Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 27
Filter
Add more filters










Publication year range
1.
J Biol Chem ; 296: 100269, 2021.
Article in English | MEDLINE | ID: mdl-33837739

ABSTRACT

ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, acrodermatitis enteropathica (AE). These mutations occur not only in the conserved transmembrane zinc transport machinery, but also in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs. How these AE-causing ECD mutations lead to ZIP4 malfunction has not be fully clarified. In this work, we characterized all seven confirmed AE-causing missense mutations in hZIP4-ECD and found that the variants exhibited completely abolished zinc transport activity in a cell-based transport assay. Although the variants were able to be expressed in HEK293T cells, they failed to traffic to the cell surface and were largely retained in the ER with immature glycosylation. When the corresponding mutations were introduced in the ECD of ZIP4 from Pteropus Alecto, a close homolog of hZIP4, the variants exhibited structural defects or reduced thermal stability, which likely accounts for intracellular mistrafficking of the AE-associated variants and as such a total loss of zinc uptake activity. This work provides a molecular pathogenic mechanism for AE.


Subject(s)
Acrodermatitis/genetics , Carrier Proteins/genetics , Cation Transport Proteins/genetics , Zinc/deficiency , Acrodermatitis/pathology , Amino Acid Sequence/genetics , Cation Transport Proteins/ultrastructure , Cell Membrane/genetics , Cell Membrane/ultrastructure , HEK293 Cells , Humans , Loss of Function Mutation/genetics , Zinc/metabolism
2.
Cell Rep ; 33(10): 108486, 2020 12 08.
Article in English | MEDLINE | ID: mdl-33296646

ABSTRACT

The mitochondrial calcium uniporter is a multi-subunit Ca2+-activated Ca2+ channel, made up of the pore-forming MCU protein, a metazoan-specific EMRE subunit, and MICU1/MICU2, which mediate Ca2+ activation. It has been established that metazoan MCU requires EMRE binding to conduct Ca2+, but how EMRE promotes MCU opening remains unclear. Here, we demonstrate that EMRE controls MCU activity via its transmembrane helix, while using an N-terminal PKP motif to strengthen binding with MCU. Opening of MCU requires hydrophobic interactions mediated by MCU residues near the pore's luminal end. Enhancing these interactions by single mutation allows human MCU to transport Ca2+ without EMRE. We further show that EMRE may facilitate MCU opening by stabilizing the open state in a conserved MCU gating mechanism, present also in non-metazoan MCU homologs. These results provide insights into the evolution of the uniporter machinery and elucidate the mechanism underlying the physiologically crucial EMRE-dependent MCU activation process.


Subject(s)
Calcium Channels/metabolism , Calcium-Binding Proteins/metabolism , Cation Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Calcium/metabolism , Calcium Channels/physiology , Calcium Channels/ultrastructure , Calcium-Binding Proteins/physiology , Calcium-Binding Proteins/ultrastructure , Cation Transport Proteins/physiology , Cation Transport Proteins/ultrastructure , HEK293 Cells , Humans , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/physiology , Mitochondrial Membrane Transport Proteins/ultrastructure , Mitochondrial Membranes/metabolism
3.
Nat Commun ; 11(1): 5686, 2020 11 10.
Article in English | MEDLINE | ID: mdl-33173040

ABSTRACT

Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H+/Fe2+ antiporter so that transport of each Fe2+ is coupled to transport of two H+ in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H+ and Fe2+. These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases.


Subject(s)
Cation Transport Proteins/ultrastructure , Cryoelectron Microscopy , Hepcidins/metabolism , Iron/metabolism , Anemia, Iron-Deficiency , Animals , Binding Sites , Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Humans , Ion Transport , Protein Binding
4.
Nature ; 586(7831): 807-811, 2020 10.
Article in English | MEDLINE | ID: mdl-32814342

ABSTRACT

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Cryoelectron Microscopy , Hepcidins/metabolism , Homeostasis , Iron/metabolism , Apoproteins/chemistry , Apoproteins/metabolism , Apoproteins/ultrastructure , Binding Sites , Cation Transport Proteins/ultrastructure , Cobalt/chemistry , Cobalt/metabolism , Hepcidins/chemistry , Humans , Iron/chemistry , Molecular Dynamics Simulation , Protein Domains , Proteolysis
5.
Elife ; 82019 12 23.
Article in English | MEDLINE | ID: mdl-31868587

