Sensor Domain of Histidine Kinase KinB of Pseudomonas: A HELIX-SWAPPED DIMER*

The overproduction of polysaccharide alginate is responsible for the formation of mucus in the lungs of cystic fibrosis patients. Histidine kinase KinB of the KinB-AlgB two-component system in Pseudomonas aeruginosa acts as a negative regulator of alginate biosynthesis. The modular architecture of KinB is similar to other histidine kinases. However, its periplasmic signal sensor domain is unique and is found only in the Pseudomonas genus. Here, we present the first crystal structures of the KinB sensor domain. The domain is a dimer in solution, and in the crystal it shows an atypical dimer of a helix-swapped four-helix bundle. A positively charged cavity is formed on the dimer interface and involves several strictly conserved residues, including Arg-60. A phosphate anion is bound asymmetrically in one of the structures. In silico docking identified several monophosphorylated sugars, including β-d-fructose 6-phosphate and β-d-mannose 6-phosphate, a precursor and an intermediate of alginate synthesis, respectively, as potential KinB ligands. Ligand binding was confirmed experimentally. Conformational transition from a symmetric to an asymmetric structure and decreasing dimer stability caused by ligand binding may be a part of the signal transduction mechanism of the KinB-AlgB two-component system.     

Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.     

Full-Length Structure of a Sensor Histidine Kinase Pinpoints Coaxial Coiled Coils as Signal Transducers and Modulators

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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.     

Unique MAP Kinase binding sites

Map kinases are drug targets for autoimmune disease, cancer, and apoptosis-related diseases. Drug discovery efforts have developed MAP kinase inhibitors directed toward the ATP binding site and neighboring "DFG-out" site, both of which are targets for inhibitors of other protein kinases. On the other hand, MAP kinases have unique substrate and small molecule binding sites that could serve as inhibition sites. The substrate and processing enzyme D-motif binding site is present in all MAP kinases, and has many features of a good small molecule binding site. Further, the MAP kinase p38alpha has a binding site near its C-terminus discovered in crystallographic studies. Finally, the MAP kinases ERK2 and p38alpha have a second substrate binding site, the FXFP binding site that is exposed in active ERK2 and the D-motif peptide induced conformation of MAP kinases. Crystallographic evidence of these latter two binding sites is presented.     

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.     

Antibacterial Agents that Inhibit Histidine Protein Kinase YycG of Bacillus subtilis

We demonstrated in vitro that YycG-YycF of Bacillus subtillis constitutes a two-component system and shows a specificity of the sensor protein for the cognate phosphorylation partner. Based on inhibition of such an autophosphorylation of YycG, we searched imidazole and zerumbone derivatives to identify the antibacterial agents such as NH125, NH126, NH127, and NH0891.     

Autophagic Cell Death in Dictyostelium Requires the Receptor Histidine Kinase DhkM

Dictyostelium constitutes a genetically tractable model for the analysis of autophagic cell death (ACD). During ACD, Dictyostelium cells first transform into paddle cells and then become round, synthesize cellulose, vacuolize, and die. Through random insertional mutagenesis, we identified the receptor histidine kinase DhkM as being essential for ACD. Surprisingly, different DhkM mutants showed distinct nonvacuolizing ACD phenotypes. One class of mutants arrested ACD at the paddle cell stage, perhaps through a dominant-negative effect. Other mutants, however, progressed further in the ACD program. They underwent rounding and cellulose synthesis but stopped before vacuolization. Moreover, they underwent clonogenic but not morphological cell death. Exogenous 8-bromo-cAMP restored vacuolization and death. A role for a membrane receptor at a late stage of the ACD pathway is puzzling, raising questions as to which ligand it is a receptor for and which moieties it phosphorylates. Together, DhkM is the most downstream-known molecule required for this model ACD, and its distinct mutants genetically separate previously undissociated late cell death events.     

Dissecting the Unique Nucleotide Specificity of Mimivirus Nucleoside Diphosphate Kinase

