Folding and assembly of TMD 6‐related segments of DMT 1 in trifluoroethanol aqueous solution

Abstract

Divalent metal-ion transporter 1 (DMT1) belongs to a large class of metal-ion transporters that drive the translocation of a wide range of divalent metal substrates across membranes toward the cytosol with couple of protons. Two highly conserved histidines in the sixth transmembrane domain (TMD6) are essential for metal transport activity in DMT1. In the present study, we determine the high-resolution structures of three 25-residue peptides, corresponding to TMD6 of the wildtype DMT1 (the segment 255-279) and its H267A and H272A mutants, in 30% TFE-d(2) aqueous solution by the combined use of circular dichroism (CD) and NMR spectroscopies. The wildtype peptide forms an 'α-helix-extended segment-α-helix' structure with two helices spanning over Gly258-Ala262 and Met265-Lys277 linked by a hinge at residues Val263-Ile264. The H267A mutation reduces the hinge to one residue (Ile264), while the H272A mutation extends the flexible region of the central part from Val263 to His267. Diffusion-ordered spectroscopy (DOSY) study demonstrates that all the peptides are self-assembly as trimer in 30% TFE-d(2) aqueous solution. The H272A substitution decreases the intermolecular interaction whereas the H267A substitution may enhance the intermolecular interaction. The specific structure of the discontinuous helix and the self-assembly feature of DMT1-TMD6 may be crucial for its biological function. The changes in conformation and intermolecular interaction induced by histidine substitution may be correlated with the deficiency of DMT1 in metal-ion permeation.