Solution Structure of the Cu(I) and Apo Forms of the Yeast Metallochaperone, Atx1^,

Abstract

The (1)H 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 (1)H and 95% of the (15)N resonances were 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 values to the mean structure of 0.37 +/- 0.07 A for the backbone and 0.83 +/- 0.08 A for all heavy atoms. The structure is constituted by four antiparallel beta strands and two alpha helices in a betaalphabetabetaalphabeta fold. Following EXAFS data [Pufahl, R., Singer, C. P., Peariso, 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 structure of the reduced apo form has also been determined with similar resolution using 1252 meaningful NOEs (rmsd values for the family to the mean structure are 0.67 +/- 0.12 A for the backbone and 1.00 +/- 0.12 A 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 site 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 pivotal role for Lys65 in the metal capture and release process is proposed.

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake

“…Sequence alignments and homology models on the six metal-binding domains of WLNP demonstrate that the Ϸ70 aa domains of N-WLNP are likely to be folded very similarly into ferredoxin-like units (16). This same fold is also found in both NMR and x-ray structures of the yeast Atx1 and human HAH1 metallochaperones (17)(18)(19)(20), the soluble cytosolic proteins that deliver copper to the copper-transporting P-type ATPases (21).…”

A copper(I) protein possibly involved in the assembly of Cu A center of bacterial cytochrome c oxidase

“…The copper ion was included in the calculations by adding new restraints obtained from EXAFS following a procedure described in ref. 18. The assessment of the structures was performed by using the program PROCHECK-NMR (19,20).…”

Unification of the Copper(I) Binding Affinities of the Metallo-chaperones Atx1, Atox1, and Related Proteins

“…They both feature the classic ferredoxin ␤␣␤␤␣␤-fold with a CXXC motif acting as a high affinity Cu I -binding site ( Fig. 1) (4,5). Homologues are found in cyanobacteria (Atx1), in Enterococcus hirae (CopZ), in Bacillus subtilis (CopZ), and in many other organisms (6).…”