Mutations That Alter the Kinase and Phosphatase Activities of the Two-Component Sensor EnvZ

Abstract

EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in Escherichia coli in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K+ P−) and the OmpR-P phosphatase-dominant state (K− P+). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.

Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein

“…HKs are multifunctional enzymes that participate in autokinase, phosphotransferase and phosphatase reactions, and their phospho-accepting histidines typically have roles in all three activities (Hsing et al, 1998). The kinase and/or phosphatase activities are regulated by sensor input and are dependent on the presence of nucleotide (reviewed in Stock et al, 2000).…”

“…The three enzymatic activities (autokinase, phosphotransfer and phosphatase) associated with the cytoplasmic region of the HK each require the participation of one or both of the DHp and CA domains (Tanaka et al, 1991;Hsing et al, 1998), suggesting that these domains can exist in different conformational states with respect to one another. Structural characterization of these signaling states has been thwarted in the dissection approach.…”

Histidine kinases: diversity of domain organization

“…Although the structure of the DHp-linked CA domain of EnvZ is yet to be solved, the evidence favouring a¯exible regulatory hinge between the two domains is strong. Mutations in this region disrupted the phosphatase activity (phospho-OmpR dephosphorylase activity of EnvZ) (Hsing et al, 1998). In the case of CheA, they are likely to help linking receptor response to the Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 633±640 Fig.…”

A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor

“…Although very little is known about the mechanism of EnvZc phosphatase activity at present, both domains A and B are considered to contribute to OmpR binding and phosphatase activity (M. Inouye, unpublished data). Also, it is known that the phosphatase activity is highly sensitive to a large number of mutations in the EnvZ molecule (Hsing et al, 1998). Therefore, the reduced phosphatase activity of EnvZc[AAB] monomer and its lower binding affinity to OmpR may be due to the following possibilities: (i) the four-helix bundle, presumed to be formed by two covalently linked domains A in the monomeric EnvZc[AssAB], may not be identical to the four-helix bundle formed by two independent domains A; and (ii) the interaction between domain B and the monomeric four-helix bundle in EnvZc[AssAB] may be slightly distorted compared with that between domain B and the dimeric four-helix bundle in EnvZc.…”

“…Signalling by multiple histidine kinases and response regulators, such as the one involved in the initiation of sporulation in Bacillus subtilis (Burbulys et al, 1991), or by hybrid signalling proteins, such as ArcB (Ishige et al, 1994), which combine both histidine kinase and response regulator modules in a single polypeptide is known (reviewed by Appleby et al, 1996). Currently more than 250 such signalling systems have been reported in prokaryotes (Hsing et al, 1998) and, recently, they have been discovered in some lower eukaryotes (see Loomis et al, 1997 for a review of eukaryotic histidine kinases).…”