title: Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study.
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.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, MA 01003-9336, USA.
authors.forename: Laila
authors.initials: L
authors.lastname: Kott
authors.forename: Emory H
authors.initials: EH
authors.lastname: Braswell
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Shrout
authors.forename: Robert M
authors.initials: RM
authors.lastname: Weis
mesh_terms: D001426:Bacterial Proteins; D020134:Catalytic Domain; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005809:Genes, Regulator; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000072236:Methyl-Accepting Chemotaxis Proteins; D010766:Phosphorylation; D010957:Plasmids; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry*; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D021122:Protein Subunits; C000607606:cheY protein, E coli; D000071677:Histidine Kinase; C000607608:cheA protein, E coli