Different conformations of the kinase-on and kinase-off signaling states in the Aer HAMP domain

HAMP domains are sensory transduction modules that connect input and output domains in diverse signaling proteins from archaea, bacteria, and lower eukaryotes. Here, we employed in vivo disulfide cross-linking to explore the structure of the HAMP domain in the Escherichia coli aerotaxis receptor Aer. Using an Aer HAMP model based on the structure of Archaeoglobus fulgidus Af1503-HAMP, the closest residue pairs at the interface of the HAMP AS-1 and AS-2' helices were determined and then replaced with cysteines and cross-linked in vivo. Except for a unique discontinuity in AS-2, the data suggest that the Aer HAMP domain forms a parallel four-helix bundle that is similar to the structure of Af1503. The HAMP discontinuity was associated with a segment of AS-2 that was recently shown to interact with the Aer-PAS sensing domain. The four-helix HAMP bundle and its discontinuity were maintained in both the kinase-on and kinase-off states of Aer, although differences in the rates of disulfide formation also indicated the existence of different HAMP conformations in the kinase-on and kinase-off states. In particular, the kinase-on state was accompanied by significantly increased disulfide formation rates at the distal end of the HAMP four-helix bundle. This indicates that HAMP signaling may be associated with a tilting of the AS-1 and AS-2' helices, which may be the signal that is transmitted to the kinase control region of Aer.     

The mechanisms of HAMP-mediated signaling in transmembrane receptors

HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.     

Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer

Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.     

Mechanistic Insights Revealed by the Crystal Structure of a Histidine Kinase with Signal Transducer and Sensor Domains

Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.     

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.     

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.     

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.     

HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter

HAMP domains, ～55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function.     

Structural Model of the Cytosolic Domain of the Plant Ethylene Receptor 1 (ETR1)

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.     

Comprehensive Analysis of HAMP Domains: Implications for Transmembrane Signal Transduction

Homodimeric receptors with one or two transmembrane (TM) segments per monomer are universal to life and represent the largest and most diverse group of cellular TM receptors. They frequently share domain types across phyla and, in some cases, have been recombined experimentally into functional chimeras (e.g., the bacterial aspartate chemoreceptor with the human insulin receptor), suggesting that they have a common mechanism. The nature of this mechanism, however, is still being debated. We have proposed a new model for transduction mechanism by axial helix rotation, based on the structure of a widespread domain, HAMP, that frequently occurs in direct continuation of the last TM segment, primarily in histidine kinases and chemoreceptors. Here we show by statistical analysis that HAMP domain sequences have biophysical properties compatible with the two conformations proposed by the model. The analysis also identifies three networks of coevolving residues, which allow the mechanism to subdivide into individual steps. The most extended of these networks is specific for membrane-bound HAMP domains and most likely accepts the signal from the TM helices. In a classification based on sequence clustering, these HAMPs form a central supercluster, surrounded by smaller clusters of divergent HAMPs, which typically combine into arrays of up to 31 consecutive copies and accept conformational input from other HAMP domains. Unexpectedly, the classification shows a division between domains of histidine kinases and those of chemoreceptors; thus, except for a few versatile lineages, HAMP domains are largely specific for one particular output domain. Within proteins using a given output domain, HAMP domains also show extensive coevolution with histidine kinases, but not with chemoreceptors. We attribute the greater capability for recombination among chemoreceptors to their acquisition of a reversible modification system, which acts as a capacitor for the initially deleterious effects of combining domains optimized in different contexts.     