ABSTRACT

RCK domains regulate the activity of K+ channels and transporters in eukaryotic and prokaryotic organisms by responding to ions or nucleotides. The mechanisms of RCK activation by Ca2+ in the eukaryotic BK and bacterial MthK K+ channels are well understood. However, the molecular details of activation in nucleotide-dependent RCK domains are not clear. Through a functional and structural analysis of the mechanism of ATP activation in KtrA, a RCK domain from the B. subtilis KtrAB cation channel, we have found that activation by nucleotide requires binding of cations to an intra-dimer interface site in the RCK dimer. In particular, divalent cations are coordinated by the γ-phosphates of bound-ATP, tethering the two subunits and stabilizing the active state conformation. Strikingly, the binding site residues are highly conserved in many different nucleotide-dependent RCK domains, indicating that divalent cations are a general cofactor in the regulatory mechanism of many nucleotide-dependent RCK domains.


Subject(s)
Bacterial Proteins/chemistry , Cation Transport Proteins/chemistry , Nucleotides/chemistry , Protein Conformation , Adenosine Triphosphate/chemistry , Bacillus subtilis/chemistry , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/ultrastructure , Binding Sites/genetics , Calcium/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/ultrastructure , Cations/chemistry , Crystallography, X-Ray , Kv1.6 Potassium Channel/chemistry , Kv1.6 Potassium Channel/ultrastructure , Nucleotides/genetics , Potassium/chemistry , Potassium/metabolism , Potassium Channels/chemistry , Potassium Channels/genetics , Potassium Channels/ultrastructure , Protein Domains/genetics , Protein Structure, Tertiary , Ribosomal Proteins
6.
Nat Commun ; 10(1): 1386, 2019 03 27.
Article in English | MEDLINE | ID: mdl-30918258

ABSTRACT

Copper (Cu) is an essential trace element for growth and development and abnormal Cu levels are associated with anemia, metabolic disease and cancer. Evolutionarily conserved from fungi to humans, the high-affinity Cu+ transporter Ctr1 is crucial for both dietary Cu uptake and peripheral distribution, yet the mechanisms for selective permeation of potentially toxic Cu+ ions across cell membranes are unknown. Here we present X-ray crystal structures of Ctr1 from Salmo salar in both Cu+-free and Cu+-bound states, revealing a homo-trimeric Cu+-selective ion channel-like architecture. Two layers of methionine triads form a selectivity filter, coordinating two bound Cu+ ions close to the extracellular entrance. These structures, together with Ctr1 functional characterization, provide a high resolution picture to understand Cu+ import across cellular membranes and suggest therapeutic opportunities for intervention in diseases characterized by inappropriate Cu accumulation.


Subject(s)
Cation Transport Proteins/ultrastructure , Copper/metabolism , Animals , Biological Transport , Cation Transport Proteins/metabolism , Cell Membrane , Copper Transporter 1 , Crystallography, X-Ray , Ion Transport , Salmo salar
7.
Elife ; 62017 05 16.
Article in English | MEDLINE | ID: mdl-28504641

ABSTRACT

Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Cation Transport Proteins/metabolism , Cation Transport Proteins/ultrastructure , Vibrio alginolyticus/enzymology , Vibrio alginolyticus/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Cation Transport Proteins/chemistry , Cryoelectron Microscopy , Electron Spin Resonance Spectroscopy , Molecular Dynamics Simulation
8.
Cell ; 164(4): 597-8, 2016 Feb 11.
Article in English | MEDLINE | ID: mdl-26871624

ABSTRACT

Ligand binding usually moves the target protein from an ensemble of inactive states to a well-defined active conformation. Matthies et al. flip this scheme around, finding that, for the magnesium channel CorA, loss of ligand binding induces an ensemble of conformations that turn the channel on.


Subject(s)
Bacterial Proteins/ultrastructure , Cation Transport Proteins/ultrastructure , Magnesium/metabolism , Thermotoga maritima/chemistry
9.
Cell ; 164(4): 747-56, 2016 Feb 11.
Article in English | MEDLINE | ID: mdl-26871634

ABSTRACT

CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD ∼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (∼ 10-25 Å) by hinge-like motions as large as ∼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.


