Histidine and aspartate phosphorylation: two-component systems and the limits of homology.

Autophosphorylating histidine kinase and response-regulator domains constitute the building blocks of two-component signaling systems. These systems use a unique phosphotransfer chemistry to regulate many aspects of bacterial physiology. Homologous systems are now being found in eukaryotes. Despite their common mechanism of phosphotransfer, the two-component systems display an extensive diversity in the arrangement of their domains, and flexibility in their roles in different signal transduction circuits.

Signal transduction. Bringing the eukaryotes up to speed.

'Two-component' signalling systems have long been known to be used very generally in bacterial signal transduction; at last, related proteins have been discovered in eukaryotes.

Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor.

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

Phycobiliprotein methylation. Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome.

The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72. This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes. An assay for the protein asparagine methylase activity was developed using [methyl-3H]S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp. PCC 7002 as substrates. This assay permitted the partial purification, from Synechococcus sp. PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72. Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp. PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity. These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72. The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp. PCC 6301. The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles. Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation. Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation. Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles. The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.

Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability.

The crystal structure of CheY protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.

Crystal structure of the beta-glycosidase from the hyperthermophile Thermosphaera aggregans: insights into its activity and thermostability.

The glycosyl hydrolases are an important group of enzymes that are responsible for cleaving a range of biologically significant carbohydrate compounds. Structural information on these enzymes has provided useful information on their molecular basis for the functional variations, while the characterization of the structural features that account for the high thermostability of proteins is of great scientific and biotechnological interest. To these ends we have determined the crystal structure of the beta-glycosidase from a hyperthermophilic archeon Thermosphaera aggregans. The structure is a (beta/alpha)8 barrel (TIM-barrel), as seen in other glycosyl hydrolase family 1 members, and forms a tetramer. Inspection of the active site and the surrounding area reveals two catalytic glutamate residues consistent with the retaining mechanism and the surrounding polar and aromatic residues consistent with a monosaccharide binding site. Comparison of this structure with its mesophilic counterparts implicates a variety of structural features that could contribute to the thermostability. These include an increased number of surface ion pairs, an increased number of internal water molecules and a decreased surface area upon forming an oligomeric quaternary structure.

Separation of phycobiliprotein subunits by reverse-phase high-pressure liquid chromatography.

Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2:1 (v/v) acetonitrile:isopropanol. The procedure was successfully applied to cyanobacterial allophycocyanin and C-phycocyanins, an unusual phycocyanin from a marine cyanobacterium, red algal B- and R-phycoerythrins, and a cryptomonad phycoerythrin. The subunit sizes in these proteins range from about 7.5 to 30 kDa. Sample recovery was in excess of 85% in all cases. On-line spectroscopic analysis with a multiple diode array detector allowed determination of the type and number of bilins carried by each subunit.

Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima.

An expressed sequence tag homologous to cheA was previously isolated by random sequencing of Thermotoga maritima cDNA clones (C. W. Kim, P. Markiewicz, J. J. Lee, C. F. Schierle, and J. H. Miller, J. Mol. Biol. 231: 960-981, 1993). Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T. maritima lambda library by using PCR. Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced. The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY. In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function. Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors. Upon induction, both proteins were synthesized at high levels. T. maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E. coli protein by heat treatment at 80 degrees C for 10 min. CheY prepared in this way was shown to be active by the demonstration of Mg(2+)-dependent autophosphorylation with [32P]acetyl phosphate. In E. coli, CheW mediates the physical coupling of the receptors to the kinase CheA. The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.

Intermolecular complementation of the kinase activity of CheA.

CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis in Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase-defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer. The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphorylation requires the presence of both carboxy-terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.

Expression of CheA fragments which define domains encoding kinase, phosphotransfer, and CheY binding activities.

The histidine protein kinase CheA is a central component of the Escherichia coli chemotaxis system. The autophosphorylation activity of CheA is controlled by membrane-bound chemoreceptors and by the CheW coupling protein. CheA phosphorylates the CheY and CheB proteins which respectively control the direction of flagellar rotation and the level of receptor adaptation, thereby regulating the cells' chemotactic response. Genes encoding three polypeptide fragments of CheA were constructed and expressed in order to better define the functional organization of the wild-type protein. These fragments allowed the identification of regions of the protein responsible for CheY binding, phosphotransfer, and kinase activity. The kinase domain was expressed as a 30-kDa polypeptide corresponding to the central portion of the wild-type protein which contains sequences homologous to other histidine kinases. It was able to phosphorylate a 15-kDa amino-terminal phosphotransfer domain which was separately expressed and purified. This latter domain is capable of phosphotransfer to CheY despite the fact that it lacks the ability to stably bind CheY. CheY was immobilized to a dextran matrix through a single cysteine residue which was introduced into the protein at a position far removed from the active site. A stable binding site for CheY was mapped to a segment between the site of autophosphorylation and the kinase domain by using surface plasmon resonance to detect binding to the immobilized CheY. The region of the kinase which tightly binds the unphosphorylated substrate may play an important role in regulating the specificity of the signal transducing system.

