Crystal structure of a trimeric form of dephosphocoenzyme A kinase from Escherichia coli

Coenzyme A (CoA) is an essential cofactor used in a wide variety of biochemical pathways. The final step in the biosynthesis of CoA is catalyzed by dephosphocoenzyme A kinase (DPCK, E.C. 2.7.1.24). Here we report the crystal structure of DPCK from Escherichia coli at 1.8 A resolution. This enzyme forms a tightly packed trimer in its crystal state, in contrast to its observed monomeric structure in solution and to the monomeric, homologous DPCK structure from Haemophilus influenzae. We have confirmed the existence of the trimeric form of the enzyme in solution using gel filtration chromatography measurements. Dephospho-CoA kinase is structurally similar to many nucleoside kinases and other P-loop-containing nucleotide triphosphate hydrolases, despite having negligible sequence similarity to these enzymes. Each monomer consists of five parallel beta-strands flanked by alpha-helices, with an ATP-binding site formed by a P-loop motif. Orthologs of the E. coli DPCK sequence exist in a wide range of organisms, including humans. Multiple alignment of orthologous DPCK sequences reveals a set of highly conserved residues in the vicinity of the nucleotide/CoA binding site.     

Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state

The pathogenic bacterium Brucella abortus codes for a multi-domain dimeric cytoplasmic histidine kinase called LOV-HK, which is a key blue light-activated virulence factor in this microorganism. The structural basis of the light activation mechanism of this protein remains unclear. In this work, full-length LOV-HK was cloned, expressed and purified. The protein was activated by light and crystallized under a controlled illumination environment. The merge of 14 individual native data sets collected on a single crystal resulted in a complete X-ray diffraction data set to a resolution of 3.70 Å with over 2 million reflections. Crystals belong to space group P2₁2₁2₁, with unit-cell parameters a = 95.96, b = 105.30, c = 164.49 Å with a dimer in the asymmetric unit. Molecular replacement with Phaser using the individual domains as search models allowed for the reconstruction of almost the whole protein. Very recently, improved LOV-HK crystals led to a 3.25-Å resolution dataset. Refinement and model building is underway. This crystal model will represent one of the very few examples of a multi-domain histidine kinase with known structure.     

Crystal structure of SecB from Escherichia coli

The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. The crystal structure of SecB from E. coli has been solved to 2.35 A resolution. The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site. Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers. Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop. The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr. The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation.     

Crystal structure of human pyridoxal kinase

Pyridoxal kinase, a member of the ribokinase superfamily, catalyzes the ATP-dependent phosphorylation reaction of vitamin B6 and is an essential enzyme in the formation of pyridoxal-5'-phosphate, a key cofactor for over 100 enzymes. Pyridoxal kinase is thus regarded as a potential target for pharmacological agents. In this paper, we report the 2.8 angstroms crystal structure of human pyridoxal kinase (HPLK) expressed in Escherichia coli. The diffraction data revealed unexpected merohedral perfect twinning along the crystallographic c axis. Taking perfect twinning into account, the structure in dimeric form was well refined according to the CNS program. Structure comparison reveals that the key 12-residue peptide over the active site in HPLK is a beta-strand/loop/beta-strand flap, while the corresponding peptide in sheep brain enzyme adopts a loop conformation. Moreover, HPLK possesses a more hydrophobic ATP-binding pocket. This structure will facilitate further biochemical studies and structure-based design of drugs related to pyridoxal kinase.     

Light-dependent dimerisation in the N-terminal sensory module of cyanobacterial phytochrome 1

Phytochromes, photoreceptors controlling important physiological processes in plants and many prokaryotes, are photochromic biliproteins. The red-absorbing Pr ground state is converted by light into the farred-absorbing Pfr which can be photoconverted back to Pr. In plants at least Pfr is the physiologically active signalling state. Here, we show that the N-terminal photochromic module of Cph1 homodimerises reversibly and independently in Pr and Pfr, Pfr-dimers being significantly more stable. Implications for the mechanism of signal transduction are discussed.     

Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli

Synthesis of the high-affinity K⁺-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. K⁺ limitation or a sudden increase in osmolarity induces the expression of kdpFABC. Due to the importance of K⁺ to maintain turgor, it has been proposed that KdpD is a turgor sensor. Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates. Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E. coli mutants defective in phospholipid biosynthesis. Surprisingly, neither the lack of the major E. coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly. However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role. These results indicate that electrostatic interactions are important for the activity of KdpD. Received: 29 March 1999 / Accepted: 26 July 1999     

Crystal structure of chondroitin polymerase from Escherichia coli K4

Elongation of glycosaminoglycan chains, such as heparan and chondroitin, is catalyzed by bi-functional glycosyltransferases, for which both 3-dimensional structures and reaction mechanisms remain unknown. The bacterial chondroitin polymerase K4CP catalyzes elongation of the chondroitin chain by alternatively transferring the GlcUA and GalNAc moiety from UDP-GlcUA and UDP-GalNAc to the non-reducing ends of the chondroitin chain. Here, we have determined the crystal structure of K4CP in the presence of UDP and UDP-GalNAc as well as with UDP and UDP-GlcUA. The structures consisted of two GT-A fold domains in which the two active sites were 60 Å apart. UDP-GalNAc and UDP-GlcUA were found at the active sites of the N-terminal and C-terminal domains, respectively. The present K4CPstructures have provided the structural basis for further investigating the molecular mechanism of biosynthesis of chondroitin chain.     

Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs that brings the N- and C-domains of PGK closer together. Here we present the 1.8A crystal structure of unliganded PGK from Thermus caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation.     

Crystal structure of the nucleotide-binding subunit DhaL of the Escherichia coli dihydroxyacetone kinase

Dihydroxyacetone (Dha) kinases are a family of sequence-related enzymes that utilize either ATP or phosphoenolpyruvate (PEP) as source of high energy phosphate. The PEP-dependent Dha kinase of Escherichia coli consists of three subunits. DhaK and DhaL are homologous to the Dha and nucleotide-binding domains of the ATP-dependent kinase of Citrobacter freundii. The DhaM subunit is a multiphosphorylprotein of the PEP:sugar phosphotransferase system (PTS). DhaL contains a tightly bound ADP as coenzyme that gets transiently phosphorylated in the double displacement of phosphate between DhaM and Dha. Here we report the 2.6A crystal structure of the E.coli DhaL subunit. DhaL folds into an eight-helix barrel of regular up-down topology with a hydrophobic core made up of eight interlocked aromatic residues and a molecule of ADP bound at the narrower end of the barrel. The alpha and beta phosphates of ADP are complexed by two Mg2+ and by a hydrogen bond to the imidazole ring of an invariant histidine. The Mg ions in turn are coordinated by three gamma-carboxyl groups of invariant aspartate residues. Water molecules complete the octahedral coordination sphere. The nucleotide is capped by an alpha-helical segment connecting helices 7 and 8 of the barrel. DhaL and the nucleotide-binding domain of the C.freundii kinase assume the same fold but display strongly different surface potentials. The latter observation and biochemical data indicate that the domains of the C.freundii Dha kinase constitute one cooperative unit and are not randomly interacting and independent like the subunits of the E.coli enzyme.     

Crystal structure and oligomeric state of the RetS signaling kinase sensory domain

The opportunistic pathogen Pseudomonas aeruginosa may cause both acute and chronic‐persistent infections in predisposed individuals. Acute infections require the presence of a functional type III secretion system (T3SS), whereas chronic P. aeruginosa infections are characterized by the formation of drug‐resistant biofilms. The T3SS and biofilm formation are reciprocally regulated by the signaling kinases LadS, RetS, and GacS. RetS downregulates biofilm formation and upregulates expression of the T3SS through a unique mechanism. RetS forms a heterodimeric complex with GacS and thus prevents GacS autophosphorylation and downstream signaling. The signals that regulate RetS are not known but RetS possesses a distinctive periplasmic sensor domain that is believed to serve as receptor for the regulatory ligand. We have determined the crystal structure of the RetS sensory domain at 2.0 Å resolution. The structure closely resembles those of carbohydrate binding modules of other proteins, suggesting that the elusive ligands are likely carbohydrate moieties. In addition to the conserved beta‐sandwich structure, the sensory domain features two alpha helices which create a unique surface topology. Protein–protein crosslinking and fluorescence energy transfer experiments also revealed that the sensory domain dimerizes with a dissociation constant of K_d = 580 ± 50 nM, a result with interesting implications for our understanding of the underlying signaling mechanism. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Dual-histidine kinases in basidiomycete fungi

Dual-histidine kinases (HKs) are complex hybrid HKs containing in a single polypeptide two HK transmitter modules (T) and two-response regulator received domains (R) that are combined in a TRTR geometry. In fungi, this protein family is limited to some particular species of the phylum Basidiomycota and absent in the other phyla. This study extends the investigation of dual-HKs to 80 fully sequenced genomes of basidiomycetes, analyzing their distribution, domain architecture and phylogenetic relationships. Moreover, similarly to dual-HKs of basidiomycetes, several species of bacteria were found that contain hybrid HKs with a TRTR domain architecture encoded in a single gene.     

Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ)

The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes. Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution. By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc. By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed. The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein. The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction. By using MBP-deltaL-EnvZc(S260C) fusion protein (deltaL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-deltaL-EnvZc(S260C)/deltaL-EnvZc(S260C/G405A) heterodimer, only the deltaL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-deltaL-EnvZc(S260C) subunit. Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer.     

