Structural features discriminating hybrid histidine kinase Rec domains from response regulator homologs

Abstract

In two-component systems, the information gathered by histidine kinases (HKs) are relayed to cognate response regulators (RRs). Thereby, the phosphoryl group of the auto-phosphorylated HK is transferred to the receiver (Rec) domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec (Rec_inter) domain that is typically part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about discriminating features of Rec_inter domains. Here we study the Rec_inter domain of the hybrid HK CckA by X-ray crystallography and NMR spectroscopy. Strikingly, all active site residues of the canonical Rec-fold are pre-arranged for phosphoryl-binding and BeF₃^- binding does not alter secondary or quaternary structure, indicating the absence of allosteric changes, the hallmark of RRs. Based on sequence-covariation and modeling, we analyze the intra-molecular DHp/Rec association in hybrid HKs.

Fig. 1. Schematic representation of two-component vs multi-component signaling pathways and the CckA–ChpT–CtrA/CpdR phosphorelay system.

a Classical two-component system comprised of a histidine kinase and a response regulator. Domain organisation and residues involved in phosphoryl transfer are indicated. b Multistep phosphorelay consisting of a hybrid histidine kinase (HHK), a histidine phosphotransferase, and a response regulator. The histidine phosphotransferase can either be an HPt protein or a pseudo-HK, as shown. c CckA–ChpT–CtrA/CpdR phosphorelay of Caulobacter crescentus. Each subunit of homo-dimeric HHK CckA (left) is comprised of two transmembrane helices (red), two Per-Arnt-Sim (PAS) domains (orange), a dimerization/histidine phosphotransfer (DHp, green) domain, a catalytic ATP binding (CA, beige) domain and a C-terminal Rec (Rec, violet) domain. The domains of ChpT are structurally related to the DHp and CA domains of CckA, although the latter is functionally degenerated. ChpT can phosphorylate two acceptors, the DNA-binding response regulator CtrA and the single-Rec protein CpdR. P∼CtrA binds as a dimer to the origin of replication to inhibit replication initiation. In parallel, CpdR phosphorylation impedes binding of CpdR to the protease complex ClpXP, which would prime the protease for CtrA degradation.

Fig. 2. Structure comparison of CckA^Rec with the canonical fold of CtrA^Rec, and with other Rec domains lacking secondary structure elements.

Cartoon representation with β-strands colored in purple and α-helices in light-blue. Non-canonical elements are indicated in cyan. PDB codes are given in brackets. a CckA^Rec displaying the classical (βα)₅, but with a disordered β4–β5 linker instead of an α4 helix. Note, that α3 is broken into two pieces (α3a and α3b) as indicated by the difference in coloring. b As reference, CtrA^Rec from B. abortus shows the canonical (βα)₅ Rec fold. c The β4–β5 linker of receiver domain Mhun_0886^Rec exhibits irregular loop structure with a small helical section. d The cryptic receiver domain ShkA^Rec1 lacks α3 and binds cyclic-di-GMP that modulates the function of the full-length protein.

Fig. 3. Crystal structure of CckA^Rec.

a Cartoon representation with β-strands colored in purple and α-helices in light-blue. CckA^Rec exhibits the canonical (ßα)₅ fold of response regulator receiver domains, but lacks on ordered α4 helix. The corresponding segment joining β4–β5 is indicated in cyan. The phospho-acceptor D623, acidic pocket residues D578, E579, and K673 implicated in phosphoryl stabilization are shown in full. Also shown are S651 and F670 (with two alternative rotamers) implicated in conformational switching in canonical Rec domains. b Detailed view of the phospho-acceptor site with 2Fo-Fc omit map contoured at 1.2 σ overlayed. Several water molecules are well resolved (red spheres) including water W739 which is neares to the canonical Mg⁺⁺ binding sites. Potential hydrogen bonds (<3.2 Å) are indicated by dashed lines. c, d Full representation of active sites of CckA^Rec (c) and activated PhoB (d) with selected H-bond and coordination interactions indicated by green, stippled lines. Note that despite the absence of Mg⁺⁺ and BeF₃^-, the active site of CckA^Rec is in the activated conformation. See also Supplementary Fig. 3a, b.

