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Title: Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase

Abstract

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. In this work, we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also highlights why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Furthermore, using standard microbiological culture equipment, we established a new method for laboratory evolution—a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.

Authors:
 [1];  [2];  [1];  [1];  [3]; ORCiD logo [4];  [5]; ORCiD logo [1]
  1. Yale Univ., New Haven, CT (United States)
  2. DS Therapeutics, Houston, TX (United States); Yale Univ., New Haven, CT (United States)
  3. Harvard Univ., Cambridge, MA (United States)
  4. Academia Sinica, Taipei (Taiwan); Yale Univ., New Haven, CT (United States)
  5. Harvard Univ., Cambridge, MA (United States); Howard Hughes Medical Inst., Cambridge, MA (United States); Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States); Yale Univ., New Haven, CT (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Institutes of Health (NIH)
OSTI Identifier:
1430347
Grant/Contract Number:  
FG02-98ER20311; AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Nature Chemical Biology
Additional Journal Information:
Journal Volume: 13; Journal Issue: 12; Journal ID: ISSN 1552-4450
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Suzuki, Tateki, Miller, Corwin, Guo, Li-Tao, Ho, Joanne M. L., Bryson, David I., Wang, Yane-Shih, Liu, David R., and Söll, Dieter. Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. United States: N. p., 2017. Web. doi:10.1038/nchembio.2497.
Suzuki, Tateki, Miller, Corwin, Guo, Li-Tao, Ho, Joanne M. L., Bryson, David I., Wang, Yane-Shih, Liu, David R., & Söll, Dieter. Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. United States. doi:10.1038/nchembio.2497.
Suzuki, Tateki, Miller, Corwin, Guo, Li-Tao, Ho, Joanne M. L., Bryson, David I., Wang, Yane-Shih, Liu, David R., and Söll, Dieter. Mon . "Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase". United States. doi:10.1038/nchembio.2497. https://www.osti.gov/servlets/purl/1430347.
@article{osti_1430347,
title = {Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase},
author = {Suzuki, Tateki and Miller, Corwin and Guo, Li-Tao and Ho, Joanne M. L. and Bryson, David I. and Wang, Yane-Shih and Liu, David R. and Söll, Dieter},
abstractNote = {Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. In this work, we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also highlights why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Furthermore, using standard microbiological culture equipment, we established a new method for laboratory evolution—a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.},
doi = {10.1038/nchembio.2497},
journal = {Nature Chemical Biology},
number = 12,
volume = 13,
place = {United States},
year = {2017},
month = {10}
}

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    Works referencing / citing this record:

    Pyrrolysine in archaea: a 22nd amino acid encoded through a genetic code expansion
    journal, November 2018

    • Brugère, Jean-François; Atkins, John F.; O'Toole, Paul W.
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