Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study

Biochim Biophys Acta. 2004 Jan 14;1696(1):131-40. doi: 10.1016/j.bbapap.2003.10.001.

Abstract

The structural domains of the Escherichia coli CheA protein resemble 'beads on a string', since the N-terminal phosphate-accepting (P) domain is joined to the CheY/CheB-binding (B) domain through a flexible linker, and the B domain is in turn joined to the C-terminal dimerization/catalytic/regulatory domains by a second intervening linker. Dimerization occurs primarily via interactions between two dimerization domains, which is required for CheA trans-autophosphorylation. In this study, sedimentation equilibrium was used to demonstrate significant subunit interactions at secondary sites in the two naturally occurring (full-length and short) forms of CheA (CheA(1-654) or CheA(L), and CheA(98-654) or CheA(S)) by contrasting the dimerization of CheA(L) and CheA(S) to CheA(T), an engineered form that lacked the P domain entirely. The estimated dimer dissociation constant (K(1,2)) for CheA(T) (3.1 microM) was weaker than K(1,2) for CheA(L) (0.49 microM), which was attributed to the P domain-catalytic domain interactions that were present in CheA(L) but not CheA(T). In contrast, CheA(S) dimerization was unexpectedly stronger (K(1,2) approximately 20 nM), which arose through interactions between two P domain remnants in the CheA(S) dimer. This conclusion was supported by the results of sedimentation equilibrium experiments conducted with P domains and P domain remnants expressed in the absence of the dimerization/catalytic/regulatory domains. The P domain remnant had a measurable tendency to self-associate; the full-length P domain did not. Hydrophobic forces probably drive this interaction, since hydrophobic amino acids buried in the intact P domain are solvent-exposed in CheA(S). Also, the nascent N-terminus of CheA(S) bound to the phosphatase (CheZ) more effectively, a conclusion based on the demonstrably greater ability of the P domain remnant to co-sediment CheZ, compared to the intact P domain.

Publication types

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

MeSH terms

  • Bacterial Proteins*
  • Catalytic Domain
  • Dimerization
  • Escherichia coli / metabolism
  • Escherichia coli Proteins
  • Genes, Regulator
  • Histidine Kinase
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Methyl-Accepting Chemotaxis Proteins
  • Phosphorylation
  • Plasmids
  • Protein Structure, Tertiary*
  • Protein Subunits / chemistry*
  • Ultracentrifugation

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Membrane Proteins
  • Methyl-Accepting Chemotaxis Proteins
  • Protein Subunits
  • cheY protein, E coli
  • Histidine Kinase
  • cheA protein, E coli