Copper(I) interaction with model peptides of WD6 and TM6 domains of Wilson ATPase: regulatory and mechanistic implications

J Inorg Biochem. 2004 Sep;98(9):1483-94. doi: 10.1016/j.jinorgbio.2004.05.013.

Abstract

With the aim to investigate the mechanism of Cu(I) transport by Wilson ATPase (ATP7B), we have studied the interaction of the peptides 2K10p (CH(3)CO-Lys-Gly-Met-Thr-Cys-Ala-Ser-Cys-Val-His-Asn-Lys-CONH(2)), and 2K8p (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), part of the sixth metal binding domain (WD6) and the sixth transmembrane segment (TM6) of Wilson ATPase, respectively, by means of CD, NMR spectroscopy and homology modeling. In addition, the interaction of Cu(I) with the 2K8p mutants 1s (CH(3)CO-Lys-Leu-Ser-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), 2s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Ser-Pro-Cys-Ser-Lys-CONH(2)) and 3s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Ser-Ser-Lys-CONH(2)), containing two cysteines in various positions, have been studied with the same methods, in order to understand the role of each cysteine in copper binding. Our studies show that the three cysteine thiolates present in the 2K8p peptide sequence act mainly as bridging ligands for Cu(I) binding, and dithiothreitol acts as an important ligand in Cu(I) ligation by 2K10p and the 2K8p mutants. Formation of oligomeric species has been evidenced for all peptides except 2s. Shift of the equilibrium between the various oligomeric species has been accomplished by reducing the Cu(I):peptide ratio. Significant shifts of proline protons upon interaction with Cu(I) have been observed for all proline containing peptides implying a possible role of proline in facilitating Cu(I) binding. These findings have been further discussed with respect to the molecular basis of copper trafficking and intermolecular interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry*
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Cation Transport Proteins / chemistry*
  • Cation Transport Proteins / metabolism*
  • Circular Dichroism
  • Copper / metabolism*
  • Copper-Transporting ATPases
  • Dithiothreitol / pharmacology
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Structural Homology, Protein
  • Structure-Activity Relationship

Substances

  • Cation Transport Proteins
  • Peptide Fragments
  • Copper
  • Adenosine Triphosphatases
  • Copper-Transporting ATPases
  • Dithiothreitol