Structural And Functional Studies Of Two Human Proteins: Human Tryptophanyl-tRNA Synthetase And Human Ras Homolog Enriched In Brain

Aminoacyl-tRNA synthetases (aaRS) are a family of ancient enzymes that charge amino acids to their cognate tRNAs in the first step of protein translation, and thus establishing the genetic codes system. During their long evolution, they have acquired various additional functions related to translation as well as other cellular processes. In particular, mammalian aaRSs usually possess extensions at N-or C-terminus which are absent in their prokaryotic counterparts. Human tryptophanyl-tRNA synthetase (hTrpRS) has an extra N-terminal domain of about 150 amino acids. Alternative splicing and proteolysis produce 3 N-terminus truncated forms of hTrpRS: mini-, T1-and T2-hTrpRS. These truncations were recently found to be active in anti-angiogenesis. The shortest fragment (T2-hTrpRS) contains the aminoacylation catalytic fragment and exhibits the most potent angiostatic activity. We determined the crystal structure of T2-hTrpRS at 2.5 ? using the multi-wavelength anomalous diffraction method. T2-hTrpRS shares a very low sequence homology of 22% with Bacillus stearothermophilus TrpRS (bTrpRS); however, their overall structures are strikingly similar. Structural comparison of T2-hTrpRS with bTrpRS reveals substantial structural differences in the substrate-binding pocket and at the entrance to the pocket that play important roles in substrate binding and tRNA binding. T2-hTrpRS has a wide opening to the active site and adopts a compact conformation similar to the closed conformation of bTrpRS. These results suggest that mammalian and bacterial TrpRSs might use different mechanisms to recognize the substrate. Modeling studies indicate that tRNA binds with the dimeric enzyme and interacts primarily with the connective polypeptide 1 of hTrpRS via its acceptor arm and the α-helical domain of hTrpRS via its anticodon loop. Our results also suggest that the angiostatic activity is likely located at the α-helical domain, which resembles the short chain cytokines.