Subject(s)
Bacterial Proteins/ultrastructure , Cation Transport Proteins/ultrastructure , Magnesium/metabolism , Thermotoga maritima/chemistry , Bacterial Proteins/chemistry , Cation Transport Proteins/chemistry , Cryoelectron Microscopy , Models, Molecular , Molecular Dynamics Simulation
10.
Biochem Biophys Res Commun ; 470(3): 663-669, 2016 Feb 12.
Article in English | MEDLINE | ID: mdl-26797276

ABSTRACT

Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.


Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/ultrastructure , Cation Transport Proteins/chemistry , Cation Transport Proteins/ultrastructure , Copper/chemistry , Models, Chemical , Models, Molecular , Binding Sites , Computer Simulation , Copper-Transporting ATPases , Enzyme Activation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Structure-Activity Relationship
11.
PLoS Comput Biol ; 11(7): e1004303, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26181442

ABSTRACT

Ion channels catalyze ionic permeation across membranes via water-filled pores. To understand how changes in intracellular magnesium concentration regulate the influx of Mg2+ into cells, we examine early events in the relaxation of Mg2+ channel CorA toward its open state using massively-repeated molecular dynamics simulations conducted either with or without regulatory ions. The pore of CorA contains a 2-nm-long hydrophobic bottleneck which remained dehydrated in most simulations. However, rapid hydration or "wetting" events concurrent with small-amplitude fluctuations in pore diameter occurred spontaneously and reversibly. In the absence of regulatory ions, wetting transitions are more likely and include a wet state that is significantly more stable and more hydrated. The free energy profile for Mg2+ permeation presents a barrier whose magnitude is anticorrelated to pore diameter and the extent of hydrophobic hydration. These findings support an allosteric mechanism whereby wetting of a hydrophobic gate couples changes in intracellular magnesium concentration to the onset of ionic conduction.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/ultrastructure , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/ultrastructure , Magnesium/chemistry , Models, Chemical , Molecular Dynamics Simulation , Water/chemistry , Hydrophobic and Hydrophilic Interactions , Ion Channel Gating , Ions/chemistry , Permeability , Wettability
12.
Biochim Biophys Acta ; 1848(10 Pt A): 2206-15, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26051127

ABSTRACT

CorA channels are responsible for the uptake of essential magnesium ions by bacteria. X-ray crystal structures have been resolved for two full-length CorA channels, each in a non-conducting state with magnesium ions bound to the protein: These structures reveal a homo-pentameric quaternary structure with approximate 5-fold rotational symmetry about a central pore axis. We report the structure of the detergent solubilized Methanocaldococcus jannaschii CorA channel determined by Cryo-Electron Microscopy and Single Particle Averaging, supported by Small Angle X-ray Scattering and X-ray crystallography. This structure also shows a pentameric channel but with a highly asymmetric domain structure. The asymmetry of the domains includes differential separations between the trans-membrane segments, which reflects mechanical coupling of the cytoplasmic domain to the trans-membrane domain. This structure therefore reveals an important aspect of the gating mechanism of CorA channels by providing an indication of how the absence of magnesium ions leads to major structural changes.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/ultrastructure , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/ultrastructure , Magnesium/chemistry , Methanocaldococcus/chemistry , Methanocaldococcus/ultrastructure , Models, Molecular , Computer Simulation , Cryoelectron Microscopy/methods , Models, Chemical , Protein Conformation
13.
Proc Natl Acad Sci U S A ; 110(6): 2140-5, 2013 Feb 05.
Article in English | MEDLINE | ID: mdl-23341604

ABSTRACT

YiiP is a dimeric Zn(2+)/H(+) antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn(2+). Starting from the X-ray structure in the presence of Zn(2+), we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.


Subject(s)
Bacterial Proteins/chemistry , Cation Transport Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cation Transport Proteins/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Sequence Homology, Amino Acid , Shewanella/genetics , Shewanella/metabolism , Zinc/metabolism
14.
J Neurosci ; 32(39): 13555-67, 2012 Sep 26.
Article in English | MEDLINE | ID: mdl-23015445

ABSTRACT

R-type calcium channels (RTCCs) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, we characterized the regional and subcellular localization of Ca(v)2.3 in mice and rats at both light and electron microscopic levels. Ca(v)2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique, or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained <20 immunogold particles, with an average density of ∼260 immunoparticles per µm(3) spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Ca(v)2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites.


