Investigating the roles of important amino acid residues in GTPase activity of Elongation Factor-Tu (EF-Tu)

Abstract

A large number of guanosine triphosphate (GTP)/ guanosine diphosphate (GDP) binding proteins (G-proteins), which usually have intrinsic (and/or stimulated) GTPase activity, are involved in a wide range of cellular regulatory and signal transduction processes. G-proteins generally exist in an active form when bound to GTP and become inactive when GTP is transformed to GDP through a hydrolysis reaction. The longer a GTP-binding protein remains in its active GTP-bound state, the longer it will transmit and amplify a certain signal. The mechanism of GTP hydrolysis is still largely unknown and of great importance to the understanding of the role of specific residues, which may lead to new and novel therapies for cancer. Experimental studies indicated the significance of some amino acid residues, e.g. His85, Asp87, Asp51, Thr62, Val20 and Ile61, for structural properties and GTPase activity of Elongation Factor-Tu (EF-Tu). In our study we aimed the comprehensive explanations of the roles of these amino acids. Our results indicated that, Switch 1 area is very unstable and dynamic when introduced into the solvent medium. Early conformational changes of this region in our simulations are showing the flexibility of the Switch 1 area. Thr62 was determined as the key factor for holding the position of Switch 1 region seen in the crystal structures coming from Thermus aquaticus and Escherichia coli species. On the other hand, Asp51 had no contribution to the position of this region. Previously, it was proposed that His85 stabilizes GTP hydrolysis transition state by orienting the attacking water molecule, especially in the ribosome induced mechanism. In our study two steps were determined for His85 activity: turning into the active site and to have a stable conformation in this site. We proposed that following factors can help His85 rotation into the active site: i) Protonation state of itself e.g. if it bears positive charge it has a tendency to stay in the active site more than the neutral form. ii) Other amino acids can help its stay in the active site e.g. His85 spent more time in the active site in the EF-Tu[D87E] mutant simulation. iii) Ribosome, probably, may bring and stabilize His85 into active site. Our findings suggested the stabilization of His85 in the active site could be achieved by Val20 and Ile61 which were previously called as the “Hydrophobic Gate” residues. These residues were thought as the barrier for His85 rotation into the active site. However, our results indicated that the mission of Val20 and Ile61 is not forming a barrier for His85 rotation, but assisting the hydrolysis when it turns into the active site by holding the His85 imidazole ring, preventing its rotation, like a clamp. This stabilization probably mostly happens in the presence of ribosome.