2ZIN

Crystal structure of the catalytic domain of pyrrolysyl-tRNA synthetase in complex with BocLys and an ATP analogue

Summary for 2ZIN

• Entry DOI: 10.2210/pdb2zin/pdb
• Related: 2E3C 2ZCD 2ZCE
• Descriptor: Pyrrolysyl-tRNA synthetase, MAGNESIUM ION, PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER, ... (6 entities in total)
• Functional Keywords: aminoacyl-trna synthetase, pyrrolysyl-trna synthetase, pyrrolysine, trna, atp analogue, non-natural amino acid, unnatural amino acid, atp-binding, cytoplasm, ligase, nucleotide-binding, protein biosynthesis, structural genomics, nppsfa, national project on protein structural and functional analyses, riken structural genomics/proteomics initiative, rsgi
• Biological source: Methanosarcina mazei
• Cellular location: Cytoplasm (By similarity): Q8PWY1
• Total number of polymer chains: 1
• Total formula weight: 34207.34

Authors

Yanagisawa, T.,Ishii, R.,Yokoyama, S.,RIKEN Structural Genomics/Proteomics Initiative (RSGI) (deposition date: 2008-02-19, release date: 2008-12-02, Last modification date: 2023-11-01)

Primary citation

Yanagisawa, T.,Ishii, R.,Fukunaga, R.,Kobayashi, T.,Sakamoto, K.,Yokoyama, S.
Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode N(varepsilon)-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification
Chem.Biol., 15:1187-1197, 2008

PubMed Abstract: Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNA(Pyl). In this study, N(epsilon)-(tert-butyloxycarbonyl)-L-lysine (BocLys) and N(epsilon)-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNA(Pyl) by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an N(epsilon)-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for N(epsilon)-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing N(epsilon)-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.

PubMed: 19022179
DOI: 10.1016/j.chembiol.2008.10.004

Experimental method

X-RAY DIFFRACTION (1.79 Å)