The structural basis of bacterial manganese import

Metal ions are essential for all forms of life. In prokaryotes, ATP-binding cassette (ABC) permeases serve as the primary import pathway for many micronutrients including the first-row transition metal manganese. However, the structural features of ionic metal transporting ABC permeases have remained undefined. Here, we present the crystal structure of the manganese transporter PsaBC from Streptococcus pneumoniae in an open-inward conformation. The type II transporter has a tightly closed transmembrane channel due to "extracellular gating" residues that prevent water permeation or ion reflux. Below these residues, the channel contains a hitherto unreported metal coordination site, which is essential for manganese translocation. Mutagenesis of the extracellular gate perturbs manganese uptake, while coordination site mutagenesis abolishes import. These structural features are highly conserved in metal-specific ABC transporters and are represented throughout the kingdoms of life. Collectively, our results define the structure of PsaBC and reveal the features required for divalent cation transport.Stephanie L. Neville, Jennie Sjöhamn, Jacinta A. Watts, Hugo MacDermott-Opeskin, Stephen J. Fairweather, Katherine Ganio, Alex Carey Hulyer, Aaron P. McGrath, Andrew J. Hayes, Tess R. Malcolm, Mark R. Davies, Norimichi Nomura, So Iwata, Megan L. O’Mara, Megan J. Maher, Christopher A. McDevit

Observation of hard scattering in photoproduction events with a large rapidity gap at HERA

Events with a large rapidity gap and total transverse energy greater than 5 GeV have been observed in quasi-real photoproduction at HERA with the ZEUS detector. The distribution of these events as a function of the $\gamma p$ centre of mass energy is consistent with diffractive scattering. For total transverse energies above 12 GeV, the hadronic final states show predominantly a two-jet structure with each jet having a transverse energy greater than 4 GeV. For the two-jet events, little energy flow is found outside the jets. This observation is consistent with the hard scattering of a quasi-real photon with a colourless object in the proton.Comment: 19 pages, latex, 4 figures appended as uuencoded fil

Comparing nonbonded metal Ion models in the divalent cation binding protein PsaA

Divalent metal cations are essential for many biological processes; however, accurately modeling divalent metal ions has proved a significant challenge for molecular dynamics force fields. Here we show that the choice of ion model influences the observed dynamics in PsaA, a metal binding protein from Streptococcus pneumoniae. We conduct extensive unbiased simulations and free energy calculations of PsaA bound to its cognate ligand Mn2+ and inhibitory ligand Zn2+ using three nonbonded ion models: a 12-6 model, a 12-6-4 model, and a multisite model. The observed coordination geometries and metal binding dynamics are sensitive to the choice of ion model, with the most dramatic differences observed in free energy calculations of ion release. We show that the conformational ensemble of Mn-bound PsaA is more similar to the crystallographic metal bound open state. This work extends the current model of PsaA metal binding and provides a framework for the rationalization of experimentally determined metal binding behavior. Our findings support the use of the 12-6-4 ion model for further simulations of divalent cation binding proteins.Hugo MacDermott-Opeskin, Christopher A. McDevitt, Megan L. O’Mar

The membrane composition defines the spatial organization and function of a major Acinetobacter baumannii drug efflux system

Acinetobacter baumannii is one of the world’s most problematic nosocomial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii’s unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. Importance: Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii may be actively acquired by A. baumannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. On a broader scope, this study presents new findings on the potential health benefits of PUFA in individuals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.Maoge Zang, Hugo MacDermott-Opeskin, Felise G. Adams, Varsha Naidu, Jack K. Waters, Ashley B. Carey, Alex Ashenden, Kimberley T. McLean, Erin B. Brazel, Jhih-Hang Jiang, Alessandra Panizza, Claudia Trappetti, James C. Paton, Anton Y. Peleg, Ingo Köper, Ian T. Paulsen, Karl A. Hassan, Megan L. O’Mara, Bart A. Eijkelkam

Structure-based interpretation of the mutagenesis database for the nucleotide binding domains of P-glycoprotein

