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John F Hunt

Publications and source records attributed to John F Hunt.

At least 37 records · Page 2Linked to original sources
PubMed2004

Concentration-dependent effects of endogenous S-nitrosoglutathione on gene regulation by specificity proteins Sp3 and Sp1.

The activities of certain nuclear regulatory proteins are modified by high concentrations of S-nitrosothiols associated with nitrosative stress. In the present study, we have studied the effect of physiological (low microM) concentrations of the endogenous S-nitrosothiol, GSNO (S-nitrosoglutathione), on the activities of nuclear regulatory proteins Sp3 and Sp1 (specificity proteins 3 and 1). Low concentrations of GSNO increased Sp3 binding, as well as Sp3-dependent transcription of the cystic fibrosis transmembrane conductance regulatory gene, cftr. However, higher GSNO levels prevented Sp3 binding, augmented Sp1 binding and prevented both cftr transcription and CFTR (cystic fibrosis transmembrane conductance regulator) expression. We conclude that low concentrations of GSNO favour Sp3 binding to 'housekeeping' genes such as cftr, whereas nitrosative stress-associated GSNO concentrations shut off Sp3-dependent transcription, possibly to redirect cellular resources. Since low micromolar concentrations of GSNO also increase the maturation and activity of a clinically common CFTR mutant, whereas higher concentrations have the opposite effect, these observations may have implications for dosing of S-nitrosylating agents used in cystic fibrosis clinical trials.

Animals↗
PubMed2003

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Adenosine Diphosphate↗
PubMed2003

Bacillus subtilis SecA ATPase exists as an antiparallel dimer in solution.

SecA ATPase promotes the biogenesis of membrane and secretory proteins into and across the cytoplasmic membrane of Eubacteria. SecA binds to translocon component SecYE and substrate proteins and undergoes ATP-dependent conformational cycles that are coupled to the stepwise translocation of proteins. Our recent crystal structure of B. subtilis SecA [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] showed two different dimer interactions in the lattice which both buried significant solvent-accessible surface area in their interface and could potentially be responsible for formation of the physiological dimer in solution. In this paper, we utilize fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues to determine the oligomeric structure of SecA protein in solution. Our results show that of the two dimers interactions observed in the crystal structure, SecA forms an antiparallel dimer in solution that maximizes the buried solvent-accessible surface area and intermolecular contacts. At the submicromolar protein concentrations used in the fluorescence experiments, we saw no evidence for the formation of higher-order oligomers of SecA based on either the alternative dimer or the 3(1) helical fiber observed in the crystal lattice. Our studies are consistent with previous ones demonstrating the existence of a dimerization determinant within the C-domain of SecA as well as those documenting the interaction of N- and C-domains of SecA. Our results also provide a valuable starting point for a determination of whether the subunit status of SecA changes during the protein translocation as well as studies designed to elucidate the conformational dynamics of this multidomain protein during its translocation cycle.

Adenosine Triphosphatases↗
PubMed2003

The 2.3-A crystal structure of the shikimate 5-dehydrogenase orthologue YdiB from Escherichia coli suggests a novel catalytic environment for an NAD-dependent dehydrogenase.

We present here the 2.3-A crystal structure of the Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase. This enzyme catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been promoted as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the protomer contains two alpha/beta domains connected by two alpha-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD+ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue in close apposition to the nicotinamide ring and a clamp over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation in all orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction.

Alcohol Oxidoreductases↗
PubMed2003

Ping-pong cross-validation in real space: a method for increasing the phasing power of a partial model without risk of model bias.

Experimental phases could only be obtained to 4.4 A resolution for crystals of the SecA translocation ATPase. Density modification of these phases exploiting the 65% solvent content of the crystal produced a map from which an approximate backbone model could be built for 80% of the structure. Combining the phases inferred from this partial model with the MIR phases and repeating the density modification produced an improved map from which a more complete backbone model could be built. However, this procedure converged before yielding a map, that allowed unambiguous sequence assignment for the majority of the protein molecule. In order to avoid the likely model bias associated with a speculative attempt at sequence assignment, a real-space cross-validation procedure was employed to facilitate completion of the crystal structure based on partial model phasing. The protein was partitioned into two disjoint sets of residues. Models in which the side chains were built for residues in one of the two sets were used for phase combination and density modification in order to produce improved electron density for interpretation of residues in the other set that had not been included in the model. Residues in the two sets were therefore omitted from the model in alternation except at sites where the side chain could be identified definitively based on phasing with the other set. This ping-pong cross-validation procedure allowed partial model phasing to be used to complete the crystal structure of SecA without being impeded by model bias. These results show that the structure of a large protein molecule can be solved with exclusively low-resolution experimental phase information based on intensive use of partial model phasing and density modification. Real-space cross-validation can be applied to reduce the risk of model bias associated with partial model phasing, streamlining this approach and expanding its range of applicability.

