title: Self-Assembly of the Second Transmembrane Domain of hCtr1 in Micelles and Interaction with Silver Ion.
abstract: Human copper transporter 1 (hCtr1) transports copper and silver by a homotrimer. The protein contains three transmembrane domains in which the second transmembrane domain (TMD2) is a key component lining the central pore of the trimer. The MXXXM motif in the C-terminal end of TMD2 plays a significant role in the function of hCtr1. In this study, we characterized the structure and assembly of isolated TMD2 of hCtr1 in sodium dodecyl sulfate (SDS) micelles and the interaction of the micelle-bound peptide with silver ion using nuclear magnetic resonance, circular dichroism, isothermal titration calorimetry and electrophoresis techniques. We detected the formation of a trimer of the isolated hCtr1-TMD2 in SDS micelles and the binding of the trimer to Ag(I) by a chemical stoichiometry of 3:2 of peptide:Ag(I). We showed that either an intensive pretreatment of the TMD2 peptide by 1,1,1,3,3,3-hexafluoro-2-propanol solvent or a conversion from methionine to leucine in the MXXXM motif changes the aggregation structure of the peptide and decreases the binding affinity by 1 order of magnitude. Our results suggest that the intrinsic interaction of the second transmembrane domain itself may be closely associated with the formation of hCtr1 pore in cellular membranes, and two methionine residues in the MXXXM motif may be important for TMD2 both in the trimeric assembly and in a higher-affinity binding to Ag(I).
authors.affiliation: †State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, PR China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Dong
authors.affiliation: †State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, PR China.
authors.forename: Yunrui
authors.initials: Y
authors.lastname: Wang
authors.affiliation: †State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, PR China.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Wang
authors.forename: Haoran
authors.initials: H
authors.lastname: Xu
authors.affiliation: †State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, PR China.
authors.forename: Liping
authors.initials: L
authors.lastname: Guan
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Li
authors.affiliation: †State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, PR China.
authors.forename: Fei
authors.initials: F
authors.lastname: Li
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002151:Calorimetry / Q000379:methods; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D000079923:Copper Transporter 1; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry; D008823:Micelles; D008969:Molecular Sequence Data; D009154:Mutation; D020005:Propanols / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012834:Silver / Q000737:chemistry / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D006845:Hydrocarbons, Fluorinated
chemical_list: D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D008715:Methionine; D008823:Micelles; D020005:Propanols; D012834:Silver; D012967:Sodium Dodecyl Sulfate; C490079:SLC31A1 protein, human; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated