Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation

Abstract

Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. Here, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light-oxygen-voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain. The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. We suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.

Keywords: cell signaling; histidine kinase; photosensory; regulation; two-component system.

Fig. 1.

EL346 is a light-regulated, monomeric HK. (A) EL346 autokinase activity under dark and lit conditions. A representative autoradiogram is shown. Data points (dark and lit conditions with closed and open circles, respectively) represent the average ± 1 SD for n = 3 measurements. (B) Molecular weight of ATP-bound EL346 in the dark and lit states, as measured by SEC-MALS. (Inset) Correlations of elution volumes and log(MW) for EL346 and standards (158, 66.5, 44, and 17 kDa). (C) Concentration dependence of EL346 autokinase activity, showing a linear relationship between protein concentration and autokinase activity, consistent with EL346 not requiring dimerization to undergo autophosphorylation.

Fig. 2.

Full-length EL346 crystal structure highlights conserved features of sensor and catalytic domains. (A, Top and Middle) Domain architecture and crystal structure of EL346FL, with domains highlighted in green (LOV), blue (DHpL) and yellow (CA). The riboflavin cofactor (RBF), His¹⁴², and AMP-PNP are represented in sticks. (Bottom) Structural comparisons with AsLOV2 (PDB ID code 2V0U) (13) and residues 199–302 of VicK chain B, with chain A shown in gray (PDB ID code 4I5S) (19). (B) Detail of selected residues in the LOV/DHpL and DHpL/CA interfaces.

Fig. 3.

DHpL inter- and intradomain interactions in the EL346FL structure. (A) DHpL and LOV interaction. The β-sheet side of the LOV domain is shown in surface representation, and the DHpL helices are presented in transparent ribbon representation with the side chains of heptad repeat residues as sticks. (B) DHpL heptad repeat. Hydrophobic residues that pack between helices α1 and α2 are indicated as gray spheres in the structure and shaded in gray in the sequences. Positions a to g in the heptad repeats are indicated in italics under the sequences. (C) DHpL/CA arrangement in the EL346FL structure showing the location of conserved residue pairs and distance between AMP-PNP and His142.

Fig. 4.

Light induces widespread conformational changes in EL346 as revealed by solution NMR spectroscopy. ¹⁵N/¹H TROSY spectra of EL346, showing the effects of illumination and AMP-PNP binding.

Fig. 5.

LOV domain photoactivation changes the DHpL/CA interface. (A) Limited trypsinolysis of EL346. (Left) SDS/PAGE of samples and molecular weight marker (M) reveals light-dependent changes in trypsinolysis pattern. (Right) Schematic of fragments corresponding to SDS/PAGE bands, using masses from electrospray ionization mass spectrometry (ESI‐MS) analysis. (B) Locations of residues selected for site-directed mutagenesis (spheres colored by domain assignment, shown on diagram representation of EL346). His142, riboflavin (RBF), and AMP-PNP are shown in sticks, as are additional residues that interact with those that were mutated. Arrowheads indicate light-independent (white) and light-dependent (black) cleavage sites observed by limited trypsinolysis. (C) Initial rate of autophosphorylation of mutants under lit and dark conditions. Assays were performed at least in duplicate, and their results are shown as the average ± 1 SD. A C55A mutant was used as a negative control for blue-light activation.

Fig. 6.

Parallels between monomeric and dimeric HK activation models. (A) In the dark state, extensive interactions between domains (orange) hold the monomeric HK EL346 in an inhibited conformation. Photoactivation of the LOV domain alters LOV/DHpL interactions, leading to signal propagation (black arrow). These changes disturb critical DHpL/CA interactions, releasing the CA domain to move from its inhibited conformation to phosphorylate His142. (B) Analogous activation model for a membrane-bound dimeric HK. Here, sensors at a distance modulate DHp structure. Signal propagation through the DHp prompts the release of inhibitory interactions between DHp and CA.