Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters

Significance Transition metals are micronutrients that all organisms use in essential metabolic processes. The ubiquitous Natural resistance-associated macrophage protein (Nramp) family facilitates the acquisition of these metal ions by transporting them across cellular membranes, including dietary iron absorption in mammals. We show that a conserved methionine, an unusual metal-binding residue found in the Nramp metal-binding site, is not essential for the transport of physiological environmentally scarce transition metals like iron and manganese. Instead, it confers selectivity against the abundant alkaline earth metals calcium and magnesium, with the tradeoff of making the toxic metal cadmium a preferred substrate. Using protein structure information, biochemical results, molecular dynamics simulations, and inorganic chemistry theory, we propose a model for how metal discrimination is enforced. Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur’s presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it—more effectively than any other amino acid—increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.