Publications by authors named "David A Nyenhuis"

7 Publications

  • Page 1 of 1

Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport.

Elife 2021 Jul 12;10. Epub 2021 Jul 12.

Chemistry, University of Virginia, Charlottesville, United States.

Outer membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse EPR measurements were made in intact cells on the Escherichia coli vitamin B transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm towards the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, and they demonstrate the importance of studying outer membrane proteins in their native environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.68548DOI Listing
July 2021

Ebola virus glycoprotein interacts with cholesterol to enhance membrane fusion and cell entry.

Nat Struct Mol Biol 2021 02 18;28(2):181-189. Epub 2021 Jan 18.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA.

Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol-TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-020-00548-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992113PMC
February 2021

Native Cell Environment Constrains Loop Structure in the Escherichia coli Cobalamin Transporter BtuB.

Biophys J 2020 10 6;119(8):1550-1557. Epub 2020 Sep 6.

Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia. Electronic address:

The extracellular loops of bacterial outer membrane (OM) transporters are thought to sample a range of conformations in the apo state but to undergo a gating motion and assume a more defined conformation upon the binding of substrate. Here, we use pulse electron paramagnetic resonance to examine the conformations of the extracellular loops of BtuB, the Escherichia coli TonB-dependent vitamin B transporter, in whole cells. Unlike previous measurements carried out in vitro, the loops assume well-defined configurations in situ that closely match the in surfo crystal structures. Moreover, there is no evidence that the loops undergo significant gating motions upon the binding of substrate. The results demonstrate that the structure of BtuB is dependent upon an intact native OM environment, in which a critical component is likely to be the extracellular lipopolysaccharide. In general, this work indicates that measurements on OM proteins in reconstituted membrane systems may not reflect the native state of the protein in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2020.08.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642247PMC
October 2020

Evidence for the Supramolecular Organization of a Bacterial Outer-Membrane Protein from In Vivo Pulse Electron Paramagnetic Resonance Spectroscopy.

J Am Chem Soc 2020 06 8;142(24):10715-10722. Epub 2020 Jun 8.

In the outer membrane of Gram-negative bacteria, membrane proteins are thought to be organized into domains or islands that play a role in the segregation, movement, and turnover of membrane components. However, there is presently limited information on the structure of these domains or the molecular interactions that mediate domain formation. In the present work, the outer membrane vitamin B transporter, BtuB, was spin-labeled, and double electron-electron resonance was used to measure the distances between proteins in intact cells. These data together with Monte Carlo simulations provide evidence for the presence of specific intermolecular contacts between BtuB monomers that could drive the formation of string-like oligomers. Moreover, the EPR data provide evidence for the location of the interacting interfaces and indicate that lipopolysaccharide mediates the contacts between BtuB monomers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.0c01754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336812PMC
June 2020

Disulfide Chaperone Knockouts Enable In Vivo Double Spin Labeling of an Outer Membrane Transporter.

Biophys J 2019 10 10;117(8):1476-1484. Epub 2019 Sep 10.

Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia. Electronic address:

Recent advances in the application of electron paramagnetic resonance spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer membrane (OM) proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli OM B transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here, we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic-facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated OMs or reconstituted systems, and the ability to label and perform electron paramagnetic resonance in cells is expected to be applicable to a range of other bacterial OM proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2019.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817557PMC
October 2019

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

Proc Natl Acad Sci U S A 2017 09 5;114(38):E7987-E7996. Epub 2017 Sep 5.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908;

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1708052114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617291PMC
September 2017

PtdInsP and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium.

Elife 2016 10 28;5. Epub 2016 Oct 28.

Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

The Ca-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from and expressed in ) binds to PtdIns(4,5)P via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca neutralizes the negative charges of the Ca-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P). These Ca-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.15886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123861PMC
October 2016