4ZIB

Crystal Structure of the C-terminal domain of PylRS mutant bound with 3-benzothienyl-l-alanine and ATP

Summary for 4ZIB

• Entry DOI: 10.2210/pdb4zib/pdb
• Descriptor: Pyrrolysine--tRNA ligase, MAGNESIUM ION, ADENOSINE-5'-TRIPHOSPHATE, ... (6 entities in total)
• Functional Keywords: aminoacyl-trna synthetases, archaeal proteins, evolution, molecular, genetic code, substrate specificity, ligase
• Biological source: Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
• Total number of polymer chains: 1
• Total formula weight: 34620.01

Authors

Nakamura, A.,Guo, L.T.,Wang, Y.S.,Soll, D. (deposition date: 2015-04-28, release date: 2016-03-02, Last modification date: 2023-11-15)

Primary citation

Englert, M.,Nakamura, A.,Wang, Y.S.,Eiler, D.,Soll, D.,Guo, L.T.
Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog.
Nucleic Acids Res., 43:11061-11067, 2015

PubMed Abstract: Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.

PubMed: 26582921
DOI: 10.1093/nar/gkv1255

Experimental method

X-RAY DIFFRACTION (2.054 Å)