Structural insights into peptide bond formation

Abstract

Perhaps the first evolutionary event in the emergence of the “protein world” from the “RNA world” was the appearance of an enzyme capable of catalyzing peptide bond formation. The peptidyl transferase center of the large ribosomal subunit, where peptide bond synthesis occurs (1–4), has two major components: an A site, which interacts with the CCA end of aminoacylated tRNAs, and a P site, where the CCA ends of peptidyl tRNAs are bound when peptide bonds form (5–7). The reaction it catalyzes is the nucleophilic attack of the α amino group of an A site-bound aminoacyl tRNA on the carbonyl carbon of the ester bond that links a nascent peptide to a tRNA in the P site. The question we wish to address is how the ribosome, an RNA–protein machine with roots in the “RNA world,” enhances the rate of peptide bond formation.

Crystal structures of the large ribosomal subunit of Haloarcula marismortui complexed with an analogue of peptide synthesis intermediate and an A site substrate analogue (8, 9) demonstrated that the peptidyl transferase center is composed entirely of RNA and confirmed that the CCA sequences of A and P site substrates interact with 23S rRNA in the manner deduced, in part, earlier from biochemical and genetic experiments (6, 7, 10, 11). The proximity of the N3 of A2486 (2451 in Escherichia coli) to the attacking α amino group and the analogue of the tetrahedral intermediate led to the suggestion that A2486 (2451) may function as a general acid/base during peptide bond formation. This hypothesis appeared to be supported by data on the pH dependence of its chemical reactivity and the finding that mutations of A2486 are dominant lethal in vivo (12). Further, the interaction between the N3 of A2486 (2451) and the phosphate oxygen of the reaction intermediate observed in the structure implied an altered pKa (8).

The hypothesis that A2486 (2451) is functioning as a general base has been tested most notably by its mutation to the three other nucleotides. Initial studies suggested that the impact of A2486 (2451) mutations on the rate of peptide bond formation is small, 10-fold or less (13–16). Furthermore, it is now clear that the chemical reactivity data that appeared to support the concept that A2486 (2451) acts as a general acid/base do not speak to its role in protein synthesis (17). However, more recent kinetic studies of 70S ribosomes that are greater than 90% active, done under conditions where the chemical step of peptide bond formation is likely to be rate limiting, demonstrate the existence of a titratable ribosomal component that affects catalysis and has a pKa of 7.4 (18). Its effects disappear when A2486 (2451) is mutated to U, and the rate of peptide bond formation catalyzed by the A2486U mutant ribosome is reduced by greater than 100-fold (18). These results could be explained either if A2486 (2451) acts as a general base or if there is an unknown pH-sensitive conformational change in the ribosome that depends on A2486 (2451).

Two concerns expressed about the relevance of these crystal structures to understanding the mechanism of peptide bond formation by the ribosome (19) have been shown to be unfounded (20). It has been argued that no inferences about the mechanism of peptide bond formation can be drawn from these crystal structures because large ribosomal subunits catalyze such reactions only in the presence of high concentrations of alcohol (21). Further, it has also been claimed that the ionic conditions in the crystals used to determine these structures were sufficiently far from physiological that the crystal structure is of limited functional relevance (19). However, the H. marismortui large subunits examined crystallographically are in fact highly active in peptide bond formation in the crystalline state, without the presence of alcohol, and manifest no sensitivity to the nature or concentration of salt (20). Furthermore, the structures obtained when peptidyl transferase substrates were diffused into preformed crystals under conditions that ensured a limited degree of reaction shows products, not substrates, bound to both the A and P sites (20). Thus, the crystal structures of the complexes previously published and those presented here are indeed relevant to the mechanism of peptide bond formation on the ribosome.

We have now determined the structure of a peptidyl-CCA bound to the P site of H. marismortui 50S ribosomal subunit and have refined the structures of its complexes with the substrates, products, and intermediates shown in Fig. ​Fig.1.1. When the structures of separately determined A and P site substrate complexes are placed together in one model, we observe that the attacking α amino group of the aminoacylated A site substrate forms hydrogen bonds with the N3 of A2486 (2451) and the 2′ OH of the A76 ribose of the P site substrate. The substrates are positioned for peptide bond formation by interactions that are entirely with the RNA component of the ribosome. From the present complex structures, it appears that the only candidates for chemical assistance in catalysis, if any occurs, are the N3 of A2486 (2451) and either the 2′ OH of the P site substrate or the 2′ OH of A2486 (2451). On the basis of this alignment, we expect that the oxyanion formed in the presumed tetrahedral carbon intermediate does not interact, as suggested (8), with A2486 (2451) but more likely points in the opposite direction. In the complexes of known structure, no ribosomal component is appropriately positioned to stabilize the oxyanion, although it cannot be excluded that U2620 (2585) moves to do so on formation of the intermediate.

Figure 1

Chemical structures of peptidyl transferase substrate analogues. (A) CCA-pcb is active as a P site substrate and binds to only the P site in the presence of the antibiotic, sparsomycin. (B) An aminoacylated RNA minihelix binds to the A site. (C) CCdA-phosphate-puromycin is an intermediate analogue containing A and P site-binding components. (D) CC-puromycin-phenylalanine-caproic acid–biotin and deacylated CCA are products of the peptidyl transferase reaction.

Acknowledgments

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