Transition metals like iron and manganese play crucial roles in various metabolic processes like photosynthesis, oxygen transport, and energy production through the electron transport chain. Transport proteins residing in cellular membranes, like the Nramps (Natural resistance associated macrophage proteins), enable the uptake and trafficking of essential transition metals. For example, in humans, NRAMP2 is indispensable for iron and manganese homeostasis, while NRAMP1 provides resistance against intracellular pathogens by competing with the pathogen’s metal transporters (including Nramps) for acquisition of essential metal ions. Interestingly, in addition to the physiological substrates iron and manganese, Nramps can also transport toxic metals like cadmium but exclude the abundant alkaline earth metals like magnesium and calcium. The Nramps therefore represent an important and fascinating system for investigating the structural and biophysical underpinnings of the transient metal-protein interactions required to transport metal ions across biological membranes.
Over the past several years, structures of bacterial Nramp homologs provided an initial understanding of the metal transport cycle revealing three main conformational states – outward-open (open only to the outside), occluded (an intermediate state closed to both sides of the membrane) and inward-open (open only to the inside). Previous studies also identified the metal-binding site, which contains conserved aspartate, asparagine, and methionine residues. However, the published structures have two important limitations: (i) most were of too low resolution to provide necessary details of metal ion coordination, and (ii) they were from different bacterial species, which hinders comparisons. Thus, little was known about the stereochemistry of metal binding and how Nramps mechanistically distinguish between transport of physiological substrates (iron and manganese) from toxic ones like cadmium.
Conformational rearrangement and distinct coordination geometry adopted by DraNramp during Mn2+ import
Our work, recently published in eLife, overcomes these limitations. We report new structures of all three known conformations that Nramps span to transport manganese, each in manganese-free and manganese-bound states. Importantly, these are of the highest resolution achieved thus far for any Nramp, and all are snapshots of the same bacterial Nramp transporter, enabling apples-to-apples comparisons in unprecedented detail. The structures reveal how the global changes in the protein are associated with changes in the geometry of the divalent Mn2+ ion coordination sphere at the metal-binding site in the center of the protein (see Figure). We also identify conserved networks of polar amino acid residues that serve as gates by stabilizing key internal movements of the protein. These are the movements that enable the protein to alternate the access to the metal-binding site from either side of the cellular membrane to transport manganese.
Moreover, we used a combination of metal ion binding and transport measurements and an additional structure to compare how the protein interacts with cadmium versus manganese. Our results show that our model bacterial Nramp transporter can distinguish the physiological substrates (like manganese) from chemically toxic ions like cadmium: the two metals bind in subtly different ways, which affects which state of the protein is most energetically favored.
Overall, our data show that the metal-binding site conserved across all Nramps is best suited to the physiological manganese substrate Mn2+ (and likely the similar iron ion Fe2+). Our new understanding of the detailed interactions of Nramp with manganese and cadmium could set the stage for the design of therapies to treat metal toxicity and prevention strategies for toxic metal accumulation in crops. Our results also lay a foundation for future studies of how the many Nramp-like transporters in bacteria evolve their substrate selectivity, for example in response to different environmental conditions.
By Shamayeeta Ray and Rachelle Gaudet
