Copper Binding

Structural studies of the prion protein have concentrated on the globular domain of the C-terminus because of its solubility when prepared from bacterial inclusion bodies (44). Further studies have found little structure in the N-terminus, a region of approx 90 amino-residues (45). Such a suggestion that a large part of this protein has no structure is surprising, as it contains what appears to be a conserved functional domain of 32 amino residues formed from 4 repeats of an octomer, each of which contains histidine. In birds and turtles, this sequence is a hexameric repeat region, but it is found in a similar position. Analysis of peptides based on this region suggested that copper might bind to this region but with a surprising low Kd of 6.7 ^M (46,47). At the time of this finding, this low affinity suggested to many that this binding was nonspecific. However, further analysis of a larger N-terminal fragment (amino residues 23-98) using equilibrium dialysis provided more rigorous support for the idea that PrPC binds copper (30). These new data suggested that the N-terminal could be saturated by 5 atoms of copper, and 3.4 atoms showed cooperativity in binding even though the first atom of copper again appeared to bind at a low Kd of 5.9 ^M. The interpretation of this is nevertheless that once one atom of copper has bound to PrPC, further binding occurs at a higher Kd, implying that perhaps the low initial Kd's reflect the artificial nature of attempting to add copper to the protein in a nonphysiological manner.

More recently, studies have continued with new peptides based on the octameric repeat region (48-53). These studies have focused on the interaction between the copper and the amino residues in the octameric repeat. Binding of copper to the repeat causes a change in conformation of amino residues outside of the repeat region itself (49). Peptides based on the repeat region appear to bind the copper in the form of Cu(II) (53) and coordinate it between the four single histidine residues of the repeats (48). The coordination of the two or four atoms of copper may require one or two histidines per atom. However, despite the suggestion that copper is bound as Cu(II), there is evidence that this copper can go through redox cycling; when this occurs, tryptophan residues within the repeats also interact with the Cu(I) (51). Although in vitro studies of peptides provide interesting insights, they are not necessarily indicative of what occurs in cells.

A very recent study by Jackson et al. (54) using NMR to monitor the binding of the first atom of Cu to an octameric-repeat region peptide has suggested that the correct Kd for this interaction is in the low femtomolar range (8 x 10-14 M). Additionally, Jackson et al. (54) also suggest that a fifth copper atom can bind at another site (involving histidines at amino residue 96 and 111 in the human sequence). Evidence for this binding has been reported (30,55,56). The Kd as reported by Jackson et al. (54) for this fifth site is also reported to be in the femtomolar region, but this was determined for a fragment of PrP in which the octameric repeat region had been deleted (PrP90-231). However, recent research from my own laboratory suggests that the Kd for the fifth site in full-length PrP is considerably less than this (27 ^M), whereas that for the first atom of Cu to bind in the octameric repeat region of full-length PrP is much lower than any previously reported (8 x 10-17 M, determined by competition assay). Regardless of the exactitude of these measurements, the implication is that PrPC is a true Cu-binding protein. The variability in the affinities determined would, however, sug-

Prion protein

Intracellular
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