Genes encoding the phycobilisome rod substructure are clustered on the Anabaena chromosome: characterization of the phycoerythrocyanin operon.

The phycoerythrocyanin (pec) operon, cloned from Anabaena sp. strain PCC 7120, encodes four genes, pecBACE, located upstream of the C-phycocyanin (cpc) operon. This pec-cpc cluster includes all the genes for the structural components of the phycobilisome rod. Oligonucleotide probes based on the amino-terminal sequence of the phycoerythrocyanin beta subunit were used to clone an 8.0-kbp EcoRI fragment which was determined, by sequencing, to partially overlap the previously cloned cpc operon. A 5.0-kbp EcoRI-ClaI fragment corresponding to the region upstream of the cpc operon was subsequently subcloned and sequenced. Five open reading frames whose polarity of transcription is parallel to that of the cpc genes were identified. pecB and pecA encode the beta and alpha subunits of phycoerythrocyanin, respectively. pecC encodes the phycoerythrocyanin-associated linker polypeptide LR34.5,PEC. The identities of these genes are confirmed by agreement with amino-terminal sequences determined from purified phycobilisome components. A gene homologous to cpcE, found downstream of pecC, has been designated pecE. The cpcE gene product is involved in the attachment of the phycocyanobilin chromophore to the alpha subunit of phycocyanin. Three transcripts were observed by Northern (RNA) analyses. The most abundant of these transcripts, 1.35 kbp, corresponds to the beta and alpha subunit genes, whereas the less-abundant transcripts, 2.3 and 3.1 kbp, correspond to pecBAC and pecBACE, respectively. Phycoerythrocyanin is strongly induced in cells cultured under low light. In parallel, all three transcripts were present at much higher levels in cells cultured under low light.

The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km.

Tyrosine-225 is hydrogen-bonded to the 3'-hydroxyl group of pyridoxal 5'-phosphate in the active site of aspartate aminotransferase. Replacement of this residue with phenylalanine (Y225F) results in a shift in the acidic limb of the pKa of the kcat/KAsp vs pH profile from 7.1 (wild-type) to 8.4 (mutant). The change in the kinetic pKa is mirrored by a similar shift in the spectrophotometrically determined pKa of the protonated internal aldimine. Thus, a major role of tyrosine-225 is to provide a hydrogen bond that stabilizes the reactive unprotonated form of the internal aldimine in the neutral pH range. The Km value for L-aspartate and the dissociation constant for alpha-methyl-DL-aspartate are respectively 20- and 37-fold lower in the mutant than in the wild-type enzyme, while the dissociation constant for maleate is much less perturbed. These results are interpreted in terms of competition between the Tyr225 hydroxyl group and the substrate or quasi-substrate amino group for the coenzyme. The value of kcat in Y225F is 450-fold less than the corresponding rate constant in wild type. The increased affinity of the mutant enzyme for substrates, combined with the lack of discrimination against deuterium in the C alpha position of L-aspartate in Y225F-catalyzed transamination [Kirsch, J. F., Toney, M. D., & Goldberg, J. M. (1990) in Protein and Pharmaceutical Engineering (Craik, C. S., Fletterick, R., Matthews, C. R., & Wells, J., Eds.) pp 105-118, Wiley-Liss, New York], suggests that the rate-determining step in the mutant is hydrolysis of the ketimine intermediate rather than C alpha-H abstraction which is partially rate-determining in wild type.

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content.

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.

Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent.

Salmonella typhimurium is a facultative intracellular pathogen capable of surviving within phagocytic cells of the reticuloendothelial system. To identify the genes important for intracellular survival, 9516 independent Tn10 insertional mutations were isolated in a virulent strain of S. typhimurium. By using an in vitro assay for survival within macrophages, 83 Tn10 mutants have been identified that have a diminished capacity for intracellular survival (designated MS or macrophage survival mutants). All of the MS mutants are less virulent than the parent strain in vivo, demonstrating that, for Salmonella, survival within the macrophage is essential for virulence. Thirty-seven of the MS mutants have been characterized as to their phenotype, including several mutations that confer sensitivity to specific microbiocidal mechanisms of the macrophage.

NMR studies of the phosphotransfer domain of the histidine kinase CheA from Escherichia coli: assignments, secondary structure, general fold, and backbone dynamics.

Multidimensional heteronuclear NMR techniques were applied to study the phosphotransfer domain, residues 1-134, of the histidine kinase CheA, from Escherichia coli, which contains the site of autophosphorylation, His48. Assignments of the backbone amide groups and side chain protons are nearly complete. Our studies show that this protein fragment consists of five alpha-helices (A-E) connected by turns. Analysis of NOE distance restraints provided by two-dimensional (2D) 1H-1H and three-dimensional (3D) 15N-edited NOESY spectra using model building and structure calculations indicates that the five helices form an antiparallel helix bundle with near-neighbor connectivity. The amino-terminal four helices are proposed to be arranged in a right-handed manner with helix E packing against helices C and D. From ideal hydrophobic helical packing and structure calculations, the site of autophosphorylation, His48, is nearly fully exposed to the solvent. We measured the NMR relaxation properties of the backbone 15N nuclei using inverse detected two-dimensional NMR spectroscopy. The protein backbone dynamics studies show that CheA1-134 is formed into a tight and compact structure with very limited flexibilities both in helices and turns. Structural implications of titration and phosphorylation experiments are briefly discussed.

