Cation Hexaammines Are Selective and Potent Inhibitors of the CorA Magnesium Transport System

Kucharski, Lisa M.; Lubbe, W.; Maguire, Michael E.

doi:10.1074/jbc.m001507200

Cited by 77 publications

(75 citation statements)

References 31 publications

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“…The inhibition of uptake of 10 mM nickel chloride by both hexa-ammine cobalt (III) chloride and magnesium ions is consistent with CorA involvement. However, hexa-ammine cobalt (III) chloride inhibition was modest compared with that reported for the Salmonella CorA (Kucharski et al, 2000). The abolition of hydrogenase activity in the cj1584c mutant after growth in media with no added nickel indicates that uptake through the ABC system is essential for hydrogenase synthesis at low environmental nickel concentrations (as is likely in the natural gut environment), but the activity of the alternative low-affinity systems during growth with excess nickel is sufficient to restore hydrogenase activity to wild-type levels.…”

Section: Discussionmentioning

confidence: 89%

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

et al. 2012

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Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K d value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wildtype strain and a decreased rate of uptake of 63 NiCl 2 . The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

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Section: Discussionmentioning

confidence: 89%

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

et al. 2012

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“…The Asn-ring, because of its geometry, apparently allows the entry of only partially hydrated Mg 2+ . Additionally, the Asn-ring might be the binding area for cobalt(III)hexamine, the known inhibitor of CorA proteins (16), which could tightly seal the selectivity filter (Fig. S3).…”

Section: Resultsmentioning

confidence: 99%

Structural insights into the mechanisms of Mg ²⁺ uptake, transport, and gating by CorA

Guskov

Nordin

Reynaud

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Despite the importance of Mg 2+ for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg 2+ transport. However, the key questions-such as, the ion selectivity, the transport pathway, and the gating mechanism-have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg 2+ during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg 2+ to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.

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“…In a recent computational study (22), removal of the ten regulatory ions from M1 and M2 led to changes in the tilt of α7, resulting in an iris-like dilation of the pore and subsequent hydration of the hydrophobic pore region between M302 and M291 (MM stretch), suggesting an allosteric mechanism coupling intracellular Mg 2+ concentration to hydrophobic gating. It has been further suggested that the acidic electrostatic nature of α5 and α6 might be used to change the position of the basic sphincter, which surrounds the tightest pore constriction formed by M291 (17 (23). NMR studies on an isolated TM domain (24) report weak and dynamic binding of hydrated Mg 2+ and [Co(NH 3 ) 6 ] 3+ to the periplasmic α7-α8 loop.…”

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confidence: 99%

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation

Pfoh

Li²,

Chakrabarti

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

135

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Magnesium ions (Mg 2+ ) are essential for life, but the mechanisms regulating their transport into and out of cells remain poorly understood. The CorA-Mrs2-Alr1 superfamily of Mg 2+ channels represents the most prevalent group of proteins enabling Mg 2+ ions to cross membranes. Thermotoga maritima CorA (TmCorA) is the only member of this protein family whose complete 3D fold is known. Here, we report the crystal structure of a mutant in the presence and absence of divalent ions and compare it with previous divalent ion-bound TmCorA structures. With Mg 2+ present, this structure shows binding of a hydrated Mg 2+ ion to the periplasmic Gly-Met-Asn (GMN) motif, revealing clues of ion selectivity in this unique channel family. In the absence of Mg 2+ , TmCorA displays an unexpected asymmetric conformation caused by radial and lateral tilts of protomers that leads to bending of the central, pore-lining helix. Molecular dynamics simulations support these movements, including a bell-like deflection. Mass spectrometric analysis confirms that major proteolytic cleavage occurs within a region that is selectively exposed by such a bell-like bending motion. Our results point to a sequential allosteric model of regulation, where intracellular Mg 2+ binding locks TmCorA in a symmetric, transport-incompetent conformation and loss of intracellular Mg 2+ causes an asymmetric, potentially influx-competent conformation of the channel.crystallography | gating | limited proteolysis | pentamer C ompared with other common biological ions (Na + , K + , Ca 2+ , Cl − ), very little is known on a molecular level about the cellular homeostasis of Mg 2+ . As the most abundant intracellular divalent cation, it stabilizes phosphate compounds (DNA, RNA, ATP) and their synthesis, is essential for the function of over 300 enzymes, and is central to photosynthesis in plants (1). In addition to antagonizing Ca 2+ signaling (2), Mg 2+ has recently been implicated as a key second messenger in T-cell activation through the MagT1 Mg 2+ channel (3). The CorA protein is the primary transport system for Mg 2+ in Bacteria and Archaea and is required for bacterial pathogenesis (4, 5). It can functionally substitute for its eukaryotic homologs Alr1 and Mrs2 (6, 7), suggesting that CorA represents an important model system for these eukaryotic Mg 2+ channels. Alr1 is the major Mg 2+ uptake system in the plasma membrane of yeast (8), and Mrs2 is present in the inner mitochondrial membranes of yeast (6), plants (9), and mammals (10). Despite Mrs2 being essential for normal mitochondrial function (11), its expression is a hallmark of embryonic stem cells (12), and Mrs2 overexpression has been linked to a multidrug resistance phenotype in cancer (13,14). Patch-clamp analysis established Mrs2 as a high-conductance (155 pS) Mg 2+ -selective channel (15), and a similar conductivity has been indicated for CorA (16).Three crystal structures of wild-type Thermotoga maritima CorA (TmCorA-WT), obtained in the presence of divalent cations (17-19), revealed a symmetric ho...

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Cation Hexaammines Are Selective and Potent Inhibitors of the CorA Magnesium Transport System

Cited by 77 publications

References 31 publications

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

Structural insights into the mechanisms of Mg ²⁺ uptake, transport, and gating by CorA

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation

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Cation Hexaammines Are Selective and Potent Inhibitors of the CorA Magnesium Transport System

Cited by 77 publications

References 31 publications

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

Structural insights into the mechanisms of Mg 2+ uptake, transport, and gating by CorA

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation

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Structural insights into the mechanisms of Mg ²⁺ uptake, transport, and gating by CorA