Talk abstracts

Talk on Saturday 10:25-10:40am submitted by Mom Das

A Tale of Two Editing Domains: Molecular Basis of Substrate Specificity of Bacterial Prolyl-tRNA Synthetase and YbaK

Sandeep Kumar (Chemistry, Center for RNA Biology, Ohio State University), Mom Das (Chemistry, Ohio State Biochemistry Program, Center for RNA Biology, Ohio State University), Christopher M. Hadad (Chemistry, Ohio State University), Karin Musier-Forsyth (Chemistry and Biochemistry, Ohio State Biochemistry Program, Center for RNA Biology, Ohio State University)

Abstract:
Prolyl-tRNA synthetases (ProRSs) mischarge cognate tRNA^Pro with the smaller noncognate amino acid Ala, and with Cys, which is of similar size as cognate Pro. Bacterial ProRSs possess a discrete editing active site (INS domain) that functions to selectively hydrolyze mischarged Ala- but fails to hydrolyze Cys-tRNA^Pro, which is edited by a free-standing domain, YbaK, via substrate sulfhydryl side chain chemistry. The molecular basis of substrate specificity of these homologous editing domains is further investigated here. Computational docking studies of E. coli (Ec) ProRS INS bound to CCA-Ala revealed that the substrate Ala binds in a small hydrophobic pocket defined by conserved residues I263, L266 and T277. This pocket is large enough to accommodate Ser- and 2-aminobutyric acid (Abu)-tRNA^Pro, but cannot hydrolyze Cys- and Pro-tRNA^Pro. Deacylation activities of INS mutants (I263A, L266A, T277S and I263A/L266A) were tested against Ala-, Pro-, Cys-, Ser- and 2Abu-tRNA substrates. All mutants showed a significant loss in Ala- and 2-Abu deacylation activity and interestingly, I263A gained Cys-deacylation specificity. The G30 residue in YbaK, which aligns with I263, was substituted with Ile to mimic the binding pocket of the INS domain. G30I YbaK failed to hydrolyze either Ala- or Cys-tRNA^Pro. Thus, restricting the binding pocket size of YbaK prevents Cys-tRNA editing, as expected, but does not allow hydrolysis of Ala. QM/MM calculations suggest that hydrolysis by INS is mediated by a nucleophilic water molecule activated by the 2^ʹOH of A76 of tRNA^Pro. In support of this idea, 2^ʹ-deoxy-A76-tRNA^Pro was severely reduced in INS Ala-deacylation activity. In conclusion, the specificity of substrate-assisted water-mediated hydrolysis by INS is largely dictated by binding pocket size. In contrast, tuning the binding pocket size of YbaK is not sufficient to alter substrate specificity, providing further support for the role of the side chain sulfhydryl in cleavage chemistry.

Keywords: Prolyl-tRNA Synthetase, YbaK, Substrate Specificity, Computational Docking, QM-MM