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

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NAM in solution. In particular, a solution of Cu(I)-(15)NAtx1 was titrated with apo-Ccc2a, and, vice versa , a solution of Cu(I)-(15)NCcc2a was titrated with apo-Atx1. By following t he N-15 and H-1 chemical shifts, a new species is detected in both experime nts. This species is the same in both titrations and is in fast exchange wi th the parent species on the NMR time scale. Nuclear relaxation data are co nsistent with the formation of an adduct. Judging from the nuclear Overhaus er effect spectroscopy patterns, the structure of Cu(I)-(15)NCcc2a in the p resence of apo-Atx1 is not significantly altered, whereas Cu(I)-(15)NAtx1 i n the presence of apo-Ccc2a experiences some changes with respect to both t he apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-(1 5)NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effe cts to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 Angstrom for the backbone and 1.11 +/- 0.11 Angstrom for the heavy at oms. N-15 and 1H chemical shifts suggest the regions of interaction that, t ogether with independent information, allow a structural model of the adduc t to be proposed. The apo form of Atx1 displays significant mobility in loo ps 1 and 5, the N-terminal part of helix alpha (1), and the C-terminal part of helix alpha (2) on the ms-ps time scale. These regions correspond to th e metal binding site. Such mobility is largely reduced in the free Cu(I)-At x1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in bot h Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is re levant as a structural and kinetic model for copper transfer from Atx1 to C cc2a in physiological conditions.