Segmental Helical Motions and Dynamical Asymmetry Modulate Histidine Kinase Autophosphorylation

Histidine kinases (HKs) are dimeric receptors that participate in most adaptive responses to environmental changes in prokaryotes. Although it is well established that stimulus perception triggers autophosphorylation in many HKs, little is known on how the input signal propagates through the HAMP domain to control the transient interaction between the histidine-containing and ATP-binding domains during the catalytic reaction. Here we report crystal structures of the full cytoplasmic region of CpxA, a prototypical HK involved in Escherichia coli response to envelope stress. The structural ensemble, which includes the Michaelis complex, unveils HK activation as a highly dynamic process, in which HAMP modulates the segmental mobility of the central HK α-helices to promote a strong conformational and dynamical asymmetry that characterizes the kinase-active state. A mechanical model based on our structural and biochemical data provides insights into HAMP-mediated signal transduction, the autophosphorylation reaction mechanism, and the symmetry-dependent control of HK kinase/phosphatase functional states.     

The CUL7/F-box and WD Repeat Domain Containing 8 (CUL7/Fbxw8) Ubiquitin Ligase Promotes Degradation of Hematopoietic Progenitor Kinase 1

HPK1, a member of mammalian Ste20-like serine/threonine kinases, is lost in >95% pancreatic cancer through proteasome-mediated degradation. However, the mechanism of HPK1 loss has not been defined. The aims of this study are to identify the ubiquitin ligase and to examine the mechanisms that targets HPK1 degradation. We found that the CUL7/Fbxw8 ubiquitin ligase targeted HPK1 for degradation via the 26 S proteasome. The ubiquitination of HPK1 required its kinase activity and autophosphorylation. Wild-type protein phosphatase 4 (PP4), but not the phosphatase-dead PP4 mutant, PP4-RL, inhibits the interaction of Fbxw8 with HPK1 and Fbxw8-mediated ubiquitination of HPK1. In addition, we showed that Thr-355 of HPK1 is a key PP4 dephosphorylation site, through which CUL7/Fbxw8 ubiquitin ligase and PP4 regulates HPK1 stability. Knockdown of Fbxw8 restores endogenous HPK1 protein expression and inhibits cell proliferation of pancreatic cancer cells. Our study demonstrated that targeted degradation of HPK1 by the CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain the activity of HPK1 and that CUL7/Fbxw8 ubiquitin ligase promotes pancreatic cancer cell proliferation. CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 degradation revealed a direct link and novel role of CUL7/Fbxw8 ubiquitin ligase in the MAPK pathway, which plays a critical role in cell proliferation and differentiation.     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase)

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.     

Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B,as Revealed by Nanobody Binding

The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell.     

Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis

After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry.We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties.     

Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein

The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADPbetaN at 1.9 A resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts.     

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.     

Protein kinase activity of rat brain hexokinase

Hexokinase 1 (HK1) purified from rat brain exhibits protein kinase activity, including autophosphorylation and phosphorylation of histone H2A. This protein kinase activity is observed only in the absence of the HK1 carbohydrate substrate, glucose. Analysis of the ATP-binding domains of the mammalian HK1 protein sequences shows significant homology with other mammalian protein kinases.     

Anti-HIV siamycin I directly inhibits autophosphorylation activity of the bacterial FsrC quorum sensor and other ATP-dependent enzyme activities

Siamycin I disrupts growth and quorum sensing in Enterococcus faecalis. Using purified intact protein, we demonstrate here that quorum membrane sensor kinase FsrC is a direct target of siamycin I, reducing pheromone-stimulated autophosphorylation activity by up to 91%. Inhibition was non-competitive with ATP as substrate. Other ATP-binding enzymes were also inhibited, including nine other membrane sensor kinases of E. faecalis, Rhodobacter sphaeroides PrrB, porcine Na(+)-dependent ATPase and the catalytic subunit of bovine protein kinase A, but not bacterial β-galactosidase, confirming targeted inhibition of a wide range of ATP dependent reactions, and elucidating a likely mechanism underlying the lethality of the inhibitor.     

