How to Switch Off a Histidine Kinase: Crystal Structure of Geobacillus stearothermophilus KinB with the inhibitor Sda

Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-Å-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to which it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.     

The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F

Histidine kinases are widely used by bacteria, fungi and plants to sense and respond to changing environmental conditions. Signals in addition to those directly sensed by the kinase are often integrated by proteins that fine-tune the biological response by modulating the activity of the kinase or its targets. The Bacillus subtilis histidine kinase KinA promotes the initiation of sporulation when nutrients are limiting, but sporulation can be delayed by two inhibitors of KinA, Sda (when DNA replication is perturbed) or KipI (under unknown conditions). We have identified residues in the dimerization/histidine-phosphotransfer (DHp) domain of KinA that are functionally important for inhibition by Sda and KipI and overlapping surface-exposed residues that lie close to or comprise the Sda binding site. Sda inhibits the intermolecular transfer of phosphate from the catalytic ATP-binding (CA) domain of KinA to the autophosphorylation site in the DHp domain when the domains are split into separate polypeptides, either by steric hindrance or by altering the conformation of the DHp domain. Sda also slows the rate of phosphotransfer from KinA∼P to its target, Spo0F, consistent with our finding that a KinA residue important for Sda function overlaps with the predicted Spo0F binding site on KinA.     

Histidine kinase regulation by a cyclophilin-like inhibitor

The sensor histidine kinase A (KinA) from Bacillus subtilis triggers a phosphorelay that activates sporulation. The antikinase KipI prevents sporulation by binding KinA and inhibiting the autophosphorylation reaction. Using neutron contrast variation, mutagenesis, and fluorescence data, we show that two KipI monomers bind via their C-domains at a conserved proline in the KinA dimerization and histidine-phosphotransfer (DHp) domain. Our crystal structure of the KipI C-domain reveals the binding motif has a distinctive hydrophobic groove formed by a five-stranded antiparallel β-sheet; a characteristic of the cyclophilin family of proteins that bind prolines and often act as cis-trans peptidyl-prolyl isomerases. We propose that the DHp domain of KinA transmits conformational signals to regulate kinase activity via this proline-mediated interaction. Given that both KinA and KipI homologues are widespread in the bacterial kingdom, this mechanism has broad significance in bacterial signal transduction.     

Expression level of a chimeric kinase governs entry into sporulation in Bacillus subtilis

Upon starvation, Bacillus subtilis cells switch from growth to sporulation. It is believed that the N-terminal sensor domain of the cytoplasmic histidine kinase KinA is responsible for detection of the sporulation-specific signal(s) that appears to be produced only under starvation conditions. Following the sensing of the signal, KinA triggers autophosphorylation of the catalytic histidine residue in the C-terminal domain to transmit the phosphate moiety, via phosphorelay, to the master regulator for sporulation, Spo0A. However, there is no direct evidence to support the function of the sensor domain, because the specific signal(s) has never been found. To investigate the role of the N-terminal sensor domain, we replaced the endogenous three-PAS repeat in the N-terminal domain of KinA with a two-PAS repeat derived from Escherichia coli and examined the function of the resulting chimeric protein. Despite the introduction of a foreign domain, we found that the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. Further, by using chemical cross-linking, we showed that the chimeric protein exists predominantly as a tetramer, mediated by the N-terminal domain, as was found for KinA. These results suggest that tetramer formation mediated by the N-terminal domain, regardless of the origin of the protein, is important and sufficient for the kinase activity catalyzed by the C-terminal domain. Taken together with our previous observations, we propose that the primary role of the N-terminal domain of KinA is to form a functional tetramer, but not for sensing an unknown signal.     

In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis

The phosphorylated form of the response regulator Spo0A (Spo0A~P) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0A~P-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.     

Modification in hydrophobic packing of HAMP domain induces a destabilization of the auto‐phosphorylation site in the histidine kinase CpxA

The histidine kinases belong to the family of two‐component systems, which serves in bacteria to couple environmental stimuli to adaptive responses. Most of the histidine kinases are homodimers, in which the HAMP and DHp domains assemble into an elongated helical region flanked by two CA domains. Recently, X‐ray crystallographic structures of the cytoplasmic region of the Escherichia coli histidine kinase CpxA were determined and a phosphotransferase‐defective mutant, M228V, located in HAMP, was identified. In the present study, we recorded 1 μs molecular dynamics trajectories to compare the behavior of the WT and M228V protein dimers. The M228V modification locally induces the appearance of larger voids within HAMP as well as a perturbation of the number of voids within DHp, thus destabilizing the HAMP and DHp hydrophobic packing. In addition, a disruption of the stacking interaction between F403 located in the lid of the CA domain involved in the auto‐phosphorylation and R296 located in the interacting DHp region, is more often observed in the presence of the M228V modification. Experimental modifications R296A and R296D of CpxA have been observed to reduce also the CpxA activity. These observations agree with the destabilization of the R296/F403 stacking, and could be the sign of the transmission of a conformational event taking place in HAMP to the auto‐phosphorylation site of histidine kinase. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 670–682, 2016.     