The analysis of the Acanthamoeba polyphaga mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 Å to 1.5 Å. Our results suggest that the mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.Mimivirus, a DNA virus infecting Acanthamoeba, is the largest and most complex virus isolated to date (8, 37). It is the first representative and prototype member of the Mimiviridae, the latest addition to the large nucleocytoplasmic DNA viruses, including the poxviruses, the phycodnaviruses, (infecting algae), the iridoviruses (infecting invertebrates and fishes), and asfarvirus (the agent of a swine fever in Africa) (18). The mimivirus''s record genome size (1.2 Mb) and gene content (911 encoded proteins), as well as the presence of genes previously thought to be specific to cellular organisms (such as aminoacyl-tRNA synthetases [3]), revived the debate about the evolutionary origin of DNA viruses and their putative role in the emergence of the eukaryote nucleus (reviewed in reference 7) or in the advent of DNA genomes (13).In this peculiar context, we found the discovery of the first virus-encoded nucleoside diphosphate kinase (NDK) within the mimivirus genome of great interest and warranting a detailed study of the structural and biochemical properties of this unique viral enzyme. Ubiquitous in cellular organisms, NDKs are responsible for the last step of 2′-deoxynucleoside triphosphate (dNTP) pathways and as such play an essential role in the replication of DNA by providing the basic precursors for its synthesis. Acting indiscriminately on ribonucleotides and deoxyribonucleotides, the cellular NDKs are also responsible for supplying energy to various essential synthetic pathways, producing NTPs for RNA synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis, and GTP for protein synthesis elongation, signal transduction, and microtubules polymerization. Besides their direct role in the above metabolic pathways, cellular NDKs have been involved in the regulation of cell growth and differentiation in vertebrates (22).Cellular NDKs are small proteins of about 150 amino acids, the sequences of which are highly conserved among the three domains of life (>40% identity). They are most often hexameric enzymes, with a few occurrences of tetrameric and dimeric NDK structures in bacteria (19, 25, 26, 31, 38). They all catalyze the transfer of a phosphate group from an NTP onto a nucleotide diphosphate (NDP) through an Mg²⁺-dependent reaction. In vivo, the phosphate donor is usually the nonlimiting ATP nucleotide.In agreement with their implication in various metabolic pathways, cellular NDKs exhibit little substrate specificity and are equally able to act on purine and pyrimidine nucleotides, in their 2′ OH and deoxyribonucleotide forms. In clear contrast, our characterization of the mimivirus NDK revealed its enhanced affinity for deoxypyrimidine nucleotides (20). This marked difference between the viral and cellular NDKs offered a good opportunity to explore the sequence and structure features governing substrate specificity. For instance, cellular NDKs exhibit a conserved loop, the Kpn loop, involved both in substrate binding and in oligomerization of the enzyme (19). Interestingly, a sequence comparison predicted this loop to be shorter in the Acanthamoeba polyphaga mimivirus NDK (NDK_apm) sequence. However, many other single-residue changes could also be involved in modifying the enzyme properties. To explore these issues, we performed a detailed structure-function analysis of the NDK_apm protein in a variety of mutated forms and substrate-enzyme complexes. Despite its markedly different sequence, the three-dimensional structure of the mimivirus NDK was found to be very similar to that of cellular enzymes. Its peculiar substrate specificity is not attributable to a single sequence feature but rather appears to result from the conjunction of several factors, suggesting the progressive optimization of an ancestral enzyme for the replication of an AT-rich (73%) genome in a thymidine-limited host environment.     

A Photochromic Histidine Kinase Rhodopsin (HKR1) That Is Bimodally Switched by Ultraviolet and Blue Light

Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (λ_max = 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (λ_max = 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light.     

The Histidine Kinase PdhS Controls Cell Cycle Progression of the Pathogenic Alphaproteobacterium Brucella abortus

Bacterial differentiation is often associated with the asymmetric localization of regulatory proteins, such as histidine kinases. PdhS is an essential and polarly localized histidine kinase in the pathogenic alphaproteobacterium Brucella abortus. After cell division, PdhS is asymmetrically segregated between the two sibling cells, highlighting a differentiation event. However, the function(s) of PdhS in the B. abortus cell cycle remains unknown. We used an original approach, the pentapeptide scanning mutagenesis method, to generate a thermosensitive allele of pdhS. We report that a B. abortus strain carrying this pdhS allele displays growth arrest and an altered DivK-yellow fluorescent protein (YFP) polar localization at the restrictive temperature. Moreover, the production of a nonphosphorylatable PdhS protein or truncated PdhS proteins leads to dominant-negative effects by generating morphological defects consistent with the inhibition of cell division. In addition, we have used a domain mapping approach combined with yeast two-hybrid and fluorescence microscopy methods to better characterize the unusual PdhS sensory domain. We have identified a fragment of the PdhS sensory domain required for protein-protein interaction (amino acids [aa] 210 to 434), a fragment sufficient for polar localization (aa 1 to 434), and a fragment (aa 527 to 661) whose production in B. abortus correlates with the generation of cell shape alterations. The data support a model in which PdhS acts as an essential regulator of cell cycle progression in B. abortus and contribute to a better understanding of the differentiation program inherited by the two sibling cells.