556786
A novel mode of dimerization via formation of a glutamate anhydride crosslink in a protein crystal structure.
Acid anhydrides are formed by removal of water, are very active, and can react with water to form the original binary components.
205373753
SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E.
Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.
19975781
Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.
The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.
24638854
Structure determination of an FMN reductase from Pseudomonas aeruginosa PA01 using sulfur anomalous signal.
The availability of high-intensity synchrotron facilities, technological advances in data-collection techniques and improved data-reduction and crystallographic software have ushered in a new era in high-throughput macromolecular crystallography. Here, the de novo automated crystal structure determination at 1.28 A resolution of an NAD(P)H-dependent FMN reductase flavoprotein from Pseudomonas aeruginosa PA01-derived protein Q9I4D4 using the anomalous signal from an unusually small number of S atoms is reported. Although this protein lacks the flavodoxin key fingerprint motif [(T/S)XTGXT], it has been confirmed to bind flavin mononucleotide and the binding site was identified via X-ray crystallography. This protein contains a novel flavin mononucleotide-binding site GSLRSGSYN, which has not been previously reported. Detailed statistics pertaining to sulfur phasing and other factors contributing to structure determination are discussed. Structural comparisons of the apoenzyme and the protein complexed with flavin mononucleotide show conformational changes on cofactor binding. NADPH-dependent activity has been confirmed with biochemical assays.
12841382
Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F
Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.
21302804
Structural analysis of a ternary complex of allantoate amidohydrolase from Escherichia coli reveals its mechanics.
Purine metabolism plays a major role in regulating the availability of purine nucleotides destined for nucleic acid synthesis. Allantoate amidohydrolase catalyzes the conversion of allantoate to (S)-ureidoglycolate, one of the crucial alternate steps in purine metabolism. The crystal structure of a ternary complex of allantoate amidohydrolase with its substrate allantoate and an allosteric effector, a sulfate ion, from Escherichia coli was determined to understand better the catalytic mechanism and substrate specificity. The 2.25 A resolution X-ray structure reveals an alpha/beta scaffold akin to zinc exopeptidases of the peptidase M20 family and lacks the (beta/alpha)(8)-barrel fold characteristic of the amidohydrolases. Arrangement of the substrate and the two co-catalytic zinc ions at the active site governs catalytic specificity for hydrolysis of N-carbamyl versus the peptide bond in exopeptidases. In its crystalline form, allantoate amidohydrolase adopts a relatively open conformation. However, structural analysis reveals the possibility of a significant movement of domains via rotation about two hinge regions upon allosteric effector and substrate binding resulting in a closed catalytically competent conformation by bringing the substrate allantoate closer to co-catalytic zinc ions. Two cis-prolyl peptide bonds found on either side of the dimerization domain in close proximity to the substrate and ligand-binding sites may be involved in protein folding and in preserving the integrity of the catalytic site.
16445532
Prevalence, determinants and prognosis of pulmonary hypertension among hemodialysis patients.
BACKGROUND
The prevalence, determinants and prognosis of pulmonary hypertension among long-term hemodialysis patients in the USA are poorly understood.


METHODS
A cross-sectional survey of prevalence and determinants of pulmonary hypertension was performed, followed by longitudinal follow-up for all-cause mortality. Pulmonary hypertension was defined as an estimated systolic pulmonary artery pressure of >35 mmHg using echocardiograms performed within an hour after the end of dialysis.


RESULTS
Prevalent in 110/288 patients (38%), the independent determinants of pulmonary hypertension were the following: left atrial diameter (odds ratio 10.1 per cm/m(2), P < 0.0001), urea reduction ratio (odds ratio 0.94 per %, P < 0.01) and vitamin D receptor activator use (odds ratio 0.41 for users, P < 0.01). Over a median follow-up of 2.15 years, 97 (34%) patients died yielding a crude mortality rate (CMR) of 114.2 per 1000 patient-years. Of these, 58 deaths occurred among 110 patients with pulmonary hypertension (53%, CMR 168.9/1000 patient-years) and 39 among 178 without pulmonary hypertension (22%, CMR 52.5/1000 patient-years) [unadjusted hazard ratio (HR) for death 2.12 (95% confidence interval 1.41-3.19), P < 0.001]. After multivariate adjustment, pulmonary hypertension remained an independent predictor for all-cause mortality [HR 2.17 (95% confidence interval 1.31-3.61), P < 0.01].


