Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase.
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-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.
Yanagisawa
Tatsuo
T
Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Ishii
Ryohei
R
Fukunaga
Ryuya
R
Kobayashi
Takatsugu
T
Sakamoto
Kensaku
K
Yokoyama
Shigeyuki
S
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017020:Methanosarcina / Q000145:classification / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism; D013329:Structure-Activity Relationship; D046249:Transfer RNA Aminoacylation
D001059:Apoproteins; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine