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Mapping critical structural elements in divalent metal‐ion transporter‐1 (DMT1)
Author(s) -
Alexander John H,
Shawki Ali,
Meller Jarek,
Mackenzie Bryan
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.609.7
Subject(s) - dmt1 , chemistry , metal , transporter , wild type , tetranitromethane , metal ions in aqueous solution , stereochemistry , biophysics , crystallography , biochemistry , mutant , divalent metal , biology , tyrosine , organic chemistry , gene
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 55 Fe 2+ 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 55 Fe 2+ transport 69 ± 6% (SE), increased 10‐fold the K 0.5 for Fe 2+ relative to wildtype, and altered substrate recognition such that Mn 2+ was excluded (whereas wildtype DMT1 transports Mn 2+ ), 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 Fe 2+ transport [ Pflügers Arch Eur J Physiol 451 , 544–558 (2006)]—to either Gly (“soft kink”) or Ala (“no kink”) inhibited 55 Fe 2+ transport 92 ± 3% and 95 ± 3% respectively. We anticipate that this work will begin to reveal structural requirements for metal coordination in DMT1.