The PrP-like protein doppel binds copper

Abstract

Doppel (Dp1) is a glycosylphosphatidylinositol-anchored protein expressed in the testis. It exhibits 26% sequence identity with the prion protein (PrP) but lacks the octarepeat region implicated as the major copper-binding domain. Contrary to expectations, Cu(II) induced a 26% reduction in the intrinsic fluorescence of Dp1(27-154) and a calculated Kd for a single-site model of 0.16 ± 0.08 μM. Other metals had minimal effects on fluorescence quenching. Matrix-assisted laser desorption ionization mass spectrometry of a Dp1 peptide revealed binding of copper (but not other metals) to the helical αB/B′-loop-αC subregion of Dp1. Fluorescence quenching and equilibrium dialysis analyses of this Dp1(101-145) peptide were compatible with a binding site of Kd = 0.4 μM. Diethylpyrocarbonate footprinting (Qin, K., Yang, Y., Mastrangelo, P., and Westaway, D. (2002) J. Biol. Chem. 277, 1981-1990) of Dp1(27-154) defined one residue/molecule was protected by copper from diethylpyrocarbonate adduct formation, and reiteration of this analysis with Dp1(101-145) suggested that His131 may contribute to Cu(II) binding. Taken together, our data indicate that the α-helical region of mouse Dp1 possesses a selective copper-binding site with a submicromolar Kd and perhaps one or more lower affinity sites. Although metallated forms of Dp1 might exist in vivo, analyses of Tg(Dp1)10329 mice were inconsistent with reports that Dp1 expression is associated with increased carbonylation and nitrosylation of brain proteins. Thus, rather than comprising an important source of free radical damage, copper binding may serve to modulate the activity, stability, or localization of the Dp1 protein.