Membrane-inserted conformation of transmembrane domain 4 of divalent-metal transporter

Divalent-metal transporter 1 (DMT1) is involved in the intestinal iron absorption and in iron transport in the transferrin cycle. It transports metal ions at low pH (approximate to5.5), but not at high pH (7.4), and the transport is a proton-coupled process. Previously it has been shown that transmembrane domain 4 (TM4) is crucial for the function of this protein. Here we provide the first direct experimental evidence for secondary-structural features and membrane insertions of a 24-residue peptide, corresponding to TM4 of DMT1 (DMT1-TM4), in various membrane-mimicking environments by the combined use of CD and NMR spectroscopies. The peptide mainly adopts an a-helical structure in trifluoroethanol, SDS and dodecylphosphocholine micelles, and dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol small unilamellar vesicles. It has been demonstrated from both Halpha secondary shifts and nuclear-Overhauser-enhancement (NOE) connectivities that the peptide is well folded into an alpha-helix from Val(8) to Lys(23) in SDS micelles at pH 4.0, whereas the N-terminus is highly flexible. The alpha-helical content estimated from NMR data is in agreement with that extracted from CD simulations. The highest helicity was observed in the anionic phospholipids {1,2-dimyristoyl-sn-glycero-3-[phosphorac-(1-glycerol)]} 1, indicating that electrostatic attraction is important for peptide binding and insertion into the membranes. The secondary-structural transition of the peptide occurred at pH 4.3 in the 2,2,2-trifluoroethanol (TFE) water mixed solvent, whereas at a higher pH value (5.6) in SDS micelles, DMT1-TM4 exhibited a more stable structure in SDS micelles than that in TFE in terms of changing the pH and temperature. PAGE did not show high-molecular-mass aggregates in SDS micelles. The position of the peptide relative to SDS micelles was probed by, the effects of 5- and 16-doxylstearic acids on the intensities of the peptide proton resonances. The results showed that the majority of the peptide is inserted into the hydrophobic interior of SDS micelles, whereas the C-terminal residues are surface-exposed. The ability of DMT1-TM4 to assume transmembrane features may be crucial for its biological function in vivo.