Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

Abstract

TheEscherichia coliribosome can incorporate a variety of non-L-α-amino acid monomers into polypeptide chains, but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives—3-aminopyridine-4-carboxylic acid (Apy),ortho-aminobenzoic acid (oABZ), andmeta-aminobenzoic acid (mABZ)—the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed byoABZ and thenmABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high resolution cryo-EM structures of the ribosome with these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A site). These structures reveal how the aromatic ring of each monomer sterically blocks positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the "proton wire" responsible for facilitating formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a clear rationale for differences in reactivity of aminobenzoic acid derivatives relative to L-α-amino acids and each other, and point to stereochemical constraints on the size and geometry of non-proteinogenic monomers that can be accepted efficiently by wild-type ribosomes.

Incorporation of Multiple β2-Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase

“…Although the structure of the PylRS variant FRSA bound to 2-benzylmalonate 27 provides an explanation for the absence of enantio-preference at the aaRS level, the preference for D-like (S)-β 2 -OH 3 in the PTC was a surprise. Metadynamics simulations and recent cryo-EM structures 46 provide evidence that the preference for tRNA charged with (S)-β 2 -OH 3 has less to do with inherent stereochemistry than with differences in the ability to induce conformational changes within the PTC necessary for favorable bond formation. A better understanding of the interactions necessary to facilitate P-site electrophile deshielding could be used to identify D-α-amino acids that are successfully elongated in vivo, another long-sought goal.…”

“…45 Indeed, recent cryo-EM structures of E. coli ribosomes with aminobenzoic acid monomers in the A-site show U2585 locked in the uninduced conformation, prohibiting access to the P-site peptidyl-tRNA. 46 Overall, the decreased pairwise distances within the RRM containing 4-acyl-tRNA fMet may indicate an inability to support the dynamic movements necessary for efficient bond formation. These differences in RRM structure provide a second explanation for the observed in vivo enantioselective preference for (S)-β 2 -OH 3 over (R)-β 2 -OH 4.…”

“…These differences in RRM structure provide a second explanation for the observed in vivo enantioselective preference for (S)-β 2 -OH 3 over (R)-β 2 -OH 4. More broadly, they emphasize that the complex mechanism of translation, including (but not limited to) EF-Tu-and tRNA-induced conformational changes and essential bound water molecules 46 must be considered in future ribosome or monomer engineering efforts.…”

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

“…295 Recent cryo-EM structures and molecular dynamic simulations of various 2-Abz derivatives within the PTC have indicated that their nucleophilic amine is located 4 Å or more away from the P site carbonyl and that their relatively rigid aromatic backbone sterically occludes optimal fit into the A and P sites, potentially disrupting the proton transfer chain. 296,297 These issues may contribute to the poor translational efficiency of these npMs. 296,297 Katoh and Suga used tRNA Pro1E2 CGG to perform single incorporation of 2-Abz and seven other derivatives into rP1-(npM) 1 at the CCG codon.…”

“…296,297 These issues may contribute to the poor translational efficiency of these npMs. 296,297 Katoh and Suga used tRNA Pro1E2 CGG to perform single incorporation of 2-Abz and seven other derivatives into rP1-(npM) 1 at the CCG codon. 295 Inclusion of EF-P increased translation efficiency, ranging from 1.4-fold to 6.8-fold.…”