Crystallographic Studies on Multiple Conformational States of Active-site Loops in Pyrrolysyl-tRNA Synthetase

Tatsuo Yanagisawa, Ryohei Ishii, Ryuya Fukunaga, Takatsugu Kobayashi, Kensaku Sakamoto, Shigeyuki Yokoyama

Research output: Contribution to journalArticlepeer-review

68 Scopus citations


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 tRNAPyl 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 β7-β8 hairpin exhibits multiple conformational states, the open, intermediate (β7-open/β8-open and β7-closed/β8-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 β7-β8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the β7-β8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNAPyl. 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 α-amino 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.

Original languageEnglish (US)
Pages (from-to)634-652
Number of pages19
JournalJournal of molecular biology
Issue number3
StatePublished - May 2 2008
Externally publishedYes


  • aminoacyl-tRNA synthetase
  • conformational changes
  • pyrrolysine
  • pyrrolysyl-tRNA synthetase
  • tRNA

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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