Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

Artem G. Evdokimov, Marlene Mekel, Kim Hutchings, Lakshmi Narasimhan, Tod Holler, Teresa McGrath, Bryan Beattie, Eric Fauman, Chunhong Yan, Holly Heaslet, Richard Walter, Barry Finzel, Jeffrey Ohren, Patrick McConnell, Timothy Braden, Fang Sun, Cindy Spessard, Craig Banotai, Loola Al-Kassim, Weijun MaPaul Wengender, Denis Kole, Norman Garceau, Peter Toogood, Jia Liu

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality-they only diffracted X-rays to 3-5 Å resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 Å or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

Original languageEnglish (US)
Pages (from-to)152-169
Number of pages18
JournalJournal of Structural Biology
Volume162
Issue number1
DOIs
StatePublished - Apr 2008

Bibliographical note

Funding Information:
The authors specifically acknowledge the mass-spectroscopic measurements made by Tracy Stevenson and Eric Lund as well as the biophysical experiments conducted by Ronald Sarver and Kimberly Huchings. Use of the IMCA-CAT beamline 17-ID at the APS was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the Center for Advanced Radiation Sources at the University of Chicago. Use of the APS was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

Keywords

  • Antibacterial drug design
  • Crystal structure
  • Phenylalanine tRNA synthetase
  • Protein engineering
  • Rational mutagenesis

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