A four-helix bundle stores copper for methane oxidation

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO). Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location

Length of Variable Numbers of Tandem Repeats in the Carboxyl Ester Lipase (CEL) Gene May Confer Susceptibility to Alcoholic Liver Cirrhosis but Not Alcoholic Chronic Pancreatitis

Background Carboxyl-ester lipase (CEL) contributes to fatty acid ethyl ester metabolism, which is implicated in alcoholic pancreatitis. The CEL gene harbours a variable number of tandem repeats (VNTR) region in exon 11. Variation in this VNTR has been linked to monogenic pancreatic disease, while conflicting results were reported for chronic pancreatitis (CP). Here, we aimed to investigate a potential association of CEL VNTR lengths with alcoholic CP. Methods Overall, 395 alcoholic CP patients, 218 patients with alcoholic liver cirrhosis (ALC) serving as controls with a comparable amount of alcohol consumed, and 327 healthy controls from Germany and the United Kingdom (UK) were analysed by determination of fragment lengths by capillary electrophoresis. Allele frequencies and genotypes of different VNTR categories were compared between the groups. Results Twelve repeats were overrepresented in UK ACP patients (P = 0.04) compared to controls, whereas twelve repeats were enriched in German ALC compared to alcoholic CP patients (P = 0.03). Frequencies of CEL VNTR lengths of 14 and 15 repeats differed between German ALC patients and healthy controls (P = 0.03 and 0.008, respectively). However, in the genotype and pooled analysis of VNTR lengths no statistical significant association was depicted. Additionally, the 16–16 genotype as well as 16 repeats were more frequent in UK ALC than in alcoholic CP patients (P = 0.034 and 0.02, respectively). In all other calculations, including pooled German and UK data, allele frequencies and genotype distributions did not differ significantly between patients and controls or between alcoholic CP and ALC. Conclusions We did not obtain evidence that CEL VNTR lengths are associated with alcoholic CP. However, our results suggest that CEL VNTR lengths might associate with ALC, a finding that needs to be clarified in larger cohorts

Investigation of Indazole Unbinding Pathways in CYP2E1 by Molecular Dynamics Simulations

Human microsomal cytochrome P450 2E1 (CYP2E1) can oxidize not only low molecular weight xenobiotic compounds such as ethanol, but also many endogenous fatty acids. The crystal structure of CYP2E1 in complex with indazole reveals that the active site is deeply buried into the protein center. Thus, the unbinding pathways and associated unbinding mechanisms remain elusive. In this study, random acceleration molecular dynamics simulations combined with steered molecular dynamics and potential of mean force calculations were performed to identify the possible unbinding pathways in CYP2E1. The results show that channel 2c and 2a are most likely the unbinding channels of CYP2E1. The former channel is located between helices G and I and the B-C loop, and the latter resides between the region formed by the F-G loop, the B-C loop and the β1 sheet. Phe298 and Phe478 act as the gate keeper during indazole unbinding along channel 2c and 2a, respectively. Previous site-directed mutagenesis experiments also supported these findings

Salmonella enterica ssp. arizonae infection in a 43-year-old Italian man with hypoglobulinemia: a case report and review of the literature

<p>Abstract</p> <p>Introduction</p> <p><it>Salmonella enterica </it>ssp. <it>arizonae </it>is an uncommon human pathogen with serious infections reported in immunocompromised hosts. In Europe, only a few cases have been described. Patients with this infection usually have a history of contact with reptiles or travel abroad. We present a case report of infection in a patient with hypoglobulinemia and a literature review.</p> <p>Case presentation</p> <p>We describe the case of a 43-year-old Caucasian Italian man with hypoglobulinemia who presented to our hospital with sepsis and diarrhea. A stool culture yielded <it>S. enterica </it>ssp. <it>arizonae</it>. Our patient was treated with oral ciprofloxacin and made a full recovery. We also present a review of the cases of <it>S. enterica </it>ssp. <it>arizonae </it>infections previously reported in Europe.</p> <p>Conclusions</p> <p>The majority of infections from <it>S. enterica </it>ssp. <it>arizonae </it>occur in patients who are immunocompromised. Data from the literature suggests that it may be difficult to eradicate the bacteria and thus, prolonged antibiotic courses are often used. It would be advisable for clinicians to investigate for pre-existing immune dysfunction if <it>S. enterica </it>ssp. <it>arizonae </it>is isolated. In Italy, although there have only been a few cases, the likely route of transmission remains unclear and requires further surveillance.</p