Functional insights from the molecular modelling of a novel two-component system

Two-component systems (TCSs) are the major signalling pathway in bacteria and represent potential drug targets. Among the 11 paired TCS proteins present in Mycobacterium tuberculosis H37Rv, the histidine kinases (HKs) Rv0600c (HK1) and Rv0601c (HK2) are annotated to phosphorylate one response regulator (RR) Rv0602c (TcrA). We wanted to establish the sequence-structure-function relationship to elucidate the mechanism of phosphotransfer using in silico methods. Sequence alignments and codon usage analysis showed that the two domains encoded by a single gene in homologous HKs have been separated into individual open-reading frames in M. tuberculosis. This is the first example where two incomplete HKs are involved in phosphorylating a single RR. The model shows that HK2 is a unique histidine phosphotransfer (HPt)-mono-domain protein, not found as lone protein in other bacteria. The secondary structure of HKs was confirmed using "far-UV" circular dichroism study of purified proteins. We propose that HK1 phosphorylates HK2 at the conserved H131 and the phosphoryl group is then transferred to D73 of TcrA.     

Insight into the sporulation phosphorelay: Crystal structure of the sensor domain of Bacillus subtilis histidine kinase,KinD

The Bacillus subtilis KinD signal‐transducing histidine kinase is a part of the sporulation phosphorelay known to regulate important developmental decisions such as sporulation and biofilm formation. We have determined crystal structures of the extracytoplasmic sensing domain of KinD, which was copurified and crystallized with a pyruvate ligand. The structure of a ligand‐binding site mutant was also determined; it was copurified and crystallized with an acetate ligand. The structure of the KinD extracytoplasmic segment is similar to that of several other sensing domains of signal transduction proteins and is composed of tandem Per‐Arnt‐Sim (PAS)‐like domains. The KinD ligand‐binding site is located on the membrane distal PAS‐like domain and appears to be highly selective; a single mutation, R131A, abolishes pyruvate binding and the mutant binds acetate instead. Differential scanning fluorimetry, using a variety of monocarboxylic and dicarboxylic acids, identified pyruvate, propionate, and butyrate but not lactate, acetate, or malate as KinD ligands. A recent report found that malate induces biofilm formation in a KinD‐dependent manner. It was suggested that malate might induce a metabolic shift and increased secretion of the KinD ligand of unknown identity. The structure and binding assays now suggests that this ligand is pyruvate and/or other small monocarboxylic acids. In summary, this study gives a first insight into the identity of a molecular ligand for one of the five phosphorelay kinases of B. subtilis.     

Identification and characterization of KvgAS,a two-component system in Klebsiella pneumoniae CG43

A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions.     

Crystal structure of Escherichia coli SufC, an ABC-type ATPase component of the SUF iron-sulfur cluster assembly machinery

SufC is an ATPase component of the SUF machinery, which is involved in the biosynthesis of Fe-S clusters. To gain insight into the function of this protein, we have determined the crystal structure of Escherichia coli SufC at 2.5A resolution. Despite the similarity of the overall structure with ABC-ATPases (nucleotide-binding domains of ABC transporters), some key differences were observed. Glu171, an invariant residue involved in ATP hydrolysis, is rotated away from the nucleotide-binding pocket to form a SufC-specific salt bridge with Lys152. Due to this salt bridge, D-loop that follows Glu171 is flipped out to the molecular surface, which may sterically inhibit the formation of an active dimer. Thus, the salt bridge may play a critical role in regulating ATPase activity and preventing wasteful ATP hydrolysis. Furthermore, SufC has a unique Q-loop structure on its surface, which may form a binding site for its partner proteins, SufB and/or SufD.     

Crystallization and characterization of polyphosphate kinase from Escherichia coli

Linear polyphosphate chains have been found to play a key role in bacterial responses to stresses and nutritional depletion, and are necessary for host infection of various pathogens. Polyphosphate kinase (PPK) is a critical enzyme responsible for polyphosphate synthesis in bacteria. PPK knockout mutations in several Gram-negative pathogens identify PPK as an ideal drug target for the development of a new class of antibacterial drugs. To reveal the catalytic mechanism and provide a structural basis for drug discovery, we have purified and crystallized full-length Escherichia coli PPK and its complex with AMP-PNP. The crystals diffract to a resolution of 2.5A and belong to the space group P4(2)2(1)2 with unit-cell parameters a=152.0, b=152.0, and c=150.0 A. Crystal structure of PPK is being determined by the Se-Met MAD experiment.     

Purification and characterization of an activated form of the protein tyrosine kinase Lck from an Escherichia coli expression system

The lymphocyte-specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye-affinity chromatography. Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near-homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine-394), which is characteristic for activated protein tyrosine kinases. Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy-terminal tyrosine residue (tyrosine-505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two-step purification procedure which yields highly active enzyme. This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction. © 1994 Wiley-Liss, Inc.     

Crystal structure of human mono-phosphorylated ERK1 at Tyr204

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204. Crystal structure demonstrated that the mono-phosphorylated ERK1 kinase possessed a novel conformation distinguishable from the un-phosphorylated (inactive) and the dual-phosphorylated (full-active) forms. The characteristic structural features in both the C-helix and the activation loop likely contribute to the basal activity of the mono-phosphorylated ERK1. The structural dissection of ERK1 compared to ERK2 suggests that the structural differences in the D-motif binding site and in the backside binding site are putative targets for development of selective ERK1/ERK2 inhibitors.