The response regulators CheB and CheY exhibit competitive binding to the kinase CheA.

The autophosphorylating kinase CheA of the bacterial chemosensory signaling pathway donates a phosphoryl group to either of two regulator proteins, CheY or the receptor methylesterase (CheB). With isothermal titration calorimetry, it was demonstrated that CheA and CheA fragment composed of amino acid residues 1-233 (CheA1-233) bound to CheY with similar dissociation constants of 2.0 and 1.2 microM at 298 K, respectively, indicating that the CheY binding site is wholly within the 1-233 amino acid locus. CheB bound to CheA1-233 with a KD of 3.2 microM, and also bound to intact CheA with the same affinity. CheY was found to complete with CheB for binding to CheA1-233, in spite of the low level of sequence identity between CheY and the regulatory domain of CheB. The competitive nature of CheY and CheB binding was determined in two independent sets of experiments: titration experiments in which either a CheB-CheA1-233 complex was titrated with CheY or CheB was titrated with a CheY-CheA1-233 complex, and competitive affinity chromatography experiments that used a Ni-NTA-chelating resin as an affinity matrix for complexes of the histidine-tagged CheA1-233 fragment and CheY or CheB. The effects of phosphorylation, binding-site mutations, and active-site mutations were also studied to probe the influence of conformational changes in CheY as a regulatory mechanism of CheY-CheA Interactions. Phosphorylated CheY, in the presence of excess EDTA, was found to have a 2-fold lower affinity for CheA1-233, and 6 mM Mg2+ further reduced the affinity of phosphorylated CheY for CheA1-233 (ca. 3-fold), although Mg2+ on its own had no effect on the interactions of either CheB or CheY with CheA1-233. The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA under physiological conditions. The idea that phosphorylation may induce a significant conformational change, reducing the strength of the CheY-CheA interaction, is supported by the relative values of the association constants measured for CheY active-site and binding-site mutants. A binding-site mutation (A103V) in CheY, which is remote from the site of phosphorylation produced a 10-fold reduction in Ka, whereas active-site mutations produced a modest (2-fold) reduction.

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ.

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phosphorylated form of the protein allow counter-clockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephosphorylation rates of these mutants. CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of cheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephosphorylation rates. Interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of interaction with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.

Comparison of a beta-glucosidase and a beta-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Purification, characterization, gene cloning, and sequence analysis.

Two distinct exo-acting, beta-specific glycosyl hydrolases were purified to homogeneity from crude cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus: a beta-glucosidase, corresponding to the one previously purified by Kengen et al. (Kengen, S. W. M., Luesink, E. J., Stams, A. J. M., and Zehnder, A. J. B. (1993) Eur. J. Biochem. 213, 305-312), and a beta-mannosidase. The beta-mannosidase and beta-glucosidase genes were isolated from a genomic library by expression screening. The nucleotide sequences predicted polypeptides with 510 and 472 amino acids corresponding to calculated molecular masses of 59.0 and 54.6 kDa for the beta-mannosidase and the beta-glucosidase, respectively. The beta-glucosidase gene was identical to that reported by Voorhorst et al. (Voorhorst, W. G. B., Eggen, R. I. L., Luesink, E. J., and deVos, W. M. (1995) J. Bacteriol. 177, 7105-7111; GenBank accession no. U37557U37557). The deduced amino acid sequences showed homology both with each other (46.5% identical) and with several other glycosyl hydrolases, including the beta-glycosidases from Sulfolobus solfataricus, Thermotoga maritima, and Caldocellum saccharolyticum. Based on these sequence similarities, the beta-mannosidase and the beta-glucosidase can both be classified as family 1 glycosyl hydrolases. In addition, the beta-mannosidase and beta-glucosidase from P. furiosus both contained the conserved active site residues found in all family 1 enzymes. The beta-mannosidase showed optimal activity at pH 7.4 and 105 degrees C. Although the enzyme had a half-life of greater than 60 h at 90 degrees C, it is much less thermostable than the beta-glucosidase, which had a reported half-life of 85 h at 100 degrees C. Km and Vmax values for the beta-mannosidase were determined to be 0.79 mM and 31.1 micromol para-nitrophenol released/min/mg with p-nitrophenyl-beta-D-mannopyranoside as substrate. The catalytic efficiency of the beta-mannosidase was significantly lower than that reported for the P. furiosus beta-glucosidase (5.3 versus 4, 500 s-1 mM-1 with p-nitrophenyl-beta-D-glucopyranoside as substrate). The kinetic differences between the two enzymes suggest that, unlike the beta-glucosidase, the primary role of the beta-mannosidase may not be disaccharide hydrolysis. Other possible roles for this enzyme are discussed.