Structural basis of metal import by Nramp family transporters.

Abstract

Transition metal ions like Mn2+ and Fe2+ play a crucial role in various metabolic processes in all living cells. Excess or deficiency of these essential nutrients leads to diseases including cancer, anemia etc. Cellular levels of these metals are thus tightly controlled and regulated. Nramps (natural resistance-associated macrophage proteins), a class of transition metal transporters present in all domains of life, regulate the levels of these divalent metal ions within cells and prevent disorders related to metal insufficiency or overload. In humans, there exists two Nramps: Nramp2 is indispensable for Fe2+ and Mn2+ homeostasis, while Nramp1 provides resistance against intracellular pathogens by competing with the pathogen's metal transporters (including Nramp homologs) for acquisition of essential metal ions. Nramp-mediated metal ion transport typically involves co-transported protons. A few structures of Nramps are reported to date which reveal their overall LeuT-fold and provide a preliminary understanding of metal binding and proton co-transport. To better understand the metal import mechanism, we resolved high-resolution structures of Deinococcus radiodurans Nramp (DraNramp) in three conformations in both Mn2+-bound and metal-free states, providing the first molecular map of the entire Mn2+ transport cycle. The structures reveal that global conformational changes during transport are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations and conserved networks of polar residues lining the inner and outer gates. A Cd2+-bound structure highlights differences in coordination geometry for Mn2+ and Cd2+. Binding studies using isothermal titration calorimetry along with transport and mutational analyses reveal that the thermodynamic landscape for binding and transporting physiological metals is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, our results lay a foundation for understanding how metal ion transporters like Nramps evolve their substrate selectivity to different ecological niches.