Dmt1 which metals does it transport

Biochem 30 , Blood 96 , a. J Cell Sci , Mol Biol Cell 9 , Blood 93 , Blood 96 , Blood 97 , Chua A, Morgan EH. J Comp Physiol B , Am J Med Sci , Nature London , Proc Soc Exp Biol Med , Elisma F, Jumarie C. Biochem Biophys Res Commun , Am J Hematol 39 , Nature Genet 16 , J Cell Physiol , Blood 81 , J Cell Biol , 82a. Blood 76 , 8a. BioMetals 10 , In: D. Armstrong ed. Totawa: Humana; pp. BioIron Cairns , Adv Cell Neurobiol 3 , Based on our structures, we suspected Gln and Ser to interact with the distal isothiourea group and Asn with the bromine atom on the aromatic ring of Br-BIT Figure 4A.

To characterize the role of residues in the predicted inhibitor binding pocket of human DMT1, we have generated several point mutants and investigated the effect of these mutations on the interaction with different inhibitors.

Due to the strong negative impact of alterations of the metal ion coordination site on transport, mutagenesis was restricted to residues lining the remainder of the binding pocket. In our experiments we wanted to target interactions of protein residues with the aromatic ring in the narrow part of the binding pocket by either shortening the side-chains in the mutants AG and QA, or by increasing their size in the mutants AV, QF, SV and LF.

Mutations that render hDMT1 inactive, most likely interfere with structural rearrangements during ion transport, as judged by the tight packing of the respective region in the inward-facing structures of SLC11 transporters Bozzi et al. Similar to WT, the addition of either compound at equivalent concentrations decreases uptake in the mutants YA and QA both located towards the extracellular entrance to the binding pocket Figures 3H and 5A,C and Figure 5—figure supplement 1B—D thus suggesting that interactions with these residues do not strongly contribute to inhibitor binding.

Conversely, the compounds had much smaller effects on the transport activity of cells expressing the mutants SV, NL and FA located deeper in the binding pocket Figures 3H and 5C,D , Figure 5—figure supplement 1B—C thus suggesting that in these cases, the mutations affected inhibitor interactions.

To further characterize the inhibitory properties of the investigated compounds, we have measured uptake at different inhibitor concentrations and found a strong reduction in potency in most cases Figure 5E , Table 4. This is consistent with the wide dimensions of the pocket in that direction that allows for a geometry-dependent placement of the aromatic ring and the attached isothiourea moiety on the distal side Figure 5D.

Despite the described species-dependent differences, our data is generally consistent with the notion that the characterized compounds inhibit both pro- and eukaryotic transporters by binding to equivalent regions. Data show mean of 15—39 replicates, errors are s. Data show mean of 6—9 replicates, errors are s. Low values, reflect a strong effect of the mutation on inhibition.

By combining chemical synthesis with X-ray crystallography and in vitro binding and transport assays on human DMT1 and its prokaryotic homologue EcoDMT, our study has revealed detailed insight into the inhibition of SLC11 transporters by aromatic bis-isothiourea-based compounds.

These compounds inhibit pro- and eukaryotic family members by a predominant competitive mechanism by binding to an outward-facing aqueous cavity leading to the transition metal ion coordination site Figures 1 , 2 , 3 and 6 which prevents substrate loading and the transition to an inward-open conformation of the transporter. We have shown that these compounds do not interact with the reactive transported substrate, which has hampered the identification of specific inhibitors in high-throughput screens Figure 1—figure supplement 1B.

We have also shown that these compounds are positively charged and thus poorly membrane permeable and most likely attracted and stabilized by the strong negative electrostatic potential in the outward-facing aqueous cavity Figure 1—figure supplement 1A , Figure 3—figure supplements 1C and 2C. Our structural studies have identified the binding mode of the inhibitors at the base of the funnel-shaped cavity, with the aromatic group snugly fitting into the pocket, thereby positioning the isothiourea group into ideal interaction distance with the aspartate of the transition metal binding site Figures 3A,B and 6B.

Although the characterization of the interaction to the metal ion binding site is experimentally challenging, since mutations at this site interfere with transport Bozzi et al. Second, the low micromolecular binding affinity of the inhibitor to the prokaryotic transporter EcoDMT observed in titration calorimetry experiments vanishes in a mutant truncating the binding site aspartate Figure 4B,C , Figure 4—figure supplement 1A,B.

Third, the interaction underlies the observed competitive mechanism that is shared by all investigated isothiourea-based compounds containing different aromatic substituents Figure 1 , Figure 1—figure supplement 3 , and fourth it underlines the strong requirement for the isothiourea group for potent inhibition.

Latter is illustrated by the inhibition of human DMT1 by compounds where either one or both isothiourea groups are modified, leading to moderately reduced potency in the first, and a strongly reduced binding affinity in the second compound Figure 1B , Figure 1—figure supplement 2E,F,G. In our proposed inhibition mechanism, the role of the aromatic group in each compound is to position the inhibitor at the base of the predominantly hydrophobic pocket in proximity to the binding site Figure 6B.

In the orthogonal direction, the funnel-shaped pocket is sufficiently wide to accommodate larger groups, which might undergo successively stronger interactions, which is illustrated by the increased potency of two compounds containing additional alkyl modification at the aromatic ring as it is the case for TMBIT and TEBIT Figures 1B and 6B ; Figure 1—figure supplement 2B,C.

This general mode of interaction might also explain the inhibition of isothiourea-based compounds with larger ring systems as it is the case for the dibenzofuran Br-DBFIT Figures 1B and 6B ; Figure 1—figure supplement 2A and related compounds characterized in a previous study Zhang et al.

