556 VOLUME 12 NUMBER 6 JUNE 2005 NATURE STRUCTURAL & MOLECULAR BIOLOGY A D-amino acid editing module coupled to the translational apparatus in archaea Shweta Dwivedi, Shobha P Kruparani & Rajan Sankaranarayanan We report the crystal structure of an archaea-specific editing domain of threonyl-tRNA synthetase that reveals a marked structural similarity to D-amino acid deacylases found in eubacteria and eukaryotes. The domain can bind D-amino acids despite a low sequence identity to other D-amino acid deacylases. These results together indicate the presence of these deacylases in all three kingdoms of life. This underlines an important role they may have played in enforcing homochirality during translation. The origin of homochirality has been debated from the day it was discovered in biological macromolecules 1 . RNA-directed fixation of homochirality of amino acids in proteins has been postulated and sup- ported by both modeling and experimental studies 2,3 . The primordial aminoacyl-tRNA synthetases (aaRSs) must have played a crucial role in further enforcement and effective perpetuation of homochirality during translation. aaRSs establish the rules of the genetic code by attaching an amino acid to the correct tRNA 4 . aaRSs that are posed with difficult sub- strate recognition problems have acquired specialized modules through which they remove the noncognate substrates attached to tRNA. Threonyl- tRNA synthetase (ThrRS) from Escherichia coli has a unique zinc ion in its active site and a tRNA-mediated editing function to ensure high fidelity during translation 5,6 . The domain responsible for editing is not conserved through evolution and most archaeal ThrRSs have a unique sequence at their N terminus. It has been shown that this domain is responsible for removing incorrectly attached serine from tRNA Thr (refs. 7,8). We determined the structure of the editing domain (Pab-NTD) of ThrRS from Pyrococcus abyssi at a resolution of 1.95 Å. The first 143 resi- dues identified in the electron density map fold into a compact domain with an approximate size of 44 × 43 × 31 Å. It belongs to the α/β class of proteins with 40 (28%) residues in an α-helix and 55 (39%) residues in a β-strand conformation (Fig. 1a). The fold can be defined as two layers of β-sheets, a three-stranded sheet and a five-stranded sheet, with two helices located adjacent to the five-stranded sheet. Two of the long strands bend to form part of both the sheets. As would be expected for thermophilic archaea, many of the 143 residues are charged and 24 of them are involved in salt bridges. In addition, the amino group of the N-terminal methionine is buried and makes salt bridges with two glutamates. Experimental details and statistics are described in Supplementary Methods and Supplementary Table 1 online. Because the sequence of Pab-NTD showed no substantial homology to any other protein of known structure, the coordinates were submitted to the Dali server to search for structurally and possibly func- tionally homologous proteins. Notably, Pab-NTD showed substantial structural homology to that of D-Tyr-tRNA Tyr deacylase (DTD) from E. coli 9 with a Z-score of 14.5. The structural superposition resulted in an r.m.s. deviation in the Cα position of 1.16 Å for 112 atoms (Fig. 1b). This marked structural similarity was also reflected in a structure- based sequence alignment with a limited 14% identity between both the proteins that could not be detected by conventional sequence-based searches (Supplementary Fig. 4 online). A recent report, however, predicted the Pab-NTD structure to be similar to that of DTD using fold recognition servers 10 . DTD from E. coli is a functional homodimer in solution, and strong dimeric interactions are observed in the crystal structure 9 . Notably, Pab-NTD also showed dimeric interactions in the crystal (Supplementary Fig. 1 online). DTD buries 17% of its total monomer surface area whereas Pab-NTD buries 10% of its monomer surface area upon dimerization. Cross-subunit interactions in the active site, as seen in DTD, were found. It is notable that Pab-NTD, a part of homodimeric ThrRS with a dimer interface belonging to the catalytic domain, has such a strong propensity for dimerization. The marked structural similarity at the monomeric level and a similar dimerization propensity strongly indicate a common evolutionary origin for the Pab-NTD homologs in archaea and DTDs found in eubacteria and eukaryotes. DTD activity is not specific for a given tRNA but for tRNAs amino- acylated with a D-amino acid 11 . Therefore, the strong structural resem- blance with DTD prompted us to test whether Pab-NTD would also bind D-amino acids, which would imply that it could be a D-amino acid deacylase as well. We followed a fluorescence-based approach by preparing a Pab-NTD–Bis-ANS complex to monitor the binding of different amino acids. The binding studies clearly showed a change in fluorescence intensity upon titration with L-serine (Fig. 2a), used as a positive control, but did not show any change with L-threonine (Fig. 2b), used as a negative control, to which Pab-NTD should not bind in accor- dance with its function. In fact, glycine did not show any binding, nor did any of the L-amino acids tested except L-cysteine. In contrast, of the 18 D-amino acids tested, most of them effected change in fluorescence intensity (Supplementary Fig. 2 online). Notably, Pab-NTD showed binding to D-serine and D-threonine (Fig. 2c,d). Crystal soaking experi- ments also indicated a clear preference for D-amino acids along with L-serine (Supplementary Fig. 3 online). These results pose key evolutionary questions regarding the presence of a D-amino acid editing module in all kingdoms of life and its identification as part of the translational machinery in archaea. aaRSs have to perpetuate homochirality by effective incorporation of molecules with single chirality (L-amino acids) in proteins. This task is essential to constitute an efficient translational machinery. Stringent quality control mechanisms at different levels, like during charging of the tRNA, formation of complex with EF-Tu, and at the ribosome, must Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. Correspondence should be addressed to R.S. (sankar@ccmb.res.in). Published online 22 May 2005; doi:10.1038/nsmb943 Figure 1 Crystal structure of Pab-NTD and a comparison with DTD. (a) Ribbon diagram of Pab-NTD. (b) Structural superposition of Pab-NTD (cyan) and DTD (pink). BRIEF COMMUNICATIONS © 2005 Nature Publishing Group http://www.nature.com/nsmb