Comparative Analysis of the Heptahelical Transmembrane Bundles of G Protein-Coupled Receptors

Background: G protein-coupled receptors represent a large family of eukaryotic membrane proteins, and are involved in almost all physiological processes in humans. Recent advances in the crystallographic study of these receptors enable a detailed comparative analysis of the commonly shared heptahelical transmembrane bundle. Systematic comparison of the bundles from a variety of receptors is indispensable for understanding not only of the structural diversification optimized for the binding of respective ligands but also of the structural conservation required for the common mechanism of activation accompanying the interaction changes among the seven helices. Methodology/Principal Findings: We have examined the bundles of 94 polypeptide chains from almost all available structures of 11 receptors, which we classified into either inactivated chain or activated chain, based on the type of bound ligand. For the inactivated chains, superposition of 200 residue bundles by secondary structure matching demonstrated that the bound ligands share a laterally limited cavity in the extracellular section of the bundle. Furthermore, a distinct feature was found for helix III of bovine rhodopsin, which might have evolved to lower its activity in the presence of 11-cis-retinal, to a level that other receptors could hardly achieve with any currently available ligands. Conclusions/Significance: Systematic analysis described here would be valuable for understanding of the rearrangement o

Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe–4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe–4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove

A half-site multimeric enzyme achieves its cooperativity without conformational changes

Cooperativity is a feature many multimeric proteins use to control activity. Here we show that the bacterial heptose isomerase GmhA displays homotropic positive and negative cooperativity among its four protomers. Most similar proteins achieve this through conformational changes: GmhA instead employs a delicate network of hydrogen bonds, and couples pairs of active sites controlled by a unique water channel. This network apparently raises the Lewis acidity of the catalytic zinc, thus increasing the activity at one active site at the cost of preventing substrate from adopting a reactive conformation at the paired negatively cooperative site – a “half-site” behavior. Our study establishes the principle that multimeric enzymes can exploit this cooperativity without conformational changes to maximize their catalytic power and control. More broadly, this subtlety by which enzymes regulate functions could be used to explore new inhibitor design strategies

Diffractive Dijet Production at sqrt(s)=630 and 1800 GeV at the Fermilab Tevatron

We report a measurement of the diffractive structure function $F_{jj}^D$ of the antiproton obtained from a study of dijet events produced in association with a leading antiproton in $\bar pp$ collisions at $\sqrt s=630$ GeV at the Fermilab Tevatron. The ratio of $F_{jj}^D$ at $\sqrt s=630$ GeV to $F_{jj}^D$ obtained from a similar measurement at $\sqrt s=1800$ GeV is compared with expectations from QCD factorization and with theoretical predictions. We also report a measurement of the $\xi$ ($x$-Pomeron) and $\beta$ ($x$ of parton in Pomeron) dependence of $F_{jj}^D$ at $\sqrt s=1800$ GeV. In the region $0.035<\xi<0.095$, $|t|<1$ GeV$^2$ and $\beta<0.5$, $F_{jj}^D(\beta,\xi)$ is found to be of the form $\beta^{-1.0\pm 0.1} \xi^{-0.9\pm 0.1}$, which obeys $\beta$-$\xi$ factorization.Comment: LaTeX, 9 pages, Submitted to Phys. Rev. Letter