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

J Struct Biol. 2008 Apr;162(1):152-69. doi: 10.1016/j.jsb.2007.11.002. Epub 2007 Nov 13.

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 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Crystallography, X-Ray / methods
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Structure
  • Mutagenesis
  • Phenylalanine-tRNA Ligase / antagonists & inhibitors
  • Phenylalanine-tRNA Ligase / chemistry*
  • Phenylalanine-tRNA Ligase / metabolism
  • Protein Engineering / methods
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Staphylococcus haemolyticus / enzymology*
  • Staphylococcus haemolyticus / genetics

Substances

  • Bacterial Proteins
  • Phenylalanine-tRNA Ligase

Associated data

  • PDB/2RHQ
  • PDB/2RHS