STRUCTURE OF THE GDP-PI COMPLEX OF GLY203-]G(I-ALPHA-1) - A MIMIC OF THE TERNARY PRODUCT COMPLEX OF G-ALPHA-CATALYZED GTP HYDROLYSIS

Background: G proteins play a vital role in transmembrane signalling e vents. In their inactive form G proteins exist as heterotrimers consis ting of an alpha subunit, complexed with GDP and a dimer of beta gamma subunits. Upon stimulation by receptors, G protein alpha subunits exc hange GDP for GTP and dissociate from beta gamma. Thus activated, alph a subunits stimulate or inhibit downstream effectors. The duration of the activated state corresponds to the single turnover rate of GTP hyd rolysis, which is typically in the range of seconds. In G(i alpha 1), the Gly203-->Ala mutation reduces the affinity of the substrate for Mg 2+, inhibits a key conformational step that occurs upon GTP binding an d consequently inhibits the release of beta gamma subunits from the GT P complex. The structure of the Gly203-->Ala mutant of G(i alpha 1) (G 203AG(i alpha 1)) bound to the slowly hydrolyzing analog of GTP (GTP g amma S) has been determined in order to elucidate the structural chang es that take place during hydrolysis. Results: We have determined the three dimensional structure of a Gly203-->Ala mutant of G(i alpha 1) a t 2.6 Angstrom resolution. Although crystals were grown in the presenc e of GTP gamma S and Mg2+, the catalytic site contains a molecule of G DP and a phosphate ion, but no Mg2+, The phosphate ion is bound to a s ite near that occupied by the gamma-phosphate of GTP gamma S in the ac tivated wild-type alpha subunit. A region of the protein, termed the S witch II helix, twists and bends to adopt a conformation that is radic ally different from that observed in other G(i alpha 1) subunit comple xes. Conclusions: Under the conditions of crystallization, the Gly203- ->Ala mutation appears to stabilize a conformation that may be similar , although perhaps not identical, to the transient ternary product com plex of G(i alpha 1)-catalyzed GTP hydrolysis. The rearrangement of th e Switch II helix avoids a potential steric conflict caused by the mut ation. However, it appears that dissociation of the gamma-phosphate fr om the pentacoordinate intermediate also requires a conformational cha nge in Switch II. Thus, a conformational rearrangement of the Switch I I helix may be required in G alpha-catalyzed GTP hydrolysis. (C) Curre nt Biology Ltd