An extended hydrophobic core induces EF-hand swapping

The structure of calbindin D(9k) with two substitutions was determined by X-ray crystallography at 1.8-A resolution. Unlike wild-type calbindin D(9k), which is a monomeric protein with two EF-hands, the structure of the mutated calbindin D(9k) reveals an intertwined dimer. In the dimer, two EF-hands of the monomers have exchanged places, and thus a 3D domain-swapped dimer has been formed. EF-hand I of molecule A is packed toward EF-hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF-hands, promotes the conversion of monomers to 3D domain-swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D(9k). Once formed, the calbindin D(9k) dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D(9k) dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.     

1H, 13C and 15N backbone and side chain resonance assignments of the N-terminal domain of the histidine kinase inhibitor KipI from Bacillus subtilis

KipI is a sporulation inhibitor in Bacillus subtilis which acts by binding to the dimerisation and histidine phosphotransfer (DHp) domain of KinA, the principle input kinase in the phosphorelay responsible for sporulation. The ¹⁵N, ¹³C and ¹H chemical shift assignments of the N-terminal domain of KipI were determined using multidimensional, multinuclear NMR experiments. The N-terminal domain has two conformers and resonance assignments have been made for both conformers.     

Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis

Bacillus subtilis is able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community.     

The crystal structure of beryllofluoride Spo0F in complex with the phosphotransferase Spo0B represents a phosphotransfer pretransition state

A number of regulatory circuits in biological systems function through the exchange of phosphoryl groups from one protein to another. Spo0F and Spo0B are components of a phosphorelay that control sporulation in the bacterium Bacillus subtilis through the exchange of a phosphoryl group. Using beryllofluoride as a mimic for phosphorylation, we trapped the interaction of the phosphorylated Spo0F with Spo0B in the crystal lattice. The transition state of phosphoryl transfer continues to be a highly debated issue, as to whether it is associative or dissociative in nature. The geometry of Spo0F binding to Spo0B favors an associative mechanism for phosphoryl transfer. In order to visualize the autophosphorylation of the histidine kinase, KinA, and the subsequent phosphoryl transfer to Spo0F, we generated in silico models representing these reaction steps.     

Small-angle X-ray scattering reveals the solution structure of a bacteriophytochrome in the catalytically active Pr state

Phytochromes are light-sensing macromolecules that are part of a two component phosphorelay system controlling gene expression. Photoconversion between the Pr and Pfr forms facilitates autophosphorylation of a histidine in the dimerization domain (DHp). We report the low-resolution structure of a bacteriophytochrome (Bph) in the catalytic (CA) Pr form in solution determined by small-angle X-ray scattering (SAXS). Ab initio modeling reveals, for the first time, the domain organization in a typical bacteriophytochrome, comprising an chromophore binding and phytochrome (PHY) N terminal domain followed by a C terminal histidine kinase domain. Homologous high-resolution structures of the light-sensing chromophore binding domain (CBD) and the cytoplasmic part of a histidine kinase sensor allows us to model 75% of the structure with the remainder comprising the phytochrome domain which has no 3D representative in the structural database. The SAXS data reveal a dimeric Y shaped macromolecule and the relative positions of the chromophores (biliverdin), autophosphorylating histidine residues and the ATP molecules in the kinase domain. SAXS data were collected from a sample in the autophosphorylating Pr form and reveal alternate conformational states for the kinase domain that can be modeled in an open (no-catalytic) and closed (catalytic) state. This model suggests how light-induced signal transduction can stimulate autophosphorylation followed by phosphotransfer to a response regulator (RR) in the two-component system.     

Characterization of the Soj/Spo0J chromosome segregation proteins and identification of putative parS sequences in Helicobacter pylori

The Soj and Spo0J proteins, together with one or more parS sequences, are crucial to chromosome segregation and the progression of cell cycle in many bacteria. In Helicobacter pylori, genes coding for Soj and a plasmid replication-partition-related protein containing a Spo0J or ParB conserved domain, together with two putative parS sites identified in this study, were found to be located within the origin-proximal 20-30% of the circular chromosome. Recombinant H. pylori Spo0J bound specifically to the two putative parS sequences and that of Bacillus subtilis. In addition, hydrolysis of ATP by H. pylori Soj was accelerated in the presence of parS and/or Spo0J. Protein-protein interactions, intracellular levels, and subcellular localization of Soj and Spo0J were analyzed through polyclonal antibodies directed against recombinant Soj and Spo0J. This study was the first implication of the existence of a functional parABS system in H. pylori.