Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 378, Issue 3, Pages 634-652Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.02.045
Keywords
pyrrolysine; tRNA; aminoacyl-tRNA synthetase; pyrrolysyl-tRNA synthetase; conformational changes
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Pyrrolysine, a lysine derivative with a bulky pyrroline ring, is the 22nd genetically encoded amino acid. In the present study, the carboxy-terminal catalytic fragment of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) was analyzed by X-ray crystallography and site-directed mutagenesis. The catalytic fragment ligated tRNA(Pyl) with pyrrolysine nearly as efficiently as the full-length PylRS. We determined the crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)bound, and AMPPNP/pyrrolysine-bound forms, and compared them with the previously-reported PylRS structures. The ordering loop and the motif-2 loop undergo conformational changes from the open states to the closed states upon AMPPNP binding. On the other hand, the beta 7-beta 8 hairpin exhibits multiple conformational states, the open, intermediate (beta 7-open/beta 8-open and beta 7-closed/beta 8-open), and closed states, which are not induced upon substrate binding. The PylRS structures with a docked tRNA suggest that the active-site pocket can accommodate the CCA terminus of tRNA when the motif-2 loop is in the closed state and the beta 7-beta 8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the beta 7-beta 8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNA(Pyl). Moreover, a structure-based mutational analysis revealed that hydrophobic residues in the amino acid-binding tunnel are important for accommodating the pyrrolysine side chain and that Asn346 is essential for anchoring the side-chain carbonyl and alpha-ammo groups of pyrrolysine. In addition, a docking model of PylRS with tRNA was constructed based on the aspartyl-tRNA synthetase/tRNA structure, and was confirmed by a mutational analysis. (C) 2008 Elsevier Ltd. All rights reserved.
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