A rotary mechanism for allostery in bacterial hybrid malic enzymes

This project was funded by BBSRC studentship 1500753 to C.J.H. and a BBSRC David Phillips fellowship to P.J.M. (BB/S010122/1).Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.Publisher PDFPeer reviewe

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.Peer reviewe

The regulation of gene expression in sulphate reducing bacteria

$Desulfovibrio desulfuricans$ ATCC27774 can use both nitrate and sulphate as terminal electron acceptors for growth. Contrary to expectations, the less favourable terminal electron acceptor, sulphate, is utilised preferentially. It has been proposed that the nap gene cluster of $D. desulfuricans$, which encodes nitrate reduction, is regulated at the transcription level by both sulphate repression and nitrate induction. Transcription factors of the CRP/FNR family have been implicated in this regulation. At the start of this study a bioinformatic analysis identified five CRP-homologues in $D. desulfuricans$, two of which, HcpR and HcpR2, were predicted to bind discrete regulatory motifs and influence the transcription of $nap$, $hcpR$ and the gene for the hybrid cluster protein, $hcp$. Physiological and qRT-PCR experiments revealed that expression of $hcpR$ is induced in the presence of nitrite whereas expression of $hcpR2$ is constitutive. Furthermore, tolerance to nitrosative stress can be pre-induced by exposure to nitrite. In electromobility shift and footprinting assays, HcpR bound to CRP-like DNA targets upstream of the $nap$ and $hcpR$ operons: HcpR2 bound to a different target upstream of $hcp$. HcpR was shown to heterolgously affect transcription at CRP-regulated promoters in an $Escherichia$ $coli$ host. The pET expression system was used to overproduce HcpR, the recombinant protein was purified and western analysis with a rabbit anti-HcpR antiserum revealed that nitric oxide is the inducer of HcpR synthesis. Once induced, significant levels of HcpR persisted in the absence of effector for many generations. The results suggest that the primary function of HcpR is auto-regulation of the $hcpR$ operon. This transcription unit includes genes similar to $nimA$ and $wrbA$ and their expression is believed to be essential for protecting $D. desulfuricans$ against nitrosative stress. Contrary to the predictions of the bioinformatic analysis, expression of $nap$ did not correlate with $hcpR$ levels. An alternative hypothesis is proposed in which $nap$ and $nrf$ are primarily regulated by a sigma-54-dependent mechanism

Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport

Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport

The Influence of Age and Sex on Genetic Associations with Adult Body Size and Shape: A Large-Scale Genome-Wide Interaction Study.

Genome-wide association studies (GWAS) have identified more than 100 genetic variants contributing to BMI, a measure of body size, or waist-to-hip ratio (adjusted for BMI, WHRadjBMI), a measure of body shape. Body size and shape change as people grow older and these changes differ substantially between men and women. To systematically screen for age- and/or sex-specific effects of genetic variants on BMI and WHRadjBMI, we performed meta-analyses of 114 studies (up to 320,485 individuals of European descent) with genome-wide chip and/or Metabochip data by the Genetic Investigation of Anthropometric Traits (GIANT) Consortium. Each study tested the association of up to ~2.8M SNPs with BMI and WHRadjBMI in four strata (men ≤50y, men >50y, women ≤50y, women >50y) and summary statistics were combined in stratum-specific meta-analyses. We then screened for variants that showed age-specific effects (G x AGE), sex-specific effects (G x SEX) or age-specific effects that differed between men and women (G x AGE x SEX). For BMI, we identified 15 loci (11 previously established for main effects, four novel) that showed significant (FDR<5%) age-specific effects, of which 11 had larger effects in younger (<50y) than in older adults (≥50y). No sex-dependent effects were identified for BMI. For WHRadjBMI, we identified 44 loci (27 previously established for main effects, 17 novel) with sex-specific effects, of which 28 showed larger effects in women than in men, five showed larger effects in men than in women, and 11 showed opposite effects between sexes. No age-dependent effects were identified for WHRadjBMI. This is the first genome-wide interaction meta-analysis to report convincing evidence of age-dependent genetic effects on BMI. In addition, we confirm the sex-specificity of genetic effects on WHRadjBMI. These results may provide further insights into the biology that underlies weight change with age or the sexually dimorphism of body shape

Genome-wide association and large-scale follow up identifies 16 new loci influencing lung function

Pulmonary function measures reflect respiratory health and are used in the diagnosis of chronic obstructive pulmonary disease. We tested genome-wide association with forced expiratory volume in 1 second and the ratio of forced expiratory volume in 1 second to forced vital capacity in 48,201 individuals of European ancestry with follow up of the top associations in up to an additional 46,411 individuals. We identified new regions showing association (combined P &lt; 5 × 10?8) with pulmonary function in or near MFAP2, TGFB2, HDAC4, RARB, MECOM (also known as EVI1), SPATA9, ARMC2, NCR3, ZKSCAN3, CDC123, C10orf11, LRP1, CCDC38, MMP15, CFDP1 and KCNE2. Identification of these 16 new loci may provide insight into the molecular mechanisms regulating pulmonary function and into molecular targets for future therapy to alleviate reduced lung function

Correction: The Influence of Age and Sex on Genetic Associations with Adult Body Size and Shape: A Large-Scale Genome-Wide Interaction Study

[This corrects the article DOI: 10.1371/journal.pgen.1005378.].status: publishe