Engineering an Osmosensor by Pivotal Histidine Positioning within Disordered Helices

Abstract

Graphical Abstract Highlights d Stabilization of disordered backbone helix promotes histidine autophosphorylation d His-Asp dyad acts as integrative node for backbone and sidechain interactions d This ''double-clamp'' switch allows dual cellular pH and osmolality sensing

EnvZ/OmpR Two-Component Signaling: An Archetype System That Can Function Noncanonically

“…In the case of EnvZ, it is poised to increase autophosphorylation in response to signaling (i.e., OmpR~P levels are relatively low in the absence of envZ) (5), whereas its close homologue CpxA is poised to regulate CpxR~P turnover (i.e., CpxR~P basal levels are high in the absence of cpxA) (62,63). Kinetic studies proposed that the HK phosphatase activity functions to limit cross-talk between highly homologous two-component systems (64), but more recent studies of co-varying residues (65) and our HDXMS experiments determined that what limits cross-talk is the sequence specificity of the RR binding domain of the HK (1,32). It is important to note that concentrations of OmpR far exceed that of EnvZ in E. coli, and RRs in general compared to HKs (7).…”

“…Membrane lipids serve to couple these two domains. A notable change in HDXMS of the full-length solubilized EnvZ incorporated into nanodiscs compared to EnvZc, was in the region of the Gly-rich motif in the ATP-binding site (32). In the presence of lipids, the rate of ATP hydrolysis in the nucleotide binding domain increased, evident as higher turnover.…”

“…This effect of lipids likely forms the basis for how EnvZ/OmpR responds to procaine (47) and other membrane perturbants (48). These agents alter the lipids, which then affect interactions with the glycine-rich loop of the ATP binding domain, stimulating ATP turnover and subsequent phosphorylation at His 243 (32). Stimulation of EnvZ autophosphorylation would lead to repression of ompF and activation of ompC (47)(48)(49).…”

“…Stimulation of EnvZ autophosphorylation would lead to repression of ompF and activation of ompC (47)(48)(49). Regions of full-length EnvZ that showed increased deuterium exchange included the HAMP, kinase, TMs and periplasmic domains (32). Inside bacterial cells, over-expression of EnvZc resulted in a higher level of βgalactosidase activity of an ompC-lacZ fusion compared to the wildtype protein (1).…”

“…Most RRs are two-domain proteins and phosphorylation of the N-terminal receiver domain alters the output of the C-terminal effector domain, which usually involves a stimulation of DNA of these two flanking moieties regulates His 243 tautomerization, rotamerization and phosphorylation. The intrinsic disorder within the locus containing the His 243 side chain affords a low basal level of autophosphorylation (32). High osmolality stabilizes the helical backbone, relieves the Ala 239 carbonyl link to enable anchoring of the His 243 imidazole by a H-bond with Asp 244 at the Nδ of His 243 , enabling phosphotransfer to the Nε of His 243 .…”