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)-15 NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15 NCcc2a was titrated with apo-Atx1. By following the 15 N and 1 H 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.

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... relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15 NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15 NAtx1 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)-15 NAtx1 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 Å for the backbone and 1.11 ؎ 0.11 Å for the heavy atoms. 15 N and 1 H 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 ␣ 1 , and the C-terminal part of helix ␣ 2 on the ms-s 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.