A pivotal role for reductive methylation in the de novo crystallization of a ternary complex composed of Yersinia pestis virulence factors YopN, SycN and YscB

Acta Crystallogr D Biol Crystallogr. 2004 Nov;60(Pt 11):1981-6. doi: 10.1107/S0907444904023005. Epub 2004 Oct 20.

Abstract

Structural studies of a ternary complex composed of the Yersina pestis virulence factors YopN, SycN and YscB were initially hampered by poor solubility of the individual proteins. Co-expression of all three proteins in Escherichia coli yielded a well behaved complex, but this sample proved to be recalcitrant to crystallization. As crystallization efforts remained fruitless, even after the proteolysis-guided engineering of a truncated YopN polypeptide, reductive methylation of lysine residues was employed to alter the surface properties of the complex. The methylated complex yielded crystals that diffracted X-rays to a maximal resolution of 1.8 A. The potential utility of reductive methylation as a remedial strategy for high-throughput structural biology was further underscored by the successful modification of a selenomethionine-substituted sample.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Crystallization
  • Crystallography, X-Ray
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism
  • Methylation
  • Molecular Chaperones / chemistry*
  • Molecular Chaperones / metabolism
  • Oxidation-Reduction
  • Protein Structure, Tertiary
  • Virulence Factors / chemistry*
  • Virulence Factors / metabolism*
  • Yersinia pestis / chemistry*
  • Yersinia pestis / metabolism*

Substances

  • Bacterial Proteins
  • Membrane Proteins
  • Molecular Chaperones
  • Virulence Factors
  • YscB protein, Yersinia pestis
  • YopN protein, Yersinia