Structure, assembly, and topology of the G185R mutant of the fourth transmembrane domain of divalent metal transporter.
The mammalian iron transporter, divalent metal transporter (DMT1), is a 12-transmembrane domain integral protein, responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Two disease-causing mutants in animals have been found and attributed to the same missense mutation (G185R), which occurs within the putative transmembrane domain 4 (TM4) of DMT1. We have characterized a synthetic 24-mer peptide, corresponding to the sequence of the TM4 of DMT1 with G185R mutation using circular dichroism (CD) and NMR spectroscopy and show that the G185R peptide assumes mainly alpha-helical conformations in various membrane-mimetic environments. Solution structures derived from NMR and molecular dynamics/simulated annealing calculations demonstrate that the peptide exhibits a highly defined alpha-helix in its middle portion, flanked by a highly flexible N-terminus and a relatively ordered C-terminus. Both the folding and location of the C-terminus in SDS micelles are regulated by pH values. Paramagnetic broadening on peptide NMR signals by spin-labeled 5- and 16-doxylstearic acids and Mn(2+) ion suggests that both the N-terminus and the helical region of the peptide are embedded in SDS micelles. Surprisingly, self-association of the peptides for both the wild type and the G185R mutant studied by CD, electrospray ionization mass spectrometry, and NMR diffusion-ordered spectroscopy demonstrated that mutation of the Gly185 to a bulky and positively charged arginine causes a different self-assembly of the peptide, e.g., from a trimer to a hexamer, which implies that the quaternary structure of integral DMT1 may be crucial for its function in vivo.
Li
Fei
F
Department of Chemistry and Open Laboratory of Chemical Biology, The University of Hong Kong, Pokfulam Road, Hong Kong, People's Republic of China.
Li
Hongyan
H
Hu
Lihong
L
Kwan
Miufan
M
Chen
Guanhua
G
He
Qing-Yu
QY
Sun
Hongzhe
H
D000595:Amino Acid Sequence; D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008345:Manganese / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D013113:Spin Labels; D013696:Temperature; D000099236:Solute Carrier Family 11, Member 2
D027682:Cation Transport Proteins; D033862:Iron-Binding Proteins; D008345:Manganese; D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D012997:Solvents; D013113:Spin Labels; D000099236:Solute Carrier Family 11, Member 2