Molecular dynamics study of enhanced autophosphorylation by S904F mutation of the RET kinase domain

The aberrant kinase activity of RET (rearranged during transfection), a transmembrane tyrosine kinase, is associated with human cancer. A point mutation caused by the replacement of solvent-front hydrophilic S904, located on the activation loop (A-loop), with a bulky hydrophobic phenylalanine residue can induce resistance to the type I kinase inhibitor vandetanib. A possible mechanism of this drug resistance is the release of a cis-autoinhibited conformation of RET for autophosphorylation, which activates RET kinase. Because the association between S904F mutation and enhanced autophosphorylation is unclear, we conducted molecular modeling analysis to compare unphosphorylated apo wild-type and S904F mutant structures. The structural compactness of the A-loop promoted ATP binding. When the A-loop is extended, the αC helix moves toward the glycine-rich loop, resulting in the protrusion of F735. The extruded F735 connects with E734 and R912 and constrains the ATP pocket entrance. Contrarily, a contracted A-loop pulls the αC helix away from the glycine-rich loop, burying F734 and making the ATP pocket accessible. The mutated F904 stabilizes the contracted A-loop and releases the autoinhibited conformation of RET, thereby facilitating autophosphorylation. We also simulated two ATP-bound systems. The binding free energies of ATP, estimated through the molecular mechanics with a generalized Born and surface area solvation approach, revealed that the S904F mutant was bound more tightly than was the wild type with the ATP. The findings support the premise of autophosphorylation promotion in the S904F mutant.     

On helicases and other motor proteins

Helicases are molecular machines that utilize energy derived from ATP hydrolysis to move along nucleic acids and to separate base-paired nucleotides. The movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. Recent structural data for the hexameric E1 helicase of papillomavirus in complex with single-stranded DNA and MgADP has provided a detailed atomic and mechanistic picture of its ATP-driven DNA translocation. The structural and mechanistic features of this helicase are compared with the hexameric helicase prototypes T7gp4 and SV40 T-antigen. The ATP-binding site architectures of these proteins are structurally similar to the sites of other prototypical ATP-driven motors such as F1-ATPase, suggesting related roles for the individual site residues in the ATPase activity.     

Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1

Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-kappaB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-kappaB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.     

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC₂; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC₂, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg²⁺. We were able to purify a full complex, RbsABC₂, in the presence of stable, transition state mimics (ATP, Mg²⁺, and VO₄); a RbsAC complex in the presence of ADP and Mg²⁺; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC₂ shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.     

Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.     

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.     

Structural analysis of the human Rad51 protein-DNA complex filament by tryptophan fluorescence scanning analysis: transmission of allosteric effects between ATP binding and DNA binding

Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction.     

Molecular model and ATPase activity of carboxyl-terminal nucleotide binding domain from human P-glycoprotein

ATP binding and hydrolysis are required for P-glycoprotein mediated multidrug resistance. To investigate the molecular mechanism involved in ATP binding and hydrolysis, a three-dimensional model of the carboxyl-terminal nucleotide binding domain (NBD2) was built by homology modeling. Modeling revealed the human P-glycoprotein ATP-binding site and the possible role of conserved Gln1118 residue. Recombinant NBD2 was overexpressed in Escherichia coli and the conserved Gln1118 residue was mutated to an alanine residue. The V_max for ATP hydrolysis by the mutant NBD2 was ～56% of the V_max of wild-type NBD2. But both proteins displayed similar affinity for ATP, with K_m of 479 and 466 µM for mutant and wild-type NBD2, respectively. These results suggest that the possible role of Gln1118 is as an activating residue for ATP hydrolysis. The molecular model also provided structural information about the interactions between NBD2 and the chemosensitizer quercetin. The complex indicated that quercetin was tightly bound to the ATP-binding site and competed for binding. The three-dimensional model of NBD2 can be used to both guide enzymological studies and provide a theoretical basis for the design of potential multidrug resistance reversers.     

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.     

Bacterial histidine kinase as signal sensor and transducer

Adaptation to an environmental stress is essential for cell survival in all organisms, from E. coli to human. To respond to changes in their surroundings, bacteria utilize two-component systems (TCSs), also known as histidyl-aspartyl phosphorelay (HAP) systems that consist of a histidine kinase (HK) sensor and a cognate response regulator (RR). While mammals developed complex signaling systems involving serine/threonine/tyrosine kinases in stress response mechanisms, bacterial TCS/HAP systems represent a simple but elegant prototype of signal transduction machineries. HKs are known as a seductive target for anti-bacterial therapeutic development, because of their significance in pathological virulence in some bacteria such as Salmonella enterica. Recent molecular and structural studies have shed light on the molecular basis of the signaling mechanism of HK sensor kinases. This review will focus on recent advancements in structural investigation of signal sensing and transducing mechanisms by HKs, which is critical to our understanding of bacterial biology and pathology.