Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase

J Biol Chem. 2001 Nov 2;276(44):41365-76. doi: 10.1074/jbc.M104807200. Epub 2001 Aug 10.

Abstract

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Binding Sites
  • Biological Transport
  • Carrier Proteins*
  • Cation Transport Proteins*
  • Copper / metabolism*
  • Copper Transport Proteins
  • Cytosol / enzymology*
  • Fungal Proteins / chemistry
  • Fungal Proteins / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Conformation
  • Saccharomyces cerevisiae Proteins*

Substances

  • ATX1 protein, S cerevisiae
  • CCC2 protein, S cerevisiae
  • Carrier Proteins
  • Cation Transport Proteins
  • Copper Transport Proteins
  • Fungal Proteins
  • Saccharomyces cerevisiae Proteins
  • Copper
  • Adenosine Triphosphatases