Mapping critical structural elements in divalent metal‐ion transporter‐1 (DMT1)

Abstract

DMT1 is essential for nonheme iron absorption and iron utilization by erythroid precursors. Our initial approach towards probing the structure‐function of DMT1 is to (1) predict critical residues based on the results of chemical modification experiments or structural models and (2) test these predictions by site‐directed mutagenesis and functional expression of human DMT1 in RNA‐injected Xenopus oocytes. The results of treatment with a battery of modifying agents point to critical roles for Tyr and Met residues since 55Fe2+ transport (at pH 5.5) was sensitive to Rose Bengal photooxidation (preincubated at pH 9.0), tetranitromethane, and cyanogen bromide. Whereas M240A behaved like wildtype, a M438A mutation inhibited 2 μM 55Fe2+ transport 69 ± 6% (SE), increased 10‐fold the K0.5 for Fe2+ relative to wildtype, and altered substrate recognition such that Mn2+ was excluded (whereas wildtype DMT1 transports Mn2+), suggesting that Met‐438 participates in metal‐ion binding. Further, we postulated that Pro (‘kinked’) residues could be involved in forming a metal‐binding pocket in DMT1. Mutating Pro‐266—selected for its proximity to His‐272, a residue known to comprise part of the H+‐coupling mechanism driving Fe2+ transport [Pflügers Arch Eur J Physiol 451, 544–558 (2006)]—to either Gly (“soft kink”) or Ala (“no kink”) inhibited 55Fe2+ transport 92 ± 3% and 95 ± 3% respectively. We anticipate that this work will begin to reveal structural requirements for metal coordination in DMT1.