INTO THE MECHANISM OF G PROTEIN GEFS

Although the mechanics differ, GEFs appear to deform their substrates in similar ways. In all cases, switch I is pulled away from switch II and the P loop, exposing the active site; dislocation of switch I also displaces a Mg2+-binding residue, but this in itself should have little affect on GDP affinity. GEFs also restructure and displace the amino terminus of switch II and consequently dismantle the γ-phosphate-binding site, at the same time moving a conserved aspartate residue that serves as a water-mediated Mg2+ ligand. Residues, either from the GEF or the G protein, are positioned to occupy and block the phosphate- and Mg2+-binding sites. To varying extents, the P loop is altered and a conserved lysine residue is extracted from the phosphate-binding site. While structural changes in the P loop may occur as a response to the evacuation of the nucleotide-binding site, it appears, especially in the case of EF-Ts, that it is GEF mediated. These rearrangements, however, leave the purine-binding site relatively undisturbed and easily accessed. Hence, incoming nucleotide can gain some purchase onto the GTP-binding site and complete the exchange reaction. Although the dissociation constants for G·GTP, G·GDP, and G·GEF are subnanomolar, that of the ternary complex with either nucleotide (GEF·G protein·GXP) is micromolar (Klebe et al. 1995xKlebe, C., Prinz, H., Wittinghofer, A., and Goody, R.S. Biochemistry. 1995; 34: 12543–12552Crossref | PubMedSee all ReferencesKlebe et al. 1995). The ternary complex is therefore a viable, yet transient intermediate in the pathway of nucleotide exchange in vivo. The delicate energetic balance between binary and ternary complexes is perhaps controlled by the relative concentrations of the two nucleotides. Clearly, structural changes, which have yet to be characterized, must attend the formation of the ternary complex. It is possible that other GEFs, in particular heterotrimeric G protein receptors, employ exchange mechanisms that differ substantially from those discussed here. Nevertheless, there is sufficient diversity among these to offer new insights into both common and unique properties of GEFs, each of which appears to have arisen independently in evolution, and each of which must catalyze nucleotide exchange in a different functional context.

Structure of Giα1·GppNHp, Autoinhibition in a Gα Protein-Substrate Complex

The structure of the G protein Giα1 complexed with the nonhydrolyzable GTP analog guanosine-5′-(βγ-imino)triphosphate (GppNHp) has been determined at a resolution of 1.5 Å. In the active site of Giα1·GppNHp, a water molecule is hydrogen bonded to the side chain of Glu43 and to an oxygen atom of the γ-phosphate group. The side chain of the essential catalytic residue Gln204 assumes a conformation which is distinctly different from that observed in complexes with either guanosine 5′-O-3-thiotriphosphate or the transition state analog GDP·AlF4 −. Hydrogen bonding and steric interactions position Gln204 such that it interacts with a presumptive nucleophilic water molecule, but cannot interact with the pentacoordinate transition state. Gln204 must be released from this auto-inhibited state to participate in catalysis. RGS proteins may accelerate the rate of GTP hydrolysis by G protein α subunits, in part, by inserting an amino acid side chain into the site occupied by Gln204, thereby destabilizing the auto-inhibited state of Gα.

Crystallographic Studies on Ascaris suum NAD-Malic Enzyme Bound to Reduced Cofactor and Identification of an Effector Site

The crystal structure of the mitochondrial NAD-malic enzyme from Ascaris suum, in a quaternary complex with NADH, tartronate, and magnesium has been determined to 2.0-Å resolution. The structure closely resembles the previously determined structure of the same enzyme in binary complex with NAD. However, a significant difference is observed within the coenzyme-binding pocket of the active site with the nicotinamide ring of NADH molecule rotating by 198° over the C-1–N-1 bond into the active site without causing significant movement of the other catalytic residues. The implications of this conformational change in the nicotinamide ring to the catalytic mechanism are discussed. The structure also reveals a binding pocket for the divalent metal ion in the active site and a binding site for tartronate located in a highly positively charged environment within the subunit interface that is distinct from the active site. The tartronate binding site, presumably an allosteric site for the activator fumarate, shows striking similarities and differences with the activator site of the human NAD-malic enzyme that has been reported recently. Thus, the structure provides additional insights into the catalytic as well as the allosteric mechanisms of the enzyme.