Two-domain reconstitution of a functional protein histidine kinase

Abstract

In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses. Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals. In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function. Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A [EnvZ(C)⅐(223-289); 67 residues] and subdomain B [EnvZ(C)⅐(290-450); 161 residues]. Subdomain A, with a high helical content, contains the autophosphorylation site, H-243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ. Subdomain B, an ␣/␤-protein, exists as a monomer. When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP. Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR. The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

“…In this class the catalytic and ATP-binding domain is separated in two regions (37). The A domain is involved in the dimerization, phosphotransfer, and phosphatase activity, while the B domain binds ATP (38,39). These histidine kinases transfer a phosphoryl group on an aspartate residue in the cognate regulator protein (OmpR in the case of EnvZ).…”

Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization

“…The cytoplasmic domain has been dissected further into a linker domain (residues 180-222), domain A (dimerization and histidine-containing domain, residues 223-289) and domain B (catalysis-assisting and ATP-binding domain, residues 290-450) [6]. Domain A and domain B are central to the autophosphorylation function of EnvZ, which mediates coupling between the environmental sensing and activation of OmpR by phosphorylation of an aspartate residue.…”

Analysis of the Role of the EnvZ Linker Region in Signal Transduction Using a Chimeric Tar/EnvZ Receptor Protein, Tez1

“…NMR structures of both domain A and domain B have been solved (4,5). Biochemical analysis has revealed that the domain A of the EnvZ is responsible for the dimerization, phosphotransfer and phosphatase functions, and domain B binds ATP and catalytically assists the enzymatic function of domain A (3,6). The spatial arrangement between these two domains appears to be crucial for the modulation of EnvZ enzymatic activities (6).…”

“…It is composed of a periplasmic domain, transmembrane domains, and a cytoplasmic domain (1,2). The cytoplasmic domain has been further dissected into three subdomains, the linker region, domain A, and domain B, in which the latter two form the catalytic core of EnvZ, harboring both kinase and phosphatase functions (3). NMR structures of both domain A and domain B have been solved (4,5).…”