Adenosine Triphosphatases↗
PubMed2003

Structural characterization of an MJ1267 ATP-binding cassette crystal with a complex pattern of twinning caused by promiscuous fiber packing.

ATP-binding cassettes represent the motor domains in ABC transporters, a superfamily of integral membrane-protein pumps that couple the hydrolysis of ATP to transmembrane solute translocation. A crystal of a Mg-ADP complex of the MJ1267 ATP-binding cassette was obtained that produced a diffraction pattern characterized by pathological streaking of the spots in the a* x b* plane. While the Laue symmetry of the diffraction pattern was P3;1m, the crystal was determined to be twinned based on intensity statistics, molecular-replacement analysis and difference Fourier analysis of an engineered single-site methylmercury derivative. The unit cell contains three similar 3(1) fibers, with two of them related by primarily translational non-crystallographic symmetry (NCS) and the third related to the first two by approximate twofold screw operations whose rotational components are very similar to the twinning operator. The promiscuous packing of these 3(1) fibers, which make both parallel and antiparallel interactions in the primary crystal lattice, can explain the twinning tendency based on the ability of the twin-related lattices to interact with one another while making only one slightly sub-optimal intermolecular contact per unit cell in the boundary region. The promiscuous fiber packing can also explain the streaking in the diffraction pattern based on the ability to form a variety of different lattices with similar inter-fiber packing interactions. The crystal structure was refined as a twin in space group P3(1) using the program CNS, yielding a free R factor of 28.9% at 2.6 A and a refined twin fraction of 0.50. The structure shows a rigid-body rotation of the ABC-transporter-specific alpha-helical subdomain (ABCalpha subdomain) in MJ1267 compared with the conformation observed for the same protein in a C2 crystal lattice; this observation suggests that the ABCalpha subdomain is flexibly attached to the F1-type ATP-binding core of the ATP-binding cassette when Mg-ADP is bound at the active site.

ATP-Binding Cassette Transporters↗
PubMed2002

Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding.

BtuF is the periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD, a member of the ATP-binding cassette (ABC) transporter superfamily of transmembrane pumps. We have determined crystal structures of Escherichia coli BtuF in the apo state at 3.0 A resolution and with vitamin B12 bound at 2.0 A resolution. The structure of BtuF is similar to that of the FhuD and TroA PBPs and is composed of two alpha/beta domains linked by a rigid alpha-helix. B12 is bound in the "base-on" or vitamin conformation in a wide acidic cleft located between these domains. The C-terminal domain shares structural homology to a B12-binding domain found in a variety of enzymes. The same surface of this domain interacts with opposite surfaces of B12 when comparing ligand-bound structures of BtuF and the homologous enzymes, a change that is probably caused by the obstruction of the face that typically interacts with this domain by the base-on conformation of vitamin B12 bound to BtuF. There is no apparent pseudo-symmetry in the surface properties of the BtuF domains flanking its B12 binding site even though the presumed transport site in the previously reported crystal structure of BtuCD is located in an intersubunit interface with 2-fold symmetry. Unwinding of an alpha-helix in the C-terminal domain of BtuF appears to be part of conformational change involving a general increase in the mobility of this domain in the apo structure compared with the B12-bound structure. As this helix is located on the surface likely to interact with BtuC, unwinding of the helix upon binding to BtuC could play a role in triggering release of B12 into the transport cavity. Furthermore, the high mobility of this domain in free BtuF could provide an entropic driving force for the subsequent release of BtuF required to complete the transport cycle.

Amino Acid Sequence↗
PubMed2002

Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA.

The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport.

Adenosine Triphosphatases↗
PubMed2002

Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA.

The SecA adenosine triphosphatase (ATPase) mediates extrusion of the amino termini of secreted proteins from the eubacterial cytosol based on cycles of reversible binding to the SecYEG translocon. We have determined the crystal structure of SecA with and without magnesium-adenosine diphosphate bound to the high-affinity ATPase site at 3.0 and 2.7 angstrom resolution, respectively. Candidate sites for preprotein binding are located on a surface containing the SecA epitopes exposed to the periplasm upon binding to SecYEG and are thus positioned to deliver preprotein to SecYEG. Comparisons with structurally related ATPases, including superfamily I and II ATP-dependent helicases, suggest that the interaction geometry of the tandem motor domains in SecA is modulated by nucleotide binding, which is shown by fluorescence anisotropy experiments to reverse an endothermic domain-dissociation reaction hypothesized to gate binding to SecYEG.

