Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways

Abstract

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.

Figure 1. Identification of coevolving amino acids in cognate pairs of histidine kinases and response regulators.

(A) Residues in histidine kinases and response regulators that strongly coevolve (adjusted MI score >3.5) are listed with lines connecting covarying pairs. Residues are numbered according to their position in E. coli EnvZ and OmpR. (B–C) Residues in histidine kinases that coevolve with residues in response regulators are shown on a primary sequence alignment of HK853 from T. maritima and EnvZ, RstB, and CpxA from E. coli. Residues in response regulators that strongly coevolve with residues in histidine kinases are shown on a primary sequence alignment of RR468 from T. maritima and OmpR, RstA, and CpxR from E. coli. Residues highly conserved across all two-component signaling proteins are shaded in grey. Coevolving residues are shown in orange and red for the kinase and regulator, respectively. Secondary structure elements, based on the co-crystal structure of HK853 and RR468 from T. maritima , are shown beneath the sequences. (D) Coevolving residues mapped onto the HK853-RR468 structure. Coevolving residues are shown by space-filling and colored as in panels A–C. The side chains of the conserved phosphorylatable histidines and aspartate are shown as magenta sticks. The HK853-RR468 complex is shown in the center with each individual molecule rotated 90° and shown separately.

Figure 2. Rewiring the specificity of response regulators.

(A) The histidine kinases EnvZ and RstB were autophosphorylated and examined for phosphotransfer to the response regulators indicated. The mutations in OmpR(MI-RstA) and OmpR(MI+loop-RstA) are listed at the top. (B) The histidine kinases EnvZ and CpxA were autophosphorylated and examined for phosphotransfer to the response regulators indicated. The mutations in OmpR(MI-CpxR) and OmpR(MI+loop-CpxR) are listed at the top. Each gel image shows phosphotransfer after 0, 10, 30, and 60 seconds. Bands corresponding to autophosphorylated kinases are labeled on the left. If phosphotransfer occurred, bands corresponding to the phosphorylated regulator appear below the kinase band.

Figure 3. Alanine-scanning mutagenesis of EnvZ.

(A) Sequence of the DHp domain of EnvZ showing the residues substituted with alanine in purple. The conserved histidine phosphorylation site is shaded in grey. Numbering and secondary structure elements indicated as in Figure 1C. (B) Autophosphorylation levels of each EnvZ alanine mutant after a 1 minute incubation, expressed as a percentage of that measured for wild-type EnvZ. For gel images, see Figure S3A. (C) Decrease in EnvZ∼P band after incubation with OmpR. Each value was expressed as a percentage of the decrease measured for wild-type EnvZ. Mutants that do not show a decrease in EnvZ∼P could be defective either in phosphotransfer or in dephosphorylation of OmpR∼P (see text for details). (D) Phosphatase activity of EnvZ alanine mutants. Each alanine mutant was tested for dephosphorylation of OmpR∼P and the rate expressed as a percentage of that measured for wild-type EnvZ. (E) Phosphotransfer from EnvZ alanine mutants to RstA. Phosphotransfer was assessed by measuring the increase in labeled RstA after a 10 second incubation. For each mutant, the increase in RstA was normalized to the autophosphorylation level for that kinase and then reported as a fold-change relative to the phosphotransfer for wild-type EnvZ to RstA. In panels B-E, the specificity residues are listed in orange, as in Figure 1C. For panels C and E, the mutant kinases were autophosphorylated for 60 minutes prior to assessing phosphotransfer. Mutants D244A and L249A did not autophosphorylate significantly enough to examine phosphotransfer. For gel images for panels C–D, see Figure S3B. For panel D, the mutant kinases were tested for dephosphorylation of OmpR∼P at 0.5, 1, and 2 minutes (Figure S4).

Figure 4. Converting the phosphotransfer specificity of EnvZ to match RstB and vice versa.

(A) Converting the phosphotransfer specificity of EnvZ to that of RstB. Wild-type EnvZ and each single, double, and triple mutant on the trajectory from EnvZ to RstB were autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 10 seconds. Wild-type RstB (far right) is shown for comparison to EnvZ(VYR). (B) Converting the phosphotransfer specificity of RstB to that of EnvZ. Wild-type RstB and each single, double, and triple mutant on the trajectory from RstB to EnvZ was autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 60 seconds. Wild-type EnvZ (far left) is shown for comparison to RstB(TLA). Arrows connect profiles of mutants differing by a single amino acid substitution.

Figure 5. Complete trajectory-scanning mutagenesis of EnvZ and OmpR.

Each histidine kinase, indicated on the far right, was autophosphorylated and tested for phosphotransfer to each of the response regulators listed across the top. Mutants of EnvZ are named according to the identity of the three specificity residues being examined; for instance, wild-type EnvZ is ‘TLA’ whereas the mutant T250V is ‘VLA’. Mutants of OmpR are named similarly. All phosphotransfer reactions were incubated for 10 seconds with the exception of RstB and CpxA, which were examined at both 10 seconds and 1 minute. Each kinase profile was composed of two separate gels that were run, exposed to phosphor screens, and scanned in parallel. The resulting two gel images were treated identically and then stitched together between OmpR(EVAPFN) and OmpR(EVATTP).

Figure 6. Hierarchical clustering of trajectory-scanning mutagenesis of EnvZ and OmpR.

Phosphotransfer profiles for each EnvZ construct examined in Figure 5 were quantified. The intensity of each response regulator band within a given kinase profile was expressed as a percentage of the maximally phosphorylated response regulator in that profile. Profiles were then clustered in two-dimensions, with the resulting tree shown for the response regulators (top) and histidine kinases (left). For each tree, the major clusters of EnvZ and OmpR mutants are designated by letters. The 1 minute time point profiles for RstB and CpxA are indicated by ‘∧’.

Figure 7. Mutational trajectories from EnvZ/OmpR to RstB/RstA.

EnvZ and OmpR can be converted by a series of single mutations to harbor the specificity residues found in RstB and RstA, respectively, without disrupting phosphotransfer in intermediate stages. (A) A series of single mutations can convert the specificity of EnvZ to match that of RstB and OmpR to match RstA. Starting with the wild type specificity residues in red text at the top, each subsequent line introduces a single mutation (shown in black text) until both sets of specificity residues have been completely changed. As noted in the text, we treated the loop as a single mutation. As shown in panel B, each kinase-regulator pair listed is capable of phosphotransfer and does not include a regulator that is phosphorylated by CpxA. (B) The complete set of intermediates between wild type OmpR (RLR/PFN) and the quadruple mutant (EVA/TTP) are listed. For wild type EnvZ (TLA), the single mutant EnvZ(TYA), the double mutant EnvZ(TYR), and the triple mutant EnvZ(VYR), the set of OmpR mutants recognized by each kinase are shaded, with a merge of all four at the bottom. Mutants that are phosphorylated by CpxA are listed in grey text, all others in black text. Bold lines connect the mutant series shown in panel A.