Whereas one isothiourea group strongly interacts with the metal ion binding site in both pro- and eukaryotic transporters, the opposite groups reside in the wider exit of the cavity in a region that is poorly conserved between different SLC11 homologues Figure 3E,G , Figure 3—figure supplement 2A. In EcoDMT it most likely undergoes no specific interactions with the protein and instead exhibits large conformational flexibility as supported by the absence of electron density for this group in the X-ray structure of the inhibitor complex and by the mostly unaltered potency in mutants of potentially interacting residues Figures 3A,B , 4 and 6B.

In contrast, mutations of equivalent positions in human DMT1 show a more pronounced effect thus pointing towards stronger inhibitor interactions distal to the metal binding site compared to EcoDMT Figures 5 and 6B.

This is generally supported by the reduced potency of an asymmetric compound binding to human DMT1 where only one of the isothiourea groups was modified Figure 1—figure supplement 2F. In this case the effect of the modification could be explained by a moderate decrease in the interaction at the distal side where interactions with the protein might be less specific and by the reduced entropy of binding of the asymmetric compound with the metal ion binding site, which demands interaction with the isothiourea group Figure 6.

A strategy to increase the potency and selectivity of compounds towards human DMT1 could thus rely on the optimization of interactions at the distal side of the binding pocket by a systematic variation of aromatic scaffolds and attached polar groups.

The values shown for WT deviate from the values shown in Figure 1—figure supplement 2 , due to small differences in the experimental setup i. A Scheme of the inhibitor binding to the outward-facing cavity of an SLC11 transporter thereby preventing substrate binding and conformational changes.

The shape of the cavity is indicated, the sidechain of the conserved aspartate of the metal ion binding site and the chemical structures of different inhibitors are shown. In summary we have provided the first detailed mechanistic insight into the pharmacology of transition metal transporters of the SLC11 family.

Our results are relevant for potential therapeutic strategies inhibiting human DMT1, which could be beneficial in cases where excessive uptake of iron in the intestine leads to iron overload disorders as observed in hereditary or secondary hemochromatosis, and our study provides a framework that might aid the improvement of these compounds to optimize both their potency and specificity.

The cell line stably over-expressing hDMT1 has been characterized previously Montalbetti et al. For uptake experiments, HEK cells were grown in clear bottom, white-well, poly-D-lysine coated 96 well plates Corning.

Briefly, culture media was removed from the wells and the cells were washed three times with uptake buffer mM NaCl, 2. After incubation, uptake solution was removed from the wells, and the cells were washed three times in ice-cold uptake buffer pH 7. Before quantification, a scintillation cocktail Mycrosinth 20, PerkinElmer was added to each well, and the cells were incubated during 1 hr at RT under constant agitation. Transport rates were quantified with:. To assess their inhibitory effect, cells were incubated with the indicated compounds at the specified concentrations during 5 min at RT prior to the addition of the uptake solution.

For the determination of IC 50 values, influx rates for each inhibitor concentration were plotted and data was fitted to a 4-parameter sigmoidal curve. Plotted influx rates correspond to the mean of the indicated biological replicates, errors are s. Each experiment was performed in duplicates for transiently transfected cells with data obtained from at least two independent transfections or triplicates for stably overexpressed WT hDMT1.

The tag was removed during purification unless specified otherwise. Protein expression was induced by addition of 0. The lysate was subjected to a low-spin centrifugation 10, g for 20 min and subsequently the membrane vesicles were harvested by ultracentrifugation , g for 1 hr.

The extracted proteins were purified by immobilized metal affinity chromatography IMAC. Peak fractions were used for reconstitution into liposomes, ITC and crystallization experiments. Crystals grew within two weeks. The two inhibitors were either added to the cryoprotection solutions at a final concentration of 5 mM or directly added as powder to the drops containing the crystals.

Anomalous data were collected at the bromine absorption edge 0. Structures were refined in Phenix Adams et al. Five percent of the reflections not used in refinement were used to calculate R free. The final refinement statistics is reported in Table 2. The electrostatic potential in the extracellular aqueous cavity harboring the inhibitor binding site was calculated by solving the linearized Poisson—Boltzmann equation in CHARMM Brooks et al. Calculations were carried out in the absence of monovalent mobile ions in the aqueous regions.

Liposomes were subjected to three freeze-thaw cycles and extruded through a nm polycarbonate filter Avestin, LiposoFast-Basic to form unilammellar vesicles.

The orientation of the transporters in proteoliposomes was determined using a reconstitution of EcoDMT-His 10 in which the C-terminally Histidine-tag preceded by a 3C protease cleavage site has not been cleaved prior to reconstitution. Initially, proteoliposomes containing a total of 2 mg lipids were extruded using a nm polycarbonate filter to generate unilammellar vesicles and split in two equal aliquots.

Purified 3C protease was subsequently added to the outside of one aliquot of the proteoliposmes and incubated for 2 hr at room temperature. After removal of the protease, the liposomes were dissolved by addition of DM at a detergent to lipid ration of 1. All 3C cleavage steps were performed with a large excess of protease to ensure completion of the reaction. Control liposomes not treated with 3C protease at the different steps were processed the same way. A sample of purified EcoDMT-His 10 was used as control to follow the removal of the His 10 -tag in a sample with unrestricted accessibility to the 3C cleavage site.

Proteoliposomes were harvested by centrifugation and washed twice with 20 volumes of buffer B without Calcein. The samples were subsequently diluted to 0.

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We thank Dr. Michael D. You can also search for this author in PubMed Google Scholar. All authors analysed data and carried out statistical analyses. All authors reviewed and approved the manuscript. Correspondence to Michael D. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions. A role for divalent metal transporter DMT1 in mitochondrial uptake of iron and manganese. Sci Rep 8, Download citation.

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