CONCLUSIONS
Among hemodialysis patients, pulmonary hypertension is common and is strongly associated with an enlarged left atrium and poor long-term survival. Reducing left atrial size such as through volume control may be an attractive target to improve pulmonary hypertension. Improving pulmonary hypertension in this group of patients may improve the dismal outcomes.
36556551
Glutathione S-transferase-dependent conjugation of leukotriene A4-methyl ester to leukotriene C4-methyl ester in mammalian skin.
The glutathione S-transferase (GST)-dependent conjugation of reduced glutathione (GSH) with leukotriene A4 (LTA4)-methyl ester in rodent and human skin was investigated. Incubation of [3H]LTA4-methyl ester (1 nmole, approximately 200,000 dpm) with cytosol prepared from rat, mouse and human skin or with affinity purified GST from rat skin cytosol in the presence of GSH resulted in the formation of LTC4-methyl ester. Maximum enzyme activity was observed in rat skin followed by mouse and human skin. With heat-denatured cytosol or in the absence of GSH, the product formation was negligible. GST purified from rat skin cytosol by GSH-agarose affinity chromatography exhibited a several-fold increase in the specific activity of enzyme with 1-chloro-2,4-dinitrobenzene (55-fold), ethacrynic acid (67-fold) and LTA4-methyl ester (12-fold) as substrates. Western blot analysis of the affinity purified GST indicated a predominant expression of the Pi class of GST isozyme followed by Mu and Alpha classes of isozymes. The formation of LTC4-methyl ester was established by its radioactivity profile on high pressure liquid chromatography and absorption spectroscopy. These results suggest that, in addition to xenobiotic metabolism, cutaneous GSTs may also be capable of metabolizing physiological substrates such as LTA4.
22757148
Involvement of singlet oxygen in chloroaluminum phthalocyanine tetrasulfonate-mediated photoenhancement of lipid peroxidation in rat epidermal microsomes.
In recent studies chloroaluminum phthalocyanine tetrasulfonate (AlPCTS) has been shown to be an effective photosensitizer for the tumor necrosis in a modality known as photodynamic therapy, but the mechanism of photodynamic effect of AlPCTS is poorly understood. In this study, in vitro incubation of rat epidermal microsomes with AlPCTS followed by exposure to red light (approximately 675 nm) resulted in an increase in ADP/iron-supported lipid peroxidation, a measure of membrane damage. This photodestructive effect was found to be dependent on both the duration of light exposure and the dose of AlPCTS. Studies employing various quenchers of reactive oxygen species revealed that scavengers of singlet oxygen (histidine, 2,5-dimethylfuran, beta-carotene and sodium azide) afforded substantial protection (up to 90%) of photoenhancement whereas the scavengers of hydrogen peroxide (catalase), superoxide anion (superoxide dismutase), and hydroxyl radical (sodium benzoate, mannitol and ethanol) were ineffective in this regard. Our data indicate that AlPCTS-mediated photodestruction mainly involves a type II reaction via singlet oxygen formation and suggest that the latter could play a significant role in the tumor necrosis evoked by AlPCTS and light.
24386028
Link between CFTR mutations and ABPA: a systematic review and meta-analysis.
Summary  There is a biological plausibility on the link between cystic fibrosis transmembrane conductance regulator (CFTR) mutations and allergic bronchopulmonary aspergillosis (ABPA). The aim of the systematic review was to investigate this link by determining the frequency of CFTR mutations in ABPA. We searched the PubMed and EmBase databases for studies reporting CFTR mutations in ABPA. We pooled the odds ratio (OR) and 95% confidence intervals (CI) from individual studies using both fixed and random effects model. Statistical heterogeneity was evaluated using the I(2) test and the Cochran-Q statistic. Publication bias was assessed using both graphical and statistical methods. Our search yielded four studies (79 ABPA, 268 controls). The odds of encountering CFTR mutation was higher in ABPA compared with the control group (OR 10.39; 95% CI, 4.35-24.79) or the asthma population (OR 5.53; 95% CI 1.62-18.82). There was no evidence of statistical heterogeneity or publication bias. There is a possible pathogenetic link between CFTR mutations and ABPA. However, because of the small numbers of patients, further studies are required to confirm this finding. Future studies should adopt a uniform methodology and should screen for the entire genetic sequence of the CFTR gene.
9534946
Tuberculous dilated cardiomyopathy: an under-recognized entity?
BackgroundTuberculosis (TB) is a common public health problem in many parts of the world. TB is generally believed to spare these four organs-heart, skeletal muscle, thyroid and pancreas. We describe a rare case of myocardial TB diagnosed on a post-mortem cardiac biopsy.Case presentationPatient presented with history suggestive of congestive heart failure. We describe the clinical presentation, investigations and outcome of this case, and review the literature on the involvement of myocardium by TB.ConclusionInvolvement of myocardium by TB is rare. However it should be suspected as a cause of congestive heart failure in any patient with features suggestive of TB. Increasing recognition of the entity and the use of endomyocardial biopsy may help us detect more cases of this "curable" form of cardiomyopathy.