scholarly journals Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter

2018 ◽  
Author(s):  
Aaron T. Bozzi ◽  
Christina M. Zimanyi ◽  
John M. Nicoludis ◽  
Brandon K. Lee ◽  
Casey H. Zhang ◽  
...  
Keyword(s):  
High Resolution ◽  
Proton Transport ◽  
Deinococcus Radiodurans ◽  
Intramolecular Rearrangements ◽  
Transport Cycle ◽  
Change Mechanisms ◽  
Multiple Conformations ◽  
Open States ◽  
Alternating Access ◽  
Primary Substrate

AbstractNramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that includes proton co-transport. We present high-resolution structures ofDeinococcus radiodurans(Dra)Nramp at complementary stages of its transport cycle to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward-to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, thus resolving the electrostatic dilemma of using a cation co-substrate to drive a cation primary substrate. Our results illustrate the flexibility of the LeuT fold to support a broad range of co-substrate coupling and conformational change mechanisms.

scholarly journals Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Aaron T Bozzi ◽  
Christina M Zimanyi ◽  
John M Nicoludis ◽  
Brandon K Lee ◽  
Casey H Zhang ◽  
...  
Keyword(s):  
High Resolution ◽  
Transition Metal ◽  
Proton Transport ◽  
Deinococcus Radiodurans ◽  
Intramolecular Rearrangements ◽  
Transport Cycle ◽  
Change Mechanisms ◽  
Multiple Conformations ◽  
Open States ◽  
Alternating Access

Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms.

High-Resolution Ion-Flux Imaging of Proton Transport Through Graphene|Nafion Membranes

2021 ◽  
Author(s):  
Cameron Bentley ◽  
Minkyung Kang ◽  
Saheed Bukola ◽  
Stephen Creager ◽  
Patrick Unwin
Keyword(s):  
High Resolution ◽  
Proton Transport ◽  
Ion Flux ◽  
Nafion Membranes

scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
H. Ronald Kaback
Keyword(s):  
Binding Site ◽  
Lactose Permease ◽  
Binding Studies ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Transport Cycle ◽  
Open Structures ◽  
Unrestricted Access ◽  
Tight Association ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

scholarly journals Crystal Structure of a ligand-bound LacY–Nanobody Complex

2018 ◽  
Vol 115 (35) ◽  
pp. 8769-8774 ◽  
Author(s):  
Hemant Kumar ◽  
Janet S. Finer-Moore ◽  
Xiaoxu Jiang ◽  
Irina Smirnova ◽  
Vladimir Kasho ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Lactose Permease ◽  
Transmembrane Helices ◽  
Helical Bundle ◽  
Flexible Loop ◽  
Sugar Binding ◽  
Open Conformation ◽  
Substrate Analog ◽  
Multiple Conformations ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb–LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

scholarly journals Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import

2019 ◽  
Vol 151 (12) ◽  
pp. 1413-1429 ◽  
Author(s):  
Aaron T. Bozzi ◽  
Lukas B. Bane ◽  
Christina M. Zimanyi ◽  
Rachelle Gaudet
Keyword(s):  
Proton Transport ◽  
Metal Uptake ◽  
Deinococcus Radiodurans ◽  
Salt Bridge ◽  
Metal Transport ◽  
Natural Resistance ◽  
Transport Pathway ◽  
Directional Bias ◽  
Proton Cotransport ◽  
Stimulation Of

Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by “coupling” proton and metal transport. Here we use the Deinococcus radiodurans (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: ΔpH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp’s structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and ΔpH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport.

scholarly journals Structural flexibility revealed by ultra-high resolution data

2014 ◽  
Vol 70 (a1) ◽  
pp. C1199-C1199
Author(s):  
Zbigniew Dauter
Keyword(s):  
High Resolution ◽  
Data Bank ◽  
Geometrical Parameters ◽  
Protein Chain ◽  
X Rays ◽  
High Resolution Data ◽  
Density Maps ◽  
Target Values ◽  
Multiple Conformations ◽  
Dna Dodecamer

Not many macromolecular crystals diffract X-rays to ultra-high resolution, defined usually as higher than 0.8 Å, and in the Protein Data Bank there are currently 43 such submissions. These structures range in size from antibiotics of about a hundred atoms to proteins with more than 3,000 independent atoms in the asymmetric unit of the crystal cell. The unprecedented data resolution reveals a great wealth of structural details, which cannot be visualized by analyses at lower resolution. The accuracy of the refined stereochemical and geometrical parameters is then comparable with values typical for small-molecular crystallography and exceeds the accuracy of the library of the standard restraint target values, routinely used in refinement of proteins and nucleotides. Somewhat unexpectedly, the very high resolution diffraction does not necessarily relates to extreme stability of the crystallized molecules, so that the obtained electron density maps reveal significant parts of the atomic models existing in multiple conformations, slightly differing from each other. For example, about 1/3 of the protein chain in the 0.65 Å structure of lysozyme [1] and majority of phosphate groups in the 0.75 Å structure of Z-DNA dodecamer [2] could be modeled in double conformations.

scholarly journals High resolution structure of the membrane embedded skeletal muscle ryanodine receptor

2021 ◽  
Author(s):  
Zephan Melville ◽  
Kookjoo Kim ◽  
Oliver B. Clarke ◽  
Andrew R. Marks
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Direct Detection ◽  
Gel Filtration ◽  
Calcium Release Channel ◽  
Release Channel ◽  
High Resolution Structure ◽  
Open States

AbstractThe type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel, though a recent study resolved the structure with limited resolution in nanodiscs1. In the present study we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, a method that improved the incorporation rate by more than 20-fold compared to a dialysis-based approach. In conjunction with new direct-detection cameras, this allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 Å, respectively. This method offers validation for detergent-based structures of the RyR and lays the groundwork for studies utilizing an electrochemical gradient mimicking the native environment, such as that of the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol of the cell at rest.

scholarly journals Effect of Cytosolic pH on Inward Currents Reveals Structural Characteristics of the Proton Transport Cycle in the Influenza A Protein M2 in Cell-Free Membrane Patches of Xenopus oocytes

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e107406 ◽  
Author(s):  
Mattia L. DiFrancesco ◽  
Ulf-Peter Hansen ◽  
Gerhard Thiel ◽  
Anna Moroni ◽  
Indra Schroeder
Keyword(s):  
Proton Transport ◽  
Influenza A ◽  
Xenopus Oocytes ◽  
Structural Characteristics ◽  
Cytosolic Ph ◽  
Transport Cycle ◽  
Inward Currents ◽  
Free Membrane

scholarly journals Transient conformers of LacY are trapped by nanobodies

2015 ◽  
Vol 112 (45) ◽  
pp. 13839-13844 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Xiaoxu Jiang ◽  
Els Pardon ◽  
Jan Steyaert ◽  
...  
Keyword(s):  
Lactose Permease ◽  
Conformational States ◽  
Dynamic Membrane ◽  
Conformational Selection ◽  
Sugar Binding ◽  
Open Conformation ◽  
Transport Cycle ◽  
Fluorescence Change ◽  
Alternating Access ◽  
Selection Of

The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H+ by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.

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