Solution structure of the Cu(I) and Apo forms of the yeast metallochaperone, Atx1

The H-1 NMR solution structure of the Cu(I)-bound form of Atx1, a 73-amino acid metallochaperone protein from the yeast Saccharomyces cerevisiae, has been determined. Ninety percent of the H-1 and 95% of the N-15 resonances w ere assigned, and 1184 meaningful NOEs and 42 (3)J(HNH alpha), and 60 (1)J( HN) residual dipolar couplings provided a family of structures with rmsd va lues to the mean structure of 0.37 +/- 0.07 Angstrom for the backbone and 0 .83 +/- 0.08 Angstrom for all heavy atoms. The structure is constituted by four antiparallel beta strands and two alpha helices in a beta alpha beta b eta alpha beta fold. Following EXAFS data [Pufahl, R., Singer, C. P., Peari so, K. L., Lin, S.-J., Schmidt, P. J., Fahrni, C. J., Cizewski Culotta, V., Penner-Hahn, J. E., and O'Halloran, T. V. (1997) Science 278, 853-856], a copper ion can be placed between two sulfur atoms of Cys15 and Cys18. The s tructure of the reduced apo form has also been determined with similar reso lution using 1252 meaningful NOEs (rmsd values for the family to the mean s tructure are 0.67 +/- 0.12 Angstrom for the backbone and 1.00 +/- 0.12 Angs trom for all heavy atoms). Comparison of the Cu(I) and apo conformations of the protein reveals that the Cu(I) binding cysteines move from a buried si te in the bound metal form to a solvent-exposed conformation on the surface of the protein after copper release. Furthermore, copper release leads to a less helical character in the metal binding site. Comparison with the Hg( II)-Atx1 solid-state structure [Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A., and O'Halloran, T. V. (1999) Structure 7, 605-617] provides insights into the copper transfer mechanism, and a pi votal role for Lys65 in the metal capture and release process is proposed.