Structure,Assembly,and Topology of the G185R Mutant of the Fourth Transmembrane Domain of Divalent Metal Transporter

摘要

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 pepttde,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 a-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 a-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.