Identification of the "missing domain" of the rat copper-transporting ATPase, atp7b: insight into the structural and metal binding characteristics of its N-terminal copper-binding domain.

Abstract

Wilson disease is an autosomal disorder of copper transport caused by mutations in the ATP7B gene encoding a copper-transporting P-type ATPase. The Long Evans Cinnamon (LEC) rat is an established animal model for Wilson disease. We have used structural homology modelling of the N-terminal copper-binding region of the rat atp7b protein (rCBD) to reveal the presence of a domain, the fourth domain (rD4), which was previously thought to be missing from rCBD. Although the CXXC motif is absent from rD4, the overall fold is preserved. Using a wide range of techniques, rCBD is shown to undergo metal-induced secondary and tertiary structural changes similar to WCBD. Competition 65Zn(II)-blot experiments with rCBD demonstrate a binding cooperativity unique to Cu(I). Far-UV circular dichroism (CD) spectra suggest significant secondary structural transformation occurring when 2-3 molar equivalents of Cu(I) is added. Near-UV CD spectra, which indicate tertiary structural transformations, show a proportional decrease in rCBD disulfide bonds upon the incremental addition of Cu(I), and a maximum 5:1 Cu(I) to protein ratio. The similarity of these results to those obtained for the Wilson disease N-terminal copper-binding region (WCBD), which has six copper-binding domains, suggests that the metal-dependent conformational changes observed in both proteins may be largely determined by the protein-protein interactions taking place between the heavy metal-associated (HMA) domains, and remain largely unaffected by the absence of one of the six CXXC copper-binding sites.