The crystal structure of the H‐ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R‐factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61‐65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma‐phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in‐line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.

v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form P21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p2lv, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio-and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDPaS and GTPaS. It has low specificity for the Sp isomer of GTP,BS. Together with the data for GDP,BS and GTP-yS these results are compared with those obtained for elongation factor (EF)-Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.

Analysis of three different Rab-RabGEF pairs reveals that RabGEFs contain the minimal targeting machinery for recruiting Rabs to specific membranes.