How important is the phosphatase activity of sensor kinases?

Abstract

In two-component signaling systems, phosphorylated response regulators (RRs) are often dephosphorylated by their partner kinases in order to control the in vivo concentration of phospho-RR (RR approximately P). This activity is easily demonstrated in vitro, but these experiments have typically used very high concentrations of the histidine kinase (HK) compared to the RR approximately P. Many two-component systems exhibit exquisite control over the ratio of HK to RR in vivo. The question thus arises as to whether the phosphatase activity of HKs is significant in vivo. This topic will be explored in the present review.

Figure 1

Crystal structure of the HK853CP-RR468 Complex [5••], PDB accession number is 3DGE. Ribbon diagram of the complex viewed with the cell membrane at the top. The DHp and CA domains are indicated. Reprinted with permission from [5••].

Figure 2

Activities underlying OmpR/EnvZ signaling. It is presumed that, at low osmolality, the level of intracellular OmpR~P is low either because the kinase activity of EnvZ is low, or because EnvZ phosphatase activity is high. At high osmolality OmpR~P levels increase either because of an increase in the EnvZ kinase activity or a decrease in EnvZ phosphatase activity.

Figure 3

EnvZc binding to OmpR using fluorescence anisotropy. The binding reactions contained 40 nM fluorescein-labeled OmpR. Closed triangles show OmpR binding to EnvZc with a K_d of 519 nM. The average K_d for EnvZc binding to OmpR from 10 independent experiments was 425 ± 127 nM. In the presence of specific DNA, EnvZc still binds OmpR, with a K_d of 568 nM (open triangles). The average K_d for EnvZc binding to OmpR C1-C2-C3 from six separate curves is 385 ± 162 nM. Reprinted with permission from [29].

Figure 4

Fluorescence resonance energy transfer (FRET) with EnvZ-GFP to fluorescent-OmpR. EnvZ-GFP was over-expressed, and spheroplasts were prepared and lysed in cold H₂O according to Osborn et al. [63]. Fluorescent-OmpR concentrations ranged from 0 to 10 µM in the presence of EnvZ-GFP. A control experiment was performed with 0 to 1000 nM unconjugated fluorophore in the presence of 250 nM EnvZ-GFP to measure the amount of non-specific interaction of the donor (GFP) and acceptor fluorophores. A curve comparing FRET with OmpR and OmpR~P is shown in the inset. K_d OmpR = 0.5 µM; OmpR~P = 1.6 µM [50]. EnvZ-GFP was a kind gift from M. Goulian. Reprinted with permission from [50].

Figure 5

Does a high k_cat compensate for a high K_d? Measurements of OmpR-stimulated ATPase activity represent the sum of the phosphorylation/phosphotransfer reactions. The affinity of EnvZ for OmpR~P is >1.5 µM [50]. Can this low affinity be overcome by a high turnover rate that could rapidly reduce OmpR~P levels? ATPase assays were carried out in a 0.6-ml volume containing 125 mM NaCl, 4 mM MgCl2, 60 mM Tris-HCl (pH 7.5), 0.75 mM EDTA, and various concentrations of EnvZ (0.015–7.5 µM) and OmpR (1.5 µM). The apparent affinity of EnvZ for ATP is 200 µM [64]. Reactions were initiated by addition of 4 mM ATP and conducted as described in [29,64]. The P_i produced in the presence of EnvZ was subtracted from the total P_i produced in the presence of OmpR. The symbol represents the mean, and error bars indicate the standard deviation of three data points obtained at each time point. The data shown are a representative experiment. At ratios that approximate in vivo levels of EnvZ and OmpR (approximately 1:35) [33], there is almost no turnover of ATP. Inset: At low ratios of EnvZ to OmpR (1:100), the kinase is still active and OmpR~P levels actually accumulate over the incubation period. Phosphotransfer rates of EnvZ~P to OmpR are extremely fast [31••]. Figure 5 was part of Kirstin Mattison’s Ph.D thesis at Oregon Health and Sciences University.