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 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.

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 and drives conformational change in an Nramp-family transition metal transporter

2020 ◽  
Vol 295 (5) ◽  
pp. 1212-1224
Author(s):  
Aaron T. Bozzi ◽  
Anne L. McCabe ◽  
Benjamin C. Barnett ◽  
Rachelle Gaudet
Keyword(s):  
Transition Metal ◽  
Conformational Change ◽  
Transmembrane Helix ◽  
Metal Release ◽  
Family Transition ◽  
Metal Transporter

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 Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters

2016 ◽  
Vol 113 (37) ◽  
pp. 10310-10315 ◽  
Author(s):  
Aaron T. Bozzi ◽  
Lukas B. Bane ◽  
Wilhelm A. Weihofen ◽  
Anne L. McCabe ◽  
Abhishek Singharoy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transition Metal ◽  
Transition Metals ◽  
Binding Site ◽  
Metal Binding ◽  
Toxic Metal ◽  
Natural Resistance ◽  
Metal Transporters ◽  
Metal Binding Site ◽  
Calcium And Magnesium

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur’s presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it—more effectively than any other amino acid—increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.

scholarly journals Regulation of Transition Metal Transport across the Yeast Plasma Membrane

1999 ◽  
Vol 274 (8) ◽  
pp. 4481-4484 ◽  
Author(s):  
Derek Radisky ◽  
Jerry Kaplan
Keyword(s):  
Plasma Membrane ◽  
Transition Metal ◽  
Metal Transport ◽  
Yeast Plasma Membrane

scholarly journals Transition metal transport

FEBS Letters ◽  
2007 ◽  
Vol 581 (12) ◽  
pp. 2263-2272 ◽  
Author(s):  
Ute Krämer ◽  
Ina N. Talke ◽  
Marc Hanikenne
Keyword(s):  
Transition Metal ◽  
Metal Transport

scholarly journals Structure and Metal Binding Properties of Chlamydia trachomatis YtgA

2019 ◽  
Vol 202 (1) ◽  
Author(s):  
Zhenyao Luo ◽  
Stephanie L. Neville ◽  
Rebecca Campbell ◽  
Jacqueline R. Morey ◽  
Shruti Menon ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Transition Metal ◽  
Metal Binding ◽  
Developed Countries ◽  
Biochemical Properties ◽  
Host Cells ◽  
Transmitted Infection ◽  
Content Type ◽  
Sexually Transmitted ◽  
Serious Disease

Chlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

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