Abstract 1325 Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome

The Escherichia coli ribosome is an attractive target for synthetic biology since it can site-specifically incorporate a variety of non-L-α-amino acid monomers into polypeptide chains. These monomers span a variety of compounds, including β-amino acids, aminobenzoic acids and malonates, which can impart distinct properties to polypeptides such as increased protease resistance and unique foldamer properties. However, most non-natural monomers are incorporated with lower efficiently than canonical L-α- amino acids, and the structural and mechanistic basis for these defects remain unknown. For instance, three aminobenzoic acid derivatives– 3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ) that are ribosomally incorporated into short polypeptides, have incorporation efficiencies that do not track with the nucleophilicity of the reactive amines. In this work, we report high resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A site). The structures reveal how the aromatic ring of the aminobenzoic acids disrupts the so called "induced fit" of the ribosome by blocking the positioning of nucleotides U2506 and U2585. They also reveal disruptions to an ordered water network responsible for facilitating amide bond formation. Together, the cryo-EM structures reported here provide a rationale for differences in reactivity between aminobenzoic acid derivatives and their lower incorporation efficiency relative to L-α-amino acids. These insights can also be used to rationally design non-proteinogenic monomers that can be accepted efficiently by wild-type ribosomes. This work was sponsored by the NSF Center for Genetically Encoded Materials, an NSF Center for Chemical Innovation (NSF CHE-2002182).