Helix bundle loops determine whether histidine kinases autophosphorylate in cis or in trans

Abstract

Bacteria frequently use two-component signal transduction pathways to sense and respond to environmental and intracellular stimuli. Upon receipt of a stimulus, a homodimeric sensor histidine kinase autophosphorylates and then transfers its phosphoryl group to a cognate response regulator. The autophosphorylation of histidine kinases has been reported to occur both in cis and in trans, but the molecular determinants dictating which mechanism is employed are unknown. Based on structural considerations, one model posits that the handedness of a loop at the base of the helical dimerization domain plays a critical role. Here, we tested this model by replacing the loop from Escherichia coli EnvZ, which autophosphorylates in trans, with the loop from three PhoR orthologs that autophosphorylate in cis. These chimeric kinases autophosphorylated in cis, indicating that this small loop is sufficient to determine autophosphorylation mechanism. Further, we report that the mechanism of autophosphorylation is conserved in orthologous sets of histidine kinases despite highly dissimilar loop sequences. These findings suggest that histidine kinases are under selective pressure to maintain their mode of autophosphorylation, but they can do so with a wide range of sequences.

Figure 1. Histidine kinases autophosphorylate in cis or in trans

(A) The cytoplasmic region of a histidine kinase (PDB ID 2C2A) consists of conserved DHp and CA domains. The histidine site of phosphorylation on the DHp domain, and the ATP analog bound by the CA domain, are shown in stick form in green and cyan, respectively. The kinase shown autophosphorylates in cis. (B) Cartoon (left) looking down the four-helix bundle (right) of the DHp-domain dimer. The α1 and α2 helices in the DHp domain are labeled, with the prime symbol (’) symbol denoting the opposite chain (top: PDB ID 3ZRX, bottom: PDB ID 2C2A). The loop at the base of the DHp domain is depicted by an arrow, and the linker between the DHp and CA domains is depicted as a wavy line to reflect the mobility of the CA domain. Depending on loop handedness in the DHp domain, the CA domain is either closer to the histidine on the same chain (cis) or the histidine on the opposite chain (trans). (C) Schematic of the assay to test in cis vs. in trans autophosphorylation. A wild-type (WT) histidine kinase homodimer is mixed with excess mutant (MUT) histidine-kinase homodimer unable to bind ATP. Autophosphorylation within the heterodimer is initiated by the addition of radiolabeled ATP, and the chains in the dimer are then separated by SDS-PAGE. In the heterodimer, either the WT or MUT chain is labeled, depending on whether the kinase autophosphorylates in cis or in trans, respectively. In addition, WT homodimer, also present in the WT+MUT mixture, undergoes autophosphorylation. To confirm autophosphorylation in cis, the kinetics of the WT and WT plus excess MUT reactions are compared.

Figure 2. Alignments of EnvZ and PhoR orthologs and loop chimeras

(A) Sequence alignment of DHp domains from EnvZ and PhoR orthologs characterized in this study. Locations of the two helices in the DHp domain, α1 and α2, and the loop connecting them are indicated. The depicted ortholog loop boundaries are based on sequence alignment to E. coli EnvZ and its loop boundary. The histidine site of phosphorylation is colored in green. Columns where one residue is conserved in more than half of 118 EnvZ or 385 PhoR orthologs are shaded in yellow. The conservation graph below each alignment measures information for each DHp position across all orthologs (see Methods). (B) Alignment of DHp domains of loop chimeras. Sequence changes relative to E. coli EnvZ are highlighted in either red (RstB) or blue (PhoR).

Figure 3. Autophosphorylation mechanism within EnvZ and PhoR orthologs is conserved

(A) Characterization of autophosphorylation mechanism for EnvZ orthologs. In each ortholog series, the first gel lane contained 5 μM wild-type (WT) kinase, the second lane contained 50 μM mutant (MUT) kinase, and the third lane contained a mixture of the two kinases. The mixture was incubated for four hours at 30 °C before initiating autophosphorylation. (B) Characterization of autophosphorylation mechanism for PhoR orthologs. The gel for each ortholog included two autophosphorylation time courses: the first for 5 μM wild-type kinase alone and the second for 5 μM wild-type kinase plus 50 μM mutant kinase. The final lane contained 50 μM mutant kinase. The autophosphorylation time is marked in each lane. Asterisks indicate possible weak in trans autophosphorylation, and solid circles mark a breakdown product found in both the WT and WT + MUT reactions. The slower mobility band seen with D. vulgaris PhoR is a PhoR dimer. Gel images were quantified and are plotted in Supplementary Fig. 2.

Figure 4. Changing DHp loop sequence changes autophosphorylation mechanism

(A) Characterization of autophosphorylation mechanism in E. coli EnvZ, E. coli RstB, and the EnvZ-RstB chimera, as in Fig. 3A. (B) Autophosphorylation time courses for E. coli EnvZ and the EnvZ-PhoR loop chimeras, as in Fig. 3B. Gel images were quantified and are plotted in Supplementary Fig. 4.

Figure 5. Phosphotransfer specificity and dimerization specificity of loop chimeras

(A) Each kinase was assayed for its ability to interact with E. coli EnvZ using a FRET competition assay. FRET signal from a complex of CFP-EnvZ and YFP-EnvZ was disrupted by interaction with unlabeled mutant (MUT) kinase (E. coli EnvZ, EnvZ-RstB chimera, EnvZ-PhoR loop chimeras, or E. coli EnvZ-E. coli PhoR*). Equilibrium dissociation constants (K_d) were fit for each kinase homodimer and for each kinase heterodimer with EnvZ. (B) E. coli EnvZ and the EnvZ loop chimeras were assayed for their ability to phosphotransfer to the response regulator (RR) E. coli OmpR. Autophosphorylated histidine kinase (HK) was incubated for the indicated times with OmpR either present (top gel) or absent (bottom gel). Each kinase was able to phosphotransfer to OmpR, as seen by decreased levels of autophosphorylated kinase upon incubation with OmpR. On the right are cartoon depictions of a response regulator interacting with the DHp domain dimer of a kinase that phosphorylates in trans (left) or in cis (right). The mode of interaction in the cis kinase is based on a crystal structure (PDB ID 3DGE), and the mode of interaction in the trans kinase is a model. (C) A DHp dimer formed between chains with different loop handedness cannot form the stereotypical DHp interface. In such a dimer, either the usual buried dimer interface (represented by solid black rectangles) will not be formed, or the arrangement of the α1 and α2 helices will be altered from what is observed in known structures.