Subject(s)
Calcium Channels, R-Type/metabolism , Calcium Channels, R-Type/ultrastructure , Cation Transport Proteins/metabolism , Cation Transport Proteins/ultrastructure , Neurons/metabolism , Neurons/ultrastructure , Analysis of Variance , Animals , Animals, Newborn , Brain/cytology , Calcium Channels, R-Type/chemistry , Calcium Channels, R-Type/deficiency , Cation Transport Proteins/chemistry , Cation Transport Proteins/deficiency , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Dendrites/metabolism , Dendrites/ultrastructure , Dendritic Spines/metabolism , Dendritic Spines/ultrastructure , Epitopes/metabolism , Female , Guinea Pigs , Imaging, Three-Dimensional , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Immunoelectron , Peptides/metabolism , Post-Synaptic Density/metabolism , Post-Synaptic Density/ultrastructure , Rats , Statistics as Topic , Statistics, Nonparametric , Subcellular Fractions/metabolism , Subcellular Fractions/ultrastructure
15.
Proc Natl Acad Sci U S A ; 107(24): 10908-13, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20534491

ABSTRACT

The trimeric human copper transporter 1 (hCTR1) is essential for copper uptake and is implicated in sensitivity to chemotherapy drugs. Using the cryoelectron microscopy (cryoEM) map of hCTR1 and evolutionary data, we constructed a Calpha-trace model of the membrane region. The model structure, supported by mutagenesis data, was used to investigate global dynamics through elastic network models. Identified as dominant hinge regions, hCTR1's MxxxM and GxxxG motifs were shown to have significant roles in functional movements characterized by the two slowest modes of motion. Both modes predicted significant changes at the wide cytoplasmic pore region; the slowest mode introduced a rotational motion around the pore central axis, whereas in the following mode the cytoplasmic parts of the helices approached and moved away from the pore center. In the most cooperative mode, the MxxxM motif in the extracellular narrow region remained static. The second mode of motion, however, predicted a cooperative rotational motion of this copper-binding motif, possibly reflecting activation at the pore's extracellular entrance. We suggest a molecular mechanism of copper transport in which this motif serves both as a gate and as a selectivity filter. We also suggest residues that are responsible for pH activation.


Subject(s)
Cation Transport Proteins/chemistry , Amino Acid Motifs , Biophysical Phenomena , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cation Transport Proteins/ultrastructure , Copper/metabolism , Copper Transporter 1 , Cryoelectron Microscopy , Elasticity , Evolution, Molecular , Humans , Hydrogen-Ion Concentration , Ion Transport , Models, Molecular , Molecular Dynamics Simulation , Motion , Mutagenesis, Site-Directed , Protein Structure, Tertiary
16.
Histol Histopathol ; 23(7): 781-7, 2008 07.
Article in English | MEDLINE | ID: mdl-18437676

ABSTRACT

The present work addresses the cellular and subcellular localization of the zinc transporter 7 (ZNT7, SLC30a7) protein and the distribution of zinc ions (Zn2+) in the mouse spinal cord. Our results indicated that the ZNT7 immunoreactive neurons were widely distributed in the Rexed's laminae of the gray matter in all spinal segments examined. The ependyma cells of the central canal and glia cells in the white matter were also shown ZNT7-positive. The ZNT7 immunoreactivity was mainly detected in the perinuclear regions of ZNT7-positive cells in the spinal gray matter. For ependyma cells, the immunoreactivity of ZNT7 was detected in the cytoplasm near the lumina of the central canal. Ultrastructural localization showed that ZNT7 was predominately present in the membrane of the Golgi stacks. The double immunofluorescence studies confirmed this result. Other intracellular organelles including the endoplasmic reticulum, mitochondria and lysosomes were devoid of ZNT7-immunostaining. The chelatable Zn2+ ions in the spinal cord were found predominantly in the terminals of the neuron rather than the cell body in the gray matter. However, overlapping distribution of chelatable Zn2+ ions and ZNT7 was found in the ependyma cells. The present study supports the notion that ZNT7 may function to supply zinc ions to the newly synthesized metalloproteins in the secretory pathway of the spinal neuron and the ependyma cell.


