Role of the arginine finger in Ras.RasGAP revealed by QM/MM calculations.

In the Ras.Ras.GAP complex, hydrolysis of guanosine triphosphate is strongly accelerated GAP as compared to Ras alone. This is largely attributed to the arginine finger R789(GAP) pointing to AlF(x) in the transition state analogue. We performed QM/MM simulations where triphosphate was treated using the quantum mechanical method of density functional theory, while the protein complex and water environment were described classically using MD. Compared to Ras, the crucial electron shift, bond stretching and distortion towards an eclipsed gamma-to-beta orientation are much more pronounced. The arginine finger is shown to act by displacing water out of the binding niche. The resulting enhanced electrostatic field catalyses the cleavage step.

Empirical rules facilitate the search for binding sites on protein surfaces.

Computational surface screening of 3D protein structures is a valuable means of finding possible docking sites for substrates, effectors and similar molecules. It can be improved by considering properties of molecules which are known to bind to protein surfaces, and thus reflect the required properties of binding sites. In-depth studies are available on drugs and lead compounds as binding partners with statistically assured properties. Here we present a simple strategy for finding binding sites, which is based on the empirical rule-of-five by Lipinski et al. for oral drugs and the rule-of-three by Congreve et al. for leads. The fast automated search with the new C-code TRIDOCK yields a preliminary set of sites, thus facilitating further investigation by visual, comparative and quantitative work. Possible binding sites are tagged by pseudo-atoms added to the structure file for molecular graphical evaluation. Usually, the strategy yields not just a few single sites, but an accumulation of several sites in known substrate binding pockets. Clusters are also found at known or putative protein-protein docking interfaces. A comparison of the activated and inactivated form of the GTPase Ras reveals clear differences and identifies a niche, which is possibly a suitable new target for compounds that bind specifically to activated Ras.