Fig. 4. NMR data of apo and BeF₃-activated CckA^Rec.

a Sequence-specific secondary chemical shifts of apo CckA^Rec (top) and BeF₃^- activated CckA^Rec (bottom) relative to the random coil values of Kjaergaard et al. Unassigned residues are marked with an asterisk. Secondary structure elements as defined by the crystal structure are indicated by thick bars, the low propensity α4 helix as defined by NMR by a thin bar. b Structure of CckA^Rec with the positions of secondary structure elements identified by NMR spectroscopy indicated by red (α-helices) and blue (β-strands). The β4–β5 loop and other parts with weak positive α-helical propensity are plotted in pink onto the structure. Unassigned residues are shown in gray. c Chemical shift perturbation of CckA^Rec upon BeF₃⁻ binding. Two significance levels of the chemical shift differences are indicated by the orange and red lines. Not assigned residues are marked with an asterisk. d Chemical shift perturbation values of panel c mapped onto CckA^Rec structure (0.2 − 0.4 ppm, orange; > 0.4 ppm, red; unassigned regions, gray). Residues of the acidic pocket are shown in full. #: Mg⁺⁺ binding site, *: BeF₃⁻ binding site. e Heteronuclar NOEs of apo CckA^Rec (black) and activated CckA^Rec (red).

Fig. 5. Phosphotransfer competent DHp/Rec association in HHKs.

Cartoon representation (DHp, gray/aquamarine; Rec, orange) with interface residues shown in full. Active histidine and aspartate residues participating in the phosphotransfer are linked by a red arrow. Alanine interface residues, which are conserved between the depicted complexes, have boxed labels. a Model of CckA^DHp/CckA^Rec based on the structure shown in panel b. The two DHp helices interacting with Rec (α1’, α2’) are distinguised by color, since they belong to different subunits of the homodimer. Also shown residues of the intramolecular D580–R593–E599–R591 network of CckA^Rec. b Structure of ChpT^DHp/CtrA^Rec (4QPJ). Note, that α1’ and α2’ of DHp belong to the same subunit, in contrast to the situation in CckA.

Fig. 6. Openbook representation of protein interfaces of the CckA–ChpT–CtrA/CpdR phosphorelay of C. crescentus.

On the left, the open book representation of the CckA DHp/Rec model (Fig. 5a) has interacting residues high-lighted by thick bonds and their carbon atoms colored according to individual subcontacts (blue, salmon, green, yellow). The (His-Asp-His-Asp) phosphoryl-transfer path between alternating DHp and Rec domains is indicated by broken arrows. Labels marked by * indicate positions that have been prediced to interact based on co-variation by Skerker et al.. Note, that the two DHp domain of CckA and ChpT superimpose with a rmsd of 1.3 Å (43 Cα positions).

Fig. 7. Comparison of CckA^Rec and CtrA^Rec sequence logos.

Overall conserved residues are marked with gray background, whereas specifically conserved CckA^Rec or CtrA^Rec residues are marked with orange and green background, respectively. The relevant part of the CckA and CtrA sequences of C. crescentus are reproduced below the logos, with intra-domain salt-bridges indicated by light-blue lines. Also indicated are inter-domain salt-bridges (orange lines; see Supplementary Fig. 3c) found in activated, dimeric CtrA^Rec structures. The quintet of functionally relevant residues (ED, D*, S/T, Y/F, K), residues co-evolving with cognate DHp domains(magenta asterisks), and residues of the sub-contact patches defined in Fig. 6 are marked on the top. The CckA^Rec and CtrA^Rec logos are based on the alignment of 132 and 571 sequences, respectively.

Fig. 8. Residue conservation in CckA^Rec and CtrA^Rec mapped onto structures.

a CckA^Rec in cartoon representation with conserved residues in full. b,c Surface representation of CckA^Rec _and CtrA^Rec _with conserved residues colored according to Fig. 7 and the active aspartate in red. The disorered β4–β5 linker of CckA^Rec is indicated by the red dashed line. The top row shows the view onto the α1 face, which would interact with DHp, the bottom row shows the view onto the 4-5-5 face, which is involved in dimerization in CtrA^Rec homologs.

Fig. 9. DHp/Rec contacts as revealed by co-variation analysis of HHK sequences.

a Co-variation scores above 3.0 between DHp (vertical axis) and part of Rec (horizontal axis) residues are shown as filled circles, with size proportional to signal strength. Residues of the sub-contact patches defined in Fig. 6 are indicated by correspondingly colored squares on the axes. Distances <6 Å in the CckA^DHp/CckA^Rec model (Fig. 5a) are indicated by filled yellow-green circles. The full co-variation matrix is given in Supplementary Fig. 6. Co-variation peaks that correspond to close distances are labeled in black (<8 Å) or red (<6 Å). Contacts that constitute potential ion-pairs are labeled in bold with further information given in panel b. b Substitution matrices for selected DHp/Rec residue pairs. Note that, e.g., R341/A592 do not form a contact in the model (due to the short A592 side-chain), but position 592 is often occupied by a D or E.