Subject(s)
Cation Transport Proteins/metabolism , Organelles/metabolism , Spinal Cord/metabolism , Animals , Biomarkers/metabolism , Cation Transport Proteins/ultrastructure , Ependyma/cytology , Ependyma/metabolism , Fluorescent Antibody Technique, Indirect , Immunoenzyme Techniques , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred Strains , Microscopy, Electron, Transmission , Neurons/cytology , Neurons/metabolism , Neurons/ultrastructure , Organelles/ultrastructure , Selenium Compounds/metabolism , Spinal Cord/ultrastructure , Zinc Compounds/metabolism
17.
J Struct Biol ; 161(3): 411-8, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17945510

ABSTRACT

The KdpFABC complex (Kdp) functions as a K+ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K+ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis.


Subject(s)
Adenosine Triphosphatases/ultrastructure , Cation Transport Proteins/ultrastructure , Escherichia coli Proteins/ultrastructure , Escherichia coli/ultrastructure , Models, Molecular , Multiprotein Complexes/ultrastructure , Sodium-Potassium-Exchanging ATPase/ultrastructure , Crystallography , Microscopy, Electron , Tomography
18.
Biochem Biophys Res Commun ; 364(3): 645-9, 2007 Dec 21.
Article in English | MEDLINE | ID: mdl-17961510

ABSTRACT

Yeast Ccc2 is a P-type ATPase responsible for transport of copper(I) from the cytosol to the trans-Golgi network. It possesses a soluble cytosolic N-terminal region containing two copper(I)-binding domains. Homologous eukaryotic copper-transporting ATPases have from one to six domains. We have expressed a fragment encompassing residues 1-150 of Ccc2, which corresponds to the two domains, and found that the second domain was substantially less structured than the first. The first domain could bind copper(I) and interact with the partner protein Atx1 at variance with the second. Similar results are found in ATPases from other organisms and may represent a general feature, whose biochemical implications are not yet fully appreciated.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/ultrastructure , Cation Transport Proteins/chemistry , Cation Transport Proteins/ultrastructure , Copper/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/ultrastructure , Saccharomyces cerevisiae/metabolism , Binding Sites , Copper Transport Proteins , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , Solubility
19.
Biophys J ; 92(12): L103-5, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17434945

ABSTRACT

Structural characterization of the bacterial channel, AmtB, provides a glimpse of how members of its family might control the protonated state of permeant ammonium to allow for its selective passage across the membrane. In a recent study, we employed a combination of simulation techniques that suggested ammonium is deprotonated and reprotonated near dehydrative phenylalanine landmarks (F107 and F31, respectively) during its passage from the periplasm to the cytoplasm. At these landmarks, ammonium is forced to maintain a critical number ( approximately 3) of hydrogen bonds, suggesting that the channel controls ammonium (de)protonation by controlling its coordination/hydration. In the work presented here, a free energy-based analysis of ammonium hydration in dilute aqueous solution indicates, explicitly, that at biological pH, the transition from ammonium (NH(4)(+)) to ammonia (NH(3)) occurs when these species are constrained to donate three hydrogen bonds or less. This result demonstrates the viability of the proposal that AmtB indirectly controls ammonium (de)protonation by directly controlling its hydration.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/ultrastructure , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/ultrastructure , Models, Chemical , Models, Molecular , Protons , Quaternary Ammonium Compounds/chemistry , Computer Simulation , Hydrogen Bonding , Ion Channel Gating , Ion Transport , Molecular Conformation , Water/chemistry
20.
Biophys J ; 92(9): L82-4, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17351012

ABSTRACT

The accessibility of water molecules to the pore of the AmtB ammonium transporter is studied using molecular dynamics simulations. Free energy calculations show that the so-called hydrophobic pore can stabilize a chain of water molecules in a well of a few kcal/mol, using a favorable electrostatic binding pocket as an anchoring point. Moreover, the structure of the water chain matches precisely the electronic density maxima observed in x-ray diffraction experiments. This result questions the general assumption that the AmtB pore only contains ammonia (NH(3)) molecules diffusing in a single file fashion. The probable presence of water molecules in the pore would influence the relative stability of NH(3) and NH(4)(+), and thus calls for a reassessment of the overall permeation mechanism in ammonium transporters.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/ultrastructure , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/ultrastructure , Ion Channel Gating , Models, Chemical , Models, Molecular , Quaternary Ammonium Compounds/chemistry , Water/chemistry , Computer Simulation , Hydrophobic and Hydrophilic Interactions , Porosity , Protein Conformation , Solubility , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...