| Natural resistance-associated macrophage protein (Nramp) is a highly conserved membrane protein family. DMT1 (also known as Nramp2, Slc11a2, DCT1) and Nramp1 (Scl11a2) are the two members of this family. DMT1 can transport divalent metal cations, and the deficiency of iron in body would lead to iron deficiency anemia. Defective Nramp1 is associated with the susceptibility to infection by intracellular pathogens and is implicated in a number of diseases known as autoimmune or inflammatory diseases. Nramp1 and Nramp2 (DMT1) are integral membrane proteins with 12 putative transmembrane domains. It has been demonstrated that the conserved motif"Asp-Pro-Gly"of TMD1 and"Met-Pro-His"of TMD6 between the discontinuous helix forms antiparallel"TM helix/extended peptide"boundary, lining a"pore"cavity which may play a key role for divalent metal ion transport. It was revealed that both the conservative substitutions of Asp86 and the mutations of Gly88 (G88A, C, V) in the signature sequence of DMT1 result in complete loss of transport activity. Other nonconserved amino acid residues in TMD1 were also found to be involved in the metal ion transport of DMT1. The studies of the structure, topology and metal ion binding of TMD1 are helpful to understand the functions of DMT1. We had studied the C-terminal part of TMD1 for metal ion binding, but the structure and metal ion binding of the full length of TMD1 are still an open question. Besides, the structures of TMD3, 4, and 6 had been studied, but three-dimentional structure of TMD2 is not reported.In our work, we studied the structures, self-assembly, topologies and the interactions with metal ions of the DMT1-TMD1 peptide and Nramp1-TMD2 peptide in SDS,DPC micelles and TFE aqueous solution,respectively. First, we studied the wildtype peptide and its D19A and D26A mutants of DMT1-TMD1. The three-dimentional structures of the peptides in model membranes were studied using NMR, CD and molecular dynamic simulation. Both the wildtype peptide and the mutants form a'helix-extended segment-helix'conformation. The results of 16-DSA and Mn2+ experiments suggest that the N-terminal part (including helix I) of the peptide TMD1 is embedded in SDS micelles, while its C-terminal part (including helix II) is close to the surface of the micelles. The addition of either Mn2+ or Co2+ in micelle solution of TMD1 not only dramatically broadens the cross-peaks associated with theβ-protons of the residues Asp19 and Asp26, but also induces evident down-field shifts of theβ-protons of the two Asp residues and theγ-protons of Glu24. The remarkable broadening of either Asp26 or Asp19 resonance remains in the NOESY spectrum of D19A or D26A, but the peak broadening of the other one is completely abolished by the substitution of Ala. The interaction of Co2+ with the peptide Nt-TMD1-loop1/2 was also characterized by observing the effect of Co2+ on the intensity and chemical shift of the peptide, and the result is identical. The metal ion binding sites are the negatively charged residues at the flexible central part and the C-terminal part of TMD1.Then we studied TMD2 of Nramp1. TMD2 adopts predominantly anα-helical structure both in micelles and in TFE aqueous solution, and the peptide is self-assembly from Leu6 to Leu22, and the peptide is self-assembly both in TFE aqueous solution and SDS micelles. The critical concentration for assembly of the peptide was obtained by the fluorescence experiments.Our study may be useful for understanding the structure and function of the integral membrane protions of Nramp.
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