Structural and Biophysical Analyses of Aspartyl-tRNA Synthetase from Homo sapiens and Triosephosphate Isomerase from Thermoplasma acidophilum

Abstract

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the aspartic acid to its specific tRNA. DRS is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unanticipated signaling pathways. The crystal structure of DRS has been determined at 2.25 Å resolution containing the anticodon binding domain, hinge region, and catalytic domain. The structure also reveals the C-terminal end of the N-helix which is considered as a unique additional domain of DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNAAsp to elongation factor 1α. From the analyses of the crystal structure and post-translational modification of DRS, I suggest that the phosphorylation of Ser146 could initiate a conformational change of the DRS dimer and provokes the separation of DRS from the MSC. This structural study provides the binding site for an interaction partner with unforeseen functions. Thermoplasma acidophilum is one of the most acidophilic organisms that utilize not only non-phosphorylative Entner-Doudoroff (ED) pathway but also Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. Triosephosphate isomerase (TPI) is structurally and functionally well-known glycolytic enzyme that plays an important role in glycolytic and gluconeogenic metabolism. Crystal structures of apo- and glycerol-3-phosphate-bound TPI from T. acidophilum (TaTPI) have been determined at 1.94 and 2.17 Å resolution. TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight β-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. Dimeric conformation of TaTPI was further confirmed by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 and helix 4 regions of thermostable tetrameric TPIs are key important tetrameric interface, forming a hydrophobic effects. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent Td value of 74.6 ℃ and maintains its overall structure with slight changes in the secondary structure contents under extremely acidic conditions. Based on the structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of Thermoplasma acidophilum TPI.