Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

Abstract

The crucial process of aminoacyl-tRNA delivery to the ribosome is energized by the GTPase reaction of the elongation factor Tu (EF-Tu). Advances in the elucidation of the structure of the EF-Tu/ribosome complex provide the rare opportunity of gaining a detailed understanding of the activation process of this system. Here, we use quantitative simulation approaches and reproduce the energetics of the GTPase reaction of EF-Tu with and without the ribosome and with several key mutants. Our study provides a novel insight into the activation process. It is found that the critical H84 residue is not likely to behave as a general base but rather contributes to an allosteric effect, which includes a major transition state stabilization by the electrostatic effect of the P loop and other regions of the protein. Our findings have general relevance to GTPase activation, including the processes that control signal transduction.enzymatic catalysis | preorganization | allostery T he elongation cycle of protein synthesis uses the elongation factor Tu (EF-Tu) with GTP to deliver aminoacyl-tRNA (aa-tRNA) to the mRNA-programmed ribosome. More specifically, the GTP-bound state of EF-Tu forms a high-affinity ternary complex with the aa-tRNA. Upon binding of the ternary complex to the ribosome, the aa-tRNA occupies the A site and, when the codon-anticodon interaction is cognate, the GTPase activity of the EF-Tu is increased significantly. The conformational change following GTP hydrolysis to GDP and a leaving phosphate group (P i ) leads to dissociation of EF-Tu from the ribosome and accommodation of the aa-tRNA on the A site for peptidyl transfer (see Fig. 1 for a schematic description; for additional details, see refs. 1-3).Breakthrough in the elucidation of the ribosome structure (2-13) and careful biochemical studies (1-3, 14-25) allow one to begin thinking about the nature of the mechanism of the elongation process. In particular, the elucidation of structure of the EF-Tu/ribosome complex (our study refers to the structure of EF-Tu in the complex as EF-Tu′) (9) opens the way for the exploration of the activation process at the molecular level. However, despite major biochemical and structural breakthroughs, a detailed explanation of how the codon recognition in the 30S subunit leads to GTP hydrolysis remains elusive. That is, although it is known that the precise positioning of H84 is critical for efficient catalysis (1,9,(11)(12)(13)(23)(24)(25)(26), the energetics of this positioning and its ultimate role in stabilizing the transition state (TS) are unclear. More precisely, it is frequently assumed that the conserved H84 is moved to a catalytic configuration and then serves as a general base, but this assumption is problematic (see ref. 1 and Reproducing the Overall Catalytic and Mutational Effects below) and has similar pitfalls as in the highly related case of the Ras-RasGAP (RasGAP) system. That is, where it was originally assumed that Q61 in the RasGAP system serves as a general base, but this assumption has been shown to ...

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

“…This mechanistic model is also compatible with recent in silico approaches that predict the catalytic histidine (His84 of Escherichia coli EF-Tu) to be protonated and to be part of an allosteric repositioning of active site residues (38). The authors suggest that for such an allosteric model, the electrostatic interaction between the positively charged histidine and the negatively charged phosphate oxygen at A2662 is important, and they predict that the removal of the latter charge would render GTP hydrolysis pH dependent.…”

“…It has been shown before that GTP hydrolysis by EF-Tu is pH independent (in the range between pH 6.5 and 8.5) and therefore a primary role of His84 (EF-Tu) as a general base in GTP hydrolysis was excluded (24). However, molecular simulation experiments lead to the suggestion that removal of the negative charge at the phosphate backbone at position A2662 would render the reaction pH dependent (38). Consequently, we decided to test ribosomes carrying the methylphosphonate in the S P configuration in activation of EF-G GTP hydrolysis at different pH values between 7.4 and 6.0.…”

Quantitative exploration of the molecular origin of the activation of GTPase

“…26) that the activation involves a histidine (His-84) as a base mechanism. On the other hand, our study that considered the structures of both the active ribosome-bound (EF-Tu′) and inactive (EF-Tu) forms (29) concluded that the His-84 cannot act as a base. We also concluded, using the 1W surfaces, that the direct effect of His-84 on the TS energy is only around 2 kcal/mol and that its effect is thus allosteric.…”

“…The calculations with the nonpolar (NP) His-84 involved significant instability, due in part to the fact (29,30) that the NP His prefers to be in the EF-Tu structure. Using constraints to keep the NP His in the EF-Tu′ structure gave different results with different constraints, with an increase in the barrier between 10.0 and −2.0 kcal/mol, depending on the constraint and the initial conditions.…”

Energetics of activation of GTP hydrolysis on the ribosome

“…Process (1) thus corresponds to His84 acting as a general base as proposed by Voorhees et al 8 , while (2) and (3) denote proton transfer to the phosphate group in a substrateassisted mechanism 9,17 in presence of either the ionized or the neutral forms of His84, respectively. The last process (4) does not involve any proton transfer but represents expulsion of metaphosphate from GTP as the limiting case of a fully dissociative path.…”