P-glycoprotein (P-gp) is the most intensively studied eukaryotic ATP binding cassette (ABC) transporter, due to its involvement in the multidrug resistance phenotype of a number of cancers. In common with most ABC transporters, P-gp is comprised of two transmembrane domains (TMDs) and two nucleotide binding domains (NBD), the latter coupling ATP hydrolysis with substrate transport (efflux in the case of P-gp). Biochemical investigations over the past twenty years have attempted to unlock mechanistic aspects of P-glycoprotein through scanning and site-directed mutagenesis of both the TMDs and the NBDs. Contemporaneously, crystallographers have elucidated the atomic structure of numerous ABC transporter NBDs, as well as the intact structure (i.e. NBDs and TMDs) of a distantly related ABC-exporter Sav1866. Significantly, the structure of P-gp remains unknown, and only low resolution electron microscopy data exists. Within the current manuscript we employ crystallographic data for homologous proteins, and a molecular model for P-gp, to perform a structural interpretation of the existing “mutagenesis database” for P-gp NBDs. Consequently, this will enable testable predictions to be made that will result in further in-roads into our understanding of this clinically important drug pump. © 2007 Elsevier B.V. All rights reserved

Study of D*(2010)+- Production in ep Collisions at HERA

We report the first observation of charmed mesons with the ZEUS detector at HERA using the decay channel D*(+) --> (D-0 --> K(-)pi(+)) pi(+) (+ c.c.). Clear signals in the mass difference Delta M=M(D*)-M(D-0) as well as in the M(K pi) distribution at the D-0 mass are found. The ep cross section for inclusive D*(+/-) production with Q(2) c $($) over bar cX)=(0.45+/-0.11(-0.22)(+0.37)) mu b at root s=296 GeV and [W]=198 GeV. The average gamma p charm cross section sigma(gamma p --> c $($) over bar cX) is found to be (6.3+/-2.2(-3.0)(+6.3)) mu b at [W]=163 GeV and (16.9+/-5.2(-8.5)(+13.9)) mu b at [W]=243 GeV. The increase of the total charm photoproduction cross section by one order of magnitude with respect to low energy data experiments is well described by QCD NLO calculations using singular gluon distributions in the proton

Exclusive rho0 production in deep inelastic electron - proton scattering at HERA

The exclusive production of $\rho^0$ mesons in deep inelastic electron-proton scattering has been studied using the ZEUS detector. Cross sections have been measured in the range $7 < Q^2 < 25$ GeV$^2$ for $\gamma^*p$ centre of mass (c.m.) energies from 40 to 130 GeV. The $\gamma^*p \to \rho^0 p$ cross section exhibits a $Q^{-(4.2 \pm 0.8 ^{+1.4}_{-0.5})}$ dependence and both longitudinally and transversely polarised $\rho^0$'s are observed. The $\gamma^*p \to \rho^0 p$ cross section rises strongly with increasing c.m. energy, when compared with NMC data at lower energy, which cannot be explained by production through soft pomeron exchange. The data are compared with perturbative QCD calculations where the rise in the cross section reflects the increase in the gluon density at low $x$. the gluon density at low $x$.Comment: 22 pages, latex, 4 figures appende

Diffractive hard photoproduction at HERA and evidence for the gluon content of the pomeron

Inclusive jet cross sections for events with a large rapidity gap with respect to the proton direction from the reaction $ep \rightarrow jet \; + \; X$ with quasi-real photons have been measured with the ZEUS detector. The cross sections refer to jets with transverse energies $E_T^{jet}>8$~GeV. The data show the characteristics of a diffractive process mediated by pomeron exchange. Assuming that the events are due to the exchange of a pomeron with partonic structure, the quark and gluon content of the pomeron is probed at a scale $\sim (E_T^{jet})^2$. A comparison of the measurements with model predictions based on QCD plus Regge phenomenology requires a contribution of partons with a hard momentum density in the pomeron. A combined analysis of the jet cross sections and recent ZEUS measurements of the diffractive structure function in deep inelastic scattering gives the first experimental evidence for the gluon content of the pomeron in diffractive hard scattering processes. The data indicate that between 30\% and 80\% of the momentum of the pomeron carried by partons is due to hard gluons.Comment: 25 pages, latex, 4 figures appended as uuencoded file. No change w.r.t. previous version (just avoiding warning latex messages while processing