Histidine kinases: diversity of domain organization

Histidine kinases play a major role in signal transduction in prokaryotes for the cellular adaptation to environmental conditions and stresses. Recent progress in the three-dimensional structure determination of two representative members of histidine kinases, EnvZ (class I) and CheA (class II), has revealed common structural features, as well as a kinase catalytic motif topologically similar to those of the ATP-binding domains of a few ATPases. They have also disclosed that there are significant differences in domain organization between class I and II histidine kinases, possibly reflecting their distinct locations, functions and regulatory mechanisms. In spite of this diversity, both class I and II histidine kinases use similar four-helix bundle motifs to relay phosphoryl groups from ATP to regulatory domains of response regulators. The previously known so-called transmitter domain of histidine kinase is further dissected into two domains: a CA (Catalytic ATP-binding) domain and a DHp (Dimerization Histidine phosphotransfer) domain for class I, or a CA domain and an HPt (Histidine-containing Phosphotransfer) domain for class II histidine kinases. From a comparative analysis of the CA domains of EnvZ, CheA and their ATPase homologues, the core elements of the CA domain have been derived. The apparent resemblance between DHp and HPt domains is only superficial, and significant differences between them are discussed.     

Structural and functional aspects of the sensor histidine kinase PrrB from Mycobacterium tuberculosis

We describe the solution structures of two- and three-domain constructs of the sensor histidine kinase PrrB from Mycobacterium tuberculosis, which allow us to locate the HAMP linker relative to the ATP binding and dimerization domains. We show that the three-domain construct is active both for autophosphorylation and for phosphotransfer to the cognate response regulator, PrrA. We also describe the high-resolution crystal structure of the catalytic domain alone, and we show that, in solution, it binds ATP. The conformational flexibility of this domain is discussed and related to other structural information.     

The HAMP domain structure implies helix rotation in transmembrane signaling

HAMP domains connect extracellular sensory with intracellular signaling domains in over 7500 proteins, including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases. The solution structure of an archaeal HAMP domain shows a homodimeric, four-helical, parallel coiled coil with unusual interhelical packing, related to the canonical packing by rotation of the helices. This suggests a model for the mechanism of signal transduction, in which HAMP alternates between the observed conformation and a canonical coiled coil. We explored this mechanism in vitro and in vivo using HAMP domain fusions with a mycobacterial adenylyl cyclase and an E. coli chemotaxis receptor. Structural and functional studies show that the equilibrium between the two forms is dependent on the side-chain size of residue 291, which is alanine in the wild-type protein.     

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 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.     

Not too loose, not too tight--just right. Biphasic control of the Tsr HAMP domain

HAMP domains communicate between input and output signalling modules in a wide variety of bacterial sensor proteins. In the Tsr chemoreceptor, they convert a signal initiated by binding of serine to the periplasmic domain of the protein into regulation of receptor control of the CheA kinase, and ultimately of the direction of flagellar rotation. In this issue, Zhou et al. report an extensive mutational analysis of the Tsr HAMP domain that shows that it can assume a number of different signalling states, which presumably correspond to a variety of different conformations. The two conformational extremes of a tightly packed and a loosely packed HAMP four‐helix bundle support only low levels of CheA activity. Thus, Tsr HAMP does not function as a simple on‐off, two‐state device but rather as a dynamic structure with biphasic control. The normal physiological operating range of Tsr is proposed to be at intermediate degrees of packing of the HAMP four‐helix bundle, but HAMP domains in other proteins could occupy different portions of the conformational spectrum.     

Transmembrane signaling of chemotaxis receptor tar: insights from molecular dynamics simulation studies

Transmembrane signaling of chemotaxis receptors has long been studied, but how the conformational change induced by ligand binding is transmitted across the bilayer membrane is still elusive at the molecular level. To tackle this problem, we carried out a total of 600-ns comparative molecular dynamics simulations (including model-building simulations) of the chemotaxis aspartate receptor Tar (a part of the periplasmic domain/transmembrane domain/HAMP domain) in explicit lipid bilayers. These simulations reveal valuable insights into the mechanistic picture of Tar transmembrane signaling. The piston-like movement of a transmembrane helix induced by ligand binding on the periplasmic side is transformed into a combination of both longitudinal and transversal movements of the helix on the cytoplasmic side as a result of different protein-lipid interactions in the ligand-off and ligand-on states of the receptor. This conformational change alters the dynamics and conformation of the HAMP domain, which is presumably a mechanism to deliver the signal from the transmembrane domain to the cytoplasmic domain. The current results are consistent with the previously suggested dynamic bundle model in which the HAMP dynamics change is a key to the signaling. The simulations provide further insights into the conformational changes relevant to the HAMP dynamics changes in atomic detail.     