Adenosine Diphosphate↗
PubMed2002

The crystal structure of the olfactory marker protein at 2.3 A resolution.

Olfactory marker protein (OMP) is a highly expressed and phylogenetically conserved cytoplasmic protein of unknown function found almost exclusively in mature olfactory sensory neurons. Electrophysiological studies of olfactory epithelia in OMP knock-out mice show strongly retarded recovery following odorant stimulation leading to an impaired response to pulsed odor stimulation. Although these studies show that OMP is a modulator of the olfactory signal-transduction cascade, its biochemical role is not established. In order to facilitate further studies on the molecular function of OMP, its crystal structure has been determined at 2.3 A resolution using multiwavelength anomalous diffraction experiments on selenium-labeled protein. OMP is observed to form a modified beta-clamshell structure with eight antiparallel beta-strands. While OMP has no significant sequence homology to proteins of known structure, it has a similar fold to a domain found in a variety of existing structures, including in a large family of viral capsid proteins. The surface of OMP is mostly convex and lacking obvious small molecule binding sites, suggesting that it is more likely to be involved in modulating protein-protein interaction than in interacting with small molecule ligands. Three highly conserved regions have been identified as leading candidates for protein-protein interaction sites in OMP. One of these sites represents a loop known to mediate ligand interactions in the structurally homologous EphB2 receptor ligand-binding domain. This site is partially buried in the crystal structure but fully exposed in the NMR solution structure of OMP due to a change in the orientation of an alpha-helix that projects outward from the structurally invariant beta-clamshell core. Gating of this conformational change by molecular interactions in the signal-transduction cascade could be used to control access to OMP's equivalent of the EphB2 ligand-interaction loop, thereby allowing OMP to function as a molecular switch.

Amino Acid Sequence↗
PubMed2002

Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters.

ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5'-(beta,gamma-imino)triphosphate and adenosine 5'-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl2, substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction.

Adenosine Diphosphate↗
PubMed2002

Acute effects of aerosolized S-nitrosoglutathione in cystic fibrosis.

S-nitrosoglutathione (GSNO), a naturally occurring constituent of airway lining fluid, enhances ciliary motility, relaxes airway smooth muscle, inhibits airway epithelial amiloride-sensitive sodium transport, and prevents pathogen replication. Remarkably, airway levels of GSNO are low in patients with cystic fibrosis (CF). We hypothesized that replacement of airway GSNO would improve gas exchange in CF. In a double-blind, placebo controlled study, we administered 0.05 ml/kg of 10 mM GSNO or phosphate buffered saline by aerosol to patients with CF and followed oxygen saturation, spirometry, respiratory rate, blood pressure, heart rate, and expired nitric oxide (NO). Nine patients received GSNO and 11 placebo. GSNO inhalation was associated with a modest but sustained increase in oxygen saturation at all time points. Expired NO increased in the low ppb range with GSNO treatment, peaking at 5 minutes but remaining above baseline at 30 minutes. There were no adverse effects. We conclude that GSNO is well tolerated in patients with CF and improves oxygenation through a mechanism that may be independent of free NO. Further, GSNO breakdown increases expired NO. We suggest that therapy aimed at restoring endogenous GSNO levels in the CF airway may merit study.

Administration, Inhalation↗
PubMed2002

Nitrogen redox balance in the cystic fibrosis airway: effects of antipseudomonal therapy.

Denitrifying bacteria metabolize nitrogen oxides through assimilatory and dissimilatory pathways. These redox reactions may affect lung physiology. We hypothesized that airway colonization with denitrifying bacteria could alter nitrogen balance in the cystic fibrosis (CF) airway. We measured airway nitrogen redox species before and after antimicrobial therapy for Pseudomonas aeruginosa in patients with CF. We also studied ammonium (NH(4)(+)) and nitric oxide (NO) metabolism in clinical strains of P. aeruginosa in vitro and in CF sputum ex vivo. Ammonium concentrations in both sputum and tracheal aspirates decreased with therapy. Nitric oxide reductase (NOR) was present in clinical strains of P. aeruginosa, which both produced NH(4)(+) and consumed NO. Further, NO consumption by CF sputum was inhibited by tobramycin ex vivo. We conclude that treatment of pseudomonal lung infections is associated with decreased NH(4)(+) concentrations in the CF airways. In epithelial cells, NH(4)(+) inhibits chloride transport, and nitrogen oxides inhibit amiloride-sensitive sodium transport and augment chloride transport. We speculate that normalization of airway nitrogen redox balance could contribute to the beneficial effects of antipseudomonal therapy on lung function in CF.

Ammonia↗
PubMed2002

Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.

Adenosine Diphosphate↗