The first discovered mammalian Nramp (Nramp1) is expressed in phagosomal membranes and likely extracts essential metals to help kill engulfed pathogens ( 2, 3). The natural resistance-associated macrophage protein (Nramp) family of metal transporters represents a common transition metal acquisition strategy conserved across all kingdoms of life ( 1). Through evolution, organisms have developed mechanisms to acquire, transport, and safely store essential metals such as manganese, iron, cobalt, and zinc. 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.Īll organisms require transition metal ions as cofactors in proteins that perform a variety of essential cellular tasks. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Moreover, the methionine sulfur’s presence makes the toxic metal cadmium a preferred substrate. 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. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. 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. Results of these studies will help in designing metal-binding motifs in proteins with varying interaction strengths.Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. Our studies suggest the factors that could explain why Met is not as frequently observed as Cys in the metal-binding motifs. Interaction of the wild-type motif with the copper ion is ~ 160 kcal/mol weaker than that of mutated motif. We also considered Met → Cys − mutation in the motif and repeated the calculations. We then considered the entire metal-binding motif with four residues and calculated the interaction energies with the copper ion. However, Cys − interactions with copper is stronger than that of Met by ~ 250 kcal/mol. On average, interactions of Met with copper ion are stronger by 13–35 kcal/mol compared to CysH. We also carried out calculations with wild-type Cys present in the same metal-binding motif. To compare the metal-binding strength, we mutated Met in silico to CysH/Cys − and performed the calculations. We used two different levels of theory (B3LYP and M06-2X) and the model compounds methyl propyl sulfide, ethanethiol and ethanethiolate were used to represent Met, CysH and Cys − respectively. In the case of Cys, both neutral (CysH) and the deprotonated form (Cys −) were considered. We performed quantum chemical calculations to find out the strength of interactions between sulfur and metal ion in both Met and Cys residues. To address methionine's lower preference in copper-binding sites in comparison to cysteine, we have considered copper-binding motifs (His-Cys-His-Met) from seven different high-resolution protein structures. Although cysteine sulfur is more frequently found as metal-binding ligand, methionine prefers to occur in copper-binding motifs of some proteins. Metals play vital role in various physiological processes and are bound to biomolecules.
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