Specificity in Substrate and Cofactor Recognition by the N-terminal Domain of the Chaperone ClpX

Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17724-9. doi: 10.1073/pnas.0601505103. Epub 2006 Nov 7.

Abstract

Clp ATPases are a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX is a representative member of these ATPases; it consists of two domains, a zinc-binding domain (ZBD) that forms dimers and a AAA+ ATP-binding domain that arranges into a hexamer. Analysis of the binding preferences of these two domains in ClpX revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of the ClpX dimeric cofactor, SspB2, on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB2 toward ZBD2 and the geometry of the SspB2-ZBD2 complex were investigated by using the newly developed quantitative optical biosensor method of dual polarization interferometry. The data suggest a model for the interaction between SspB2 and the ClpX hexamer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATPases Associated with Diverse Cellular Activities
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Coenzymes*
  • DNA Mutational Analysis
  • Endopeptidase Clp / chemistry
  • Endopeptidase Clp / genetics
  • Endopeptidase Clp / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Models, Molecular
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Substrate Specificity

Substances

  • Carrier Proteins
  • Coenzymes
  • Escherichia coli Proteins
  • Molecular Chaperones
  • sspB protein, E coli
  • Endopeptidase Clp
  • Adenosine Triphosphatases
  • ClpX protein, E coli
  • ATPases Associated with Diverse Cellular Activities