Structural plasticity and catalysis regulation of a thermosensor histidine kinase

Abstract

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

Fig. 1.

Three distinct conformational states of DesKC. Cartoon representations of the DesK homodimers, with the two α-helical hairpins from the DHp domain highlighted in pink (α1) and blue (α2), the ABDs colored in yellow, and bound nucleotides in green. (A) Overall structures of DesKC_Δ174 (Top) and E188b (Bottom), with mobile ABDs. (B) Structure of DesKC-P (Top), similar to E188a (Bottom), rotated approximately 60° around the vertical axis with respect to (A) for clarity. (C) Structures of V188a (Top) and V188b (Bottom). (D) Superposition of the 11 independent ABDs seen in all DesKC variants. The bound nucleotide is shown in cyan, with the adenine ring stacked against F324 (in yellow). The Mg²⁺ ion (in red) contacts the nucleotide phosphates and two residues (E289 and N293, in green) that belong to the conserved N box (17). The ATP-lid (residues 321–334) shows the largest structural differences and is partially disordered in many crystal structures. (E) Hydrophobic residues (CPK spheres) of one helical hairpin that, upon dimerization, forms the core of the 4-HB in DesKC_Δ174. For each residue, its a/d position within the heptad repeats and the percentage of members of the HisKA_3 subfamily having a hydrophobic residue (AVLMI) at the same position are indicated in parenthesis.

Fig. 2.

Pronounced helical bending in phosphorylated DesKC. (A) The pattern of intrahelical H bonds in monomer A of DesKC-P (Top) and E188a (Bottom) around the phosphorylatable histidine is modified as a consequence of a helical bulge, facilitated by the presence of a conserved glycine (G192). Note that the carbonyl oxygen of D189 (arrow) is not involved in H bond interactions. Other side-chains are omitted for clarity. (B) Close view of the interaction between helix α1 and the ABD from the opposite monomer in E188a.

Fig. 3.

Extensive intradomain interactions in DesKC_H188V. (A) Close view of the interaction between the ABDs and the DHp domain in DesKC_H188V. Key residues are colored according to the type of interaction (electrostatic in red, hydrophobic in blue, and H-bonding in green). (B) Cartoon representation (Right) of the two-helical coiled-coil formed by the homodimerization of residues 160–180. Core hydrophobic residues are shown in stick representation. Molecular surface representation (Left) of the same helical region for one monomer, showing the exposed hydrophobic patch. (C) Overall view of the parallel coiled-coil and the 4-HB in DesKC_H188V. The molecular surface color-coded according to electrostatic charges is shown for helix α1 in one monomer. The side-chains of I183 and L187, which were part of the DHp core in DesKC_Δ174, are now at the outer surface of the domain (engaged in interactions with the ABD domain, not shown).

Fig. 4.

Biochemical characterization of DesK proteoliposomes. Left (DesK inserted in liposomes) and right (soluble DesKC) panels show (A) autokinase, (B) phosphatase, and (C) phosphotransferase activities at the indicated times (min), assayed at 25 °C (Top) and 37 °C (Bottom). To perform DesK phosphotransferase activity assays, the protein was first allowed to autophosphorylate at 25 °C and subsequently incubated with DesR at both temperatures. The bands corresponding to each phosphorylated protein are indicated, see SI Text and Fig. S7 for further details.

Fig. 5.

Interhelical rearrangements in the DHp domain modulate intradomain and protein-protein interactions. (A) Cartoon representation of the helical rotations taking place in the 4-HB. The DHp helices from one monomer of DesKC_H188V (carbon atoms in yellow) and DesKC_Δ174 (C atoms in magenta) are superimposed. Key residues in interdomain contacts are labeled and selected bond distances marked. Black dotted arrows connect the same residue on both structures, red dotted arrows highlight the cogwheel rotation and the green dotted arrows indicate the tilt of the two-helix hairpin. (B) Phosphatase activity of DesK_DHp proteoliposomes assayed at 25 °C and 37 °C (see SI Text for details).

Fig. 6.

Proposed model of catalysis regulation. The observed structures of the DesKC homodimer (shown as surface models with helix α1 highlighted) are ascribed to the three functional states of the kinase: phosphatase-competent (DesK*), kinase-competent (DesK), and phosphotransferase-competent (DesK-P). The corresponding reactions (P, K, PT) are indicated in the lower panel.