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.

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.

Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum.

Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown. To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote Cenarchaeum symbiosum found living in association with a marine sponge, Axinella mexicana. Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from C. symbiosum. Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed. Homologous regions from the two variants were sequenced and compared in detail. The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region. Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.

Molecular cloning and functional expression of a protein-serine/threonine phosphatase from the hyperthermophilic archaeon Pyrodictium abyssi TAG11.

An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon Pyrodictium abyssi TAG11 and named Py-PP1. Py-PP1 was expressed in Escherichia coli, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo.

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.

The complete genome of the hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus was one of the earliest diverging, and is one of the most thermophilic, bacteria known. It can grow on hydrogen, oxygen, carbon dioxide, and mineral salts. The complex metabolic machinery needed for A. aeolicus to function as a chemolithoautotroph (an organism which uses an inorganic carbon source for biosynthesis and an inorganic chemical energy source) is encoded within a genome that is only one-third the size of the E. coli genome. Metabolic flexibility seems to be reduced as a result of the limited genome size. The use of oxygen (albeit at very low concentrations) as an electron acceptor is allowed by the presence of a complex respiratory apparatus. Although this organism grows at 95 degrees C, the extreme thermal limit of the Bacteria, only a few specific indications of thermophily are apparent from the genome. Here we describe the complete genome sequence of 1,551,335 base pairs of this evolutionarily and physiologically interesting organism.

Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum.

Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.

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.

Phosphotransfer and CheY-binding domains of the histidine autokinase CheA are joined by a flexible linker.

Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli. This fragment (CheA1-233) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region. Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA1-233 remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA124-257. Relaxation properties of the backbone 15N nuclei were measured to study the rotational correlations of the two domains and properties of the linker region. Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model. The experimental T1 and T2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible. Dynamics data of CheA1-233 were also compared with those of CheA1-134. Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions. These observations suggest a model for the graded regulation of CheA autophosphorylation activity. In this model, the various activity states of the receptor are generated by controlling the access of the mean position of the kinase domain to the phosphotransfer domain. This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values.

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.

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.

Localized perturbations in CheY structure monitored by NMR identify a CheA binding interface.

Phosphotransfer between the autophosphorylating histidine kinase CheA and the response regulator CheY represents a crucial step in the bacterial chemotaxis signal transduction pathway. The 15N-1H correlation spectrum of CheY complexed with an amino-terminal fragment of CheA exhibits specific localized differences in chemical shifts when compared to the spectrum of uncomplexed CheY. When mapped onto the three-dimensional structure of CheY, these changes define a region distinct from the active site. A single amino-acid substitution within this binding region on CheY, alanine to valine at position 103, significantly decreases the affinity of CheY for CheA. The binding face described by these changes partially overlaps a flagellar switch binding surface previously defined by mutagenesis.

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.

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.

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.

Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance.

We have used surface plasmon resonance biosensor technology to monitor the assembly and dynamics of a signal transduction complex which controls chemotaxis in Escherichia coli. A quaternary complex formed which consisted of the response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar. Using various experimental conditions and mutant proteins, we have shown that the complex dissociates under conditions that favour phosphorylation of CheY. Direct physical analysis of interactions among proteins in this signal transduction pathway provides evidence for a previously unrecognized binding interaction between the kinase and its substrate. This interaction may be important for enhancing substrate specificity and preventing 'crosstalk' with other systems. The approach is generally applicable to furthering our understanding of how signalling complexes transduce intracellular messages.

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.