The prion protein is a combined zinc and copper binding protein: Zn2+ alters the distribution of Cu2+ coordination modes

Abstract

PrP binds copper in the highly conserved, unstructured N-terminal half of the protein. The octarepeat region consists of 4 tandem repeats of PHGGGWGQ and binds four equivalents of copper at full occupancy. Adjacent to the octarepeats are two additional histidines that may also bind copper. We recently showed that when the octarepeat region is titrated with Cu²⁺, the copper binding mode depends on the number of equivalents of copper bound. In addition to copper, other metals have been associated with PrP, however zinc is the only metal other than copper that induces PrP endocytosis, inhibits fibril formation and promotes inter-molecular interactions. In this work we show that even large excesses of zinc (> 1mM) are unable to displace copper from either the octarepeat region or the full-length protein. However, EPR reveals that physiologically relevant levels of zinc significantly alter the distribution of copper among the available binding modes. Diethyl pyrocarbonate (DEPC) modification and Mass Spectrometry is used to identify the octarepeat region as the zinc binding site and to confirm that the affinity of PrP for zinc is ~200 μM. PrP can simultaneously bind both copper and zinc by shifting to binding modes that minimize the ratio of histidines to copper.

Figure 1. Effect of zinc on the distribution of the components of the copper EPR spectrum

a)PrP(23–28,57–91) w/1 eq copper titrated with zinc b) PrP(23–28,57–91) with and without 300 μM Zn titrated with copper c) recombinant PrP(23–231) w/2 eq copper titrated with zinc d) PrP(23–231) with and without 300 μM Zn titrated with copper. Total copper bound (black), Component 3 (blue), 1+2 (red) and non-octarepeat (green). Copper and zinc (solid), copper only (dotted).

Figure 2. Models representing metal binding in the N-terminal domain of PrP

Top row (High Zinc); Zinc (red) is bound by the octarepeat region (left) while non-octarepeat sites (H96 and H111) are available for copper binding (blue, middle). Copper at high concentration will displace zinc from octarepeats to form up to 4 eq of Component 1 (right). Bottom row (Low Zinc); Copper (blue) is bound by the octarepeats in Component 3 when copper is low (left), with increasing copper loads the non-octarepeat sites (middle). High copper (right column) results in Component 1 copper binding by the octarepeats. Approximate molar metal concentrations are shown in the arrows. Octarepeat structures based on data from Chattopadhyay et al.