scholarly journals Proton co-transport and voltage dependence enforce unidirectional metal transport in an Nramp transporter

2018 ◽  
Author(s):  
Aaron T. Bozzi ◽  
Lukas B. Bane ◽  
Christina M. Zimanyi ◽  
Rachelle Gaudet
Keyword(s):  
Metal Binding ◽  
Voltage Dependence ◽  
Mechanistic Model ◽  
Salt Bridge ◽  
Metal Transport ◽  
Natural Resistance ◽  
Metal Type ◽  
Proton Symport ◽  
Macrophage Protein ◽  
Stimulation Of

AbstractSecondary transporters harness electrochemical energy to move substrate through structurally-enforced co-substrate “coupling”. We untangle the “proton-metal coupling” behavior by a Natural resistance-associated macrophage protein (Nramp) transporter into two distinct phenomena: ΔpH stimulation of metal transport and metal stimulation of proton co-transport. Surprisingly, metal type dictates co-transport stoichiometry, leading to manganese-proton symport but cadmium uniport. Additionally, the membrane potential affects both the kinetics and thermodynamics of metal transport. A conserved salt-bridge network near the metal-binding site imparts voltage dependence and enables proton co-transport, properties that allow this Nramp transporter to maximize metal uptake and prevent deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp metal-proton symport in which, in addition to substrate gradients determining directionality as in canonical secondary transport, synergy between protein structure and physiological voltage enforces unidirectional substrate movement. Our results illustrate a functional advantage that arises from deviations from the traditional model of symport.

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 Transmembrane helix 6b links proton- and metal-release pathways to drive conformational change in an Nramp transition metal transporter

2019 ◽  
Author(s):  
Aaron T. Bozzi ◽  
Anne L. McCabe ◽  
Benjamin C. Barnett ◽  
Rachelle Gaudet
Keyword(s):  
Transition Metal ◽  
Conformational Change ◽  
Metal Binding ◽  
Deinococcus Radiodurans ◽  
Transmembrane Helix ◽  
Metal Transport ◽  
Natural Resistance ◽  
Metal Release ◽  
Transport Pathway ◽  
Exit Route

ABSTRACTThe natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton co-transporters found in organisms from bacteria to humans. Recent structures ofDeinococcus radiodurans(Dra)Nramp in multiple conformations revealed the intramolecular rearrangements required for alternating access. Here we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp—one lining the wide intracellular vestibule for metal release, the other forming a narrow proton-transport pathway—are essential for metal transport. We further show that mutagenic or post-translational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impedes normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, H232 and H237. Different mutagenic perturbations for H232, just below the metal-binding site along the proton-exit route, differentially affect DraNramp’s conformational state, suggesting H232 serves as a pivot point for conformational change. In contrast, any tested replacement for H237, lining the metal-exit route, locks the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement to the inward-open state upon metal binding and facilitate the return of the empty transporter to an outward-open state upon metal release.

scholarly journals Functional analysis of the human NRAMP family expressed in fission yeast

1999 ◽  
Vol 344 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Mitsuaki TABUCHI ◽  
Tsutomu YOSHIDA ◽  
Kaoru TAKEGAWA ◽  
Fumio KISHI
Keyword(s):  
Fission Yeast ◽  
Positional Cloning ◽  
Metal Transport ◽  
Divalent Metal ◽  
Natural Resistance ◽  
Functional Difference ◽  
Transport Activity ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
Macrophage Protein

The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the positional cloning of this locus identified the Nramp1 (natural resistance-associated macrophage protein) gene. Nramp2 was initially isolated as a homologue of Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutation which was subsequently identified in rats suffering from hereditary iron-deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear. To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human NRAMP1 and NRAMP2 with each other. The functional characteristics of wild-type NRAMP1, NRAMP2 and their chimeras were determined by expression in the divalent metal transporter-disrupted strain of fission yeast, pdt1δ, and we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1δ. Replacement of the N-terminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not solely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2. These findings indicate that NRAMP1 itself cannot represent the divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts.

scholarly journals Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis

Membranes ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 255
Author(s):  
Matthias Gantner ◽  
Theodoros Laftsoglou ◽  
Honglin Rong ◽  
Vincent L. G. Postis ◽  
Lars J. C. Jeuken
Keyword(s):  
Transition Metals ◽  
Enterococcus Faecalis ◽  
Fluorescent Dyes ◽  
Metal Transport ◽  
Natural Resistance ◽  
Experimental Conditions ◽  
Metal Transporters ◽  
Supported Membrane ◽  
Essential Trace Elements ◽  
Macrophage Protein

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.

scholarly journals Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter

2019 ◽  
Vol 295 (5) ◽  
pp. 1212-1224 ◽  
Author(s):  
Aaron T. Bozzi ◽  
Anne L. McCabe ◽  
Benjamin C. Barnett ◽  
Rachelle Gaudet
Keyword(s):  
Transition Metal ◽  
Binding Site ◽  
Metal Binding ◽  
Transmembrane Helix ◽  
Metal Transport ◽  
Natural Resistance ◽  
Metal Release ◽  
Transport Pathway ◽  
Metal Binding Site ◽  
Exit Route

The natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton cotransporters that are present in many organisms from bacteria to humans. Recent structures of Deinococcus radiodurans Nramp (DraNramp) in multiple conformations revealed the intramolecular rearrangements required for alternating access of the metal-binding site to the external or cytosolic environment. Here, using recombinant proteins and metal transport and cysteine accessibility assays, we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp, one lining the wide intracellular vestibule for metal release and the other forming a narrow proton transport pathway, are essential for metal transport. We further show that mutagenic or posttranslational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impede normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, His232 and His237. We found that different mutagenic perturbations of His232, just below the metal-binding site along the proton exit route, differentially affect DraNramp's conformational state, suggesting that His232 serves as a pivot point for conformational changes. In contrast, any replacement of His237, lining the metal exit route, locked the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement of DraNramp to the inward-open state upon metal binding and facilitate return of the empty transporter to an outward-open state upon metal release.

Evaluation of clinical efficacy of Derinat® in form of spray in practice of district therapist

2019 ◽  
Vol 1 (9) ◽  
pp. 38-46
Author(s):  
A. P. Babkin ◽  
A. A. Zuikova ◽  
O. N. Krasnorutskaya ◽  
Yu. A. Kotova ◽  
D. Yu. Bugrimov ◽  
...  
Keyword(s):  
Clinical Efficacy ◽  
Respiratory Diseases ◽  
Viral Infections ◽  
Pharmacological Action ◽  
The Body ◽  
Natural Resistance ◽  
Acute Respiratory Diseases ◽  
T Helpers ◽  
T Killers ◽  
Stimulation Of

The widespread worldwide spread of acute respiratory diseases is an urgent problem in health care. Expressed polyetiology of respiratory diseases does not allow to limit the use of specific vaccine preparations and dictates the need to use to combat them a variety of non-specific means that stimulate the natural resistance of the human body. The main pharmacological action of sodium deoxyribonucleate is the stimulation of phagocytic activity of T-helpers and T-killers, increasing the functional activity of neutrophils and monocytes/ macrophages, providing regeneration and repair processes in the epithelial component of antiviral protection of the body. Based on the above, the study of the clinical efficacy of Derinat® in the form of spray in the treatment of acute respiratory viral infections is relevant.

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