A M Berghuis1, E Lee, A S Raw, A G Gilman, S R Sprang. 1. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9050, USA. sprang@howie.swmed.edu
Abstract
BACKGROUND: G proteins play a vital role in transmembrane signalling events. In their inactive form G proteins exist as heterotrimers consisting of an alpha subunit, complexed with GDP and a dimer of betagamma subunits. Upon stimulation by receptors, G protein alpha subunits exchange GDP for GTP and dissociate from betagamma . Thus activated, alphasubunits stimulate or inhibit downstream effectors. The duration of the activated state corresponds to the single turnover rate of GTP hydrolysis, which is typically in the range of seconds. In Gialpha1, the Gly203-->Ala mutation reduces the affinity of the substrate for Mg2+, inhibits a key conformational step that occurs upon GTP binding and consequently inhibits the release of betagamma subunits from the GTP complex. The structure of the Gly203-->Ala mutant of Gialpha1 (G203AGialpha1) bound to the slowly hydrolyzing analog of GTP (GTPgammaS) has been determined in order to elucidate the structural changes that take place during hydrolysis. RESULTS: We have determined the three dimensional structure of a Gly203-->Ala mutant of Gialpha1 at 2.6 A resolution. Although crystals were grown in the presence of GTPgammaS and Mg2+, the catalytic site contains a molecule of GDP and a phosphate ion, but no Mg2+. The phosphate ion is bound to a site near that occupied by the gamma-phosphate of GTPgammaS in the activated wild-type alpha subunit. A region of the protein, termed the Switch II helix, twists and bends to adopt a conformation that is radically different from that observed in other Gialpha1 subunit complexes. 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 complex of Gialpha1-catalyzed GTP hydrolysis. The rearrangement of the Switch II helix avoids a potential steric conflict caused by the mutation. However, it appears that dissociation of the gamma-phosphate from the pentacoordinate intermediate also requires a conformational change in Switch II. Thus, a conformational rearrangement of the Switch II helix may be required in Galpha-catalyzed GTP hydrolysis.
BACKGROUND: G proteins play a vital role in transmembrane signalling events. In their inactive form G proteins exist as heterotrimers consisting of an alpha subunit, complexed with GDP and a dimer of betagamma subunits. Upon stimulation by receptors, G protein alpha subunits exchange GDP for GTP and dissociate from betagamma . Thus activated, alphasubunits stimulate or inhibit downstream effectors. The duration of the activated state corresponds to the single turnover rate of GTP hydrolysis, which is typically in the range of seconds. In Gialpha1, the Gly203-->Ala mutation reduces the affinity of the substrate for Mg2+, inhibits a key conformational step that occurs upon GTP binding and consequently inhibits the release of betagamma subunits from the GTP complex. The structure of the Gly203-->Ala mutant of Gialpha1 (G203AGialpha1) bound to the slowly hydrolyzing analog of GTP (GTPgammaS) has been determined in order to elucidate the structural changes that take place during hydrolysis. RESULTS: We have determined the three dimensional structure of a Gly203-->Ala mutant of Gialpha1 at 2.6 A resolution. Although crystals were grown in the presence of GTPgammaS and Mg2+, the catalytic site contains a molecule of GDP and a phosphate ion, but no Mg2+. The phosphate ion is bound to a site near that occupied by the gamma-phosphate of GTPgammaS in the activated wild-type alpha subunit. A region of the protein, termed the Switch II helix, twists and bends to adopt a conformation that is radically different from that observed in other Gialpha1 subunit complexes. 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 complex of Gialpha1-catalyzed GTP hydrolysis. The rearrangement of the Switch II helix avoids a potential steric conflict caused by the mutation. However, it appears that dissociation of the gamma-phosphate from the pentacoordinate intermediate also requires a conformational change in Switch II. Thus, a conformational rearrangement of the Switch II helix may be required in Galpha-catalyzed GTP hydrolysis.
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