Determinants of homodimerization specificity in histidine kinases

Abstract

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.

Figure 1. The Cytoplasmic Domains of E. coli Histidine Kinases EnvZ and RstB Specifically Homodimerize

(A) Schematic of in vitro FRET competition assay. FRET signal from a complex of CFP-kinase A and YFP-kinase A is reduced by interaction with an unlabeled competitor kinase. (B) FRET competition assay for homo-association and hetero-association of EnvZ_HDC and RstB_HDC. Protein concentrations were 0.5 µM CFP-EnvZ_HDC and 0.5 µM YFP-EnvZ_HDC (left panel) or 0.5 µM CFP-RstB_HDC and 0.5 µM YFP-RstB_HDC (right panel). The curves are fit as described in the methods. Cartoons of EnvZ and RstB show the HAMP, DHp and CA domains. (C) Pull-down assay with purified EnvZ_HDC and RstB_HDC. A mixture of FLAG-labeled kinase and MBP-labeled kinase was incubated and complexes were isolated using anti-FLAG beads. Odd lanes show inputs and even lanes show elutions. The following mixtures were assayed: FLAG-EnvZ_HDC + MBP-EnvZ_HDC (lane 2), FLAG-RstB_HDC + MBP-RstB_HDC (lane 4), FLAG-EnvZ_HDC + MBP-RstB_HDC (lane 6), FLAG-RstB_HDC + MBP-EnvZ_HDC (lane 8). Non-specific binding to beads was assessed for MBP-EnvZ_HDC (lane 10) and MBP-RstB_HDC (lane 12).

Figure 2. The DHp Domains of EnvZ and RstB Homodimerize

FRET competition assay for homo-association and hetero-association of EnvZ_DC (left panel) and RstB_DC (right panel). CFP and YFP fusion protein concentrations were 20 µM.

Figure 3. Covarying Pairs in the EnvZ Dimerization Interface

(A) Residues in EnvZ that highly covary and are within 5.5 Å across the dimerization interface are listed (at left) and shown on the NMR structure of EnvZ (at right). Covarying residues in helices α1 and α2 are connected by lines. Cluster 1 residues are shown in green, cluster 2 residues are shown in purple and all other covarying, interchain residues are shown in grey. The conserved histidine is shown in orange. (B) Close-up views of clusters 1 and 2. The chimera-1-region backbone (residues 256–265) is in red; clusters 1 and 2 are outside that region.

Figure 4. EnvZ-RstB Chimeric Proteins Isolate Dimerization Specificity to the DHp Domain Base

(A) Sequence alignment of the DHp domains of EnvZ, RstB and chimeras 1–4. Amino acids mutated, relative to EnvZ, to make the chimeras are shown in bold. The columns highlighted in color correspond to covarying residues in cluster 1 (I252, T256, I270) and cluster 2 (A255, L266, S269). The highlighted column in orange corresponds to the highly conserved histidine. Locations of the two helices in the DHp domain are indicated. (B) Models of chimeras 1–4 showing residues from EnvZ in blue and residues from RstB in red. The EnvZ NMR structure is used as a template. The chimeras are fused to the EnvZ CA domain, which is omitted in the models. (C) Interactions between EnvZ_DC or chimeras 1–4 with EnvZ_HDC (5 µM each of the CFP and YFP fusions).

Figure 5. Phosphotransfer Specificity for Dimerization Specificity Switch Mutants

Each kinase (HK) was autophosphorylated with radiolabeled ATP and either incubated alone (−) or assayed for phosphotransfer to a response regulator (RR). (A) EnvZ_DC and RstB_HDC phosphorylated their cognate response regulators, OmpR and RstA, respectively. Chimera 2 showed a switch in phosphotransfer specificity relative to chimera 1. (B) Phosphotransfer specificity of EnvZ_DC dimerization specificity switch mutants Mut 1–3, defined as in the figure. (C) View from the base of the four-helix bundle in EnvZ, with positions affecting interaction specificity shown in space-filling form. Cluster 1 positions 252, 256, and 270, and cluster 2 position 266 (blue) are buried in the dimerization interface. Positions 250, 254, 255, and 269, previously identified to affect histidine kinase-response regulator phosphotransfer specificity (red) are solvent-exposed. The residues are mapped onto the EnvZ DHp domain.