Structural and Functional Studies Into Nramp Divalent Metal Transporter Mechanisms

Abstract

The Nramp (natural resistance associated macrophage protein) family of transition metal ion transporters is an important class of secondary active transporters involved in metal homeostasis used by all branches of life. Two Nramp paralogs in mammals are required for the dietary uptake and endosomal recycling of non-heme iron, as well as in the innate immune response to intracellular bacterial infections. Mutations in or mis-regulation of Nramps are associated with iron disorders and immune pathologies causing disease in mammals, and Nramps have been implicated in heavy metal poisoning. The molecular explanations underlying disease phenotypes and metal selectivity by Nramps were poorly understood. Here I present the second X-ray crystal structure of a bacterial Nramp homolog, a mechanism for two disease-causing missense mutations in mammalian Nramps, and detail the role of Nramp’s metal-binding site methionine residue in metal selectivity. Using Deinococcus radiodurans Nramp (DraNramp) as a model system, I determined its crystal structure and successfully resolved the N-terminal half of TM1, which was absent from the previously determined Staphylococcus capitis Nramp structure. By using the DraNramp structure, in vitro proteoliposome reconstitutions, in vivo cell-based cobalt transport assays, and cysteine accessibility experiments, I explain that that a glycine-to-arginine disease-mutation in the N- terminal half of TM1 acts as a steric wedge locking the transporter in the inward state inhibiting its function, while another glycine-to-arginine disease-mutation in TM4 shifts the transporter to the outward state and impairs metal transport in a charge-dependent manner. Using many of the same methods, it is also shown here that Nramp’s metal-binding methionine prevents transport of hard alkaline earth metals including magnesium and calcium, is not required for the transport of softer transition metals, and promotes high-affinity transport of cadmium and manganese. These results provide molecular explanations for the disease phenotypes found in mammals bearing the glycine-to-arginine mutations, and also explain how Nramps specifically discriminate against the highly abundant divalent metals calcium and magnesium, and yet at the same time remain promiscuous in their ability to transport other divalent metals including the toxic metal cadmium.