Grb7-SH2 domain dimerisation is affected by a single point mutation

Eur Biophys J. 2005 Jul;34(5):454-60. doi: 10.1007/s00249-005-0480-1. Epub 2005 Apr 20.

Abstract

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is co-overexpressed and forms a tight complex with the ErbB2 receptor in a number of breast tumours and breast cancer cell lines. The interaction of Grb7 with the ErbB2 receptor is mediated via its Src homology 2 (SH2) domain. Whilst most SH2 domains exist as monomers, recently reported studies have suggested that the Grb7-SH2 domain exists as a homodimer. The self-association properties of the Grb7-SH2 domain were therefore studied using sedimentation equilibrium ultracentrifugation. Analysis of the data demonstrated that the Grb7-SH2 domain is dimeric with a dissociation constant of approximately 11 muM. We also demonstrate, using size-exclusion chromatography, that mutation of phenylalanine 511 to an arginine produces a monomeric form of the Grb7-SH2 domain. This mutation represents the first step in the engineering of a Grb7-SH2 domain with good solution properties for further biophysical and structural investigation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arginine / chemistry
  • Biophysics / methods
  • Cell Line, Tumor
  • Chromatography
  • Dimerization
  • Dose-Response Relationship, Drug
  • Escherichia coli / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Models, Statistical
  • Molecular Conformation
  • Mutagenesis, Site-Directed
  • Mutation
  • Phenylalanine / chemistry
  • Plasmids / metabolism
  • Point Mutation*
  • Protein Binding
  • Protein Engineering
  • Protein Structure, Tertiary
  • Ultracentrifugation
  • src Homology Domains

Substances

  • Phenylalanine
  • Arginine