Signal transmission through the HtrII transducer alters the interaction of two alpha-helices in the HAMP domain

A conformational change of the transducer HtrII upon photoexcitation of the associated photoreceptor sensory rhodopsin II (SRII) was investigated by monitoring the kinetics of volume changes and the diffusion coefficient (D) of the complex during the photochemical reaction cycle. To localize the region of the transducer responsible, we truncated it at various positions in the cytoplasmic HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain. The truncations do not alter receptor binding, which is dependent primarily on membrane-embedded domain interactions. We found that the light-induced reduction in D occurs in transducers of lengths 120 and 157 residues (Tr120 and Tr157), which are both predicted to contain a HAMP domain consisting of two amphipathic α-helices (AS-1 and AS-2). In contrast, the change in D was abolished in a transducer of 114 amino acid residues (Tr114), which lacks a distal portion of the second α-helix AS-2. The volume changes in SRII-Tr114 are comparable in amplitude and kinetics with those in SRII-Tr120 and SRII-Tr157, confirming the integrity of the complex, which was previously concluded from the similar SRII binding affinity and similar blocking of SRII proton transport by full-length HtrII and Tr114. Our results indicate that a substantial conformational change occurs in the HAMP domain during SRII-HtrII signaling. The data presented here are the first demonstration of stimulus-induced conformational changes of a HAMP domain and provide evidence that the presence of AS-2 is crucial for the conformational alterations. The reduction in diffusion coefficient is likely to due to structural changes in the AS-1 and AS-2 helices such that hydrogen bonding with the surrounding water molecules is increased, thereby increasing friction with the solvent. Similar structural changes may be a general feature in HAMP domain switching, which occurs in diverse signaling proteins, including sensor kinases, taxis receptors/transducers, adenylyl cyclases, and phosphatases.     

Mutational analysis of the connector segment in the HAMP domain of Tsr, the Escherichia coli serine chemoreceptor

HAMP domains are approximately 50-residue motifs, found in many bacterial signaling proteins, that consist of two amphiphilic helices joined by a nonhelical connector segment. The HAMP domain of Tsr, the serine chemoreceptor of Escherichia coli, receives transmembrane input signals from the periplasmic serine binding domain and in turn modulates output signals from the Tsr kinase control domain to elicit chemotactic responses. We created random amino acid replacements at each of the 14 connector residues of Tsr-HAMP to identify those that are critical for Tsr function. In all, we surveyed 179 connector missense mutants and identified three critical residues (G235, L237, and I241) at which most replacements destroyed Tsr function and another important residue (G245) at which most replacements impaired Tsr function. The region surrounding G245 tolerated 1-residue deletions and insertions of up to 10 glycines, suggesting a role as a relatively nonspecific, flexible linker. The critical connector residues are consistent with a structural model of the Tsr-HAMP domain based on the solution structure of an isolated thermophile HAMP domain (M. Hulko, F. Berndt, M. Gruber, J. U. Linder, V. Truffault, A. Schultz, J. Martin, J. E. Schultz, A. N. Lupas, and M. Coles, Cell 126:929-940, 2006) in which G235 defines a critical turn at the C terminus of the first helix and L237 and I241 pack against the helices, perhaps to stabilize alternative HAMP signaling conformations. Most I241 lesions locked Tsr signal output in the kinase-on mode, implying that this residue is responsible mainly for stabilizing the kinase-off signaling state. In contrast, lesions at L237 resulted in a variety of aberrant output patterns, suggesting a role in toggling output between signaling states.