CuII binding sites located at His-96 and His-111 of the human prion protein: thermodynamic and spectroscopic studies on model peptides

Abstract

The prion protein (PrP) is a Cu(2+)-binding cell-surface glycoprotein. Using PrP peptide fragments, by means of potentiometric, spectroscopic and thermodynamic techniques, we have shown that Cu(2+) ions bind to the region comprising His-96, His-111 and the octarepeat domain within residues 60-91. Cu(2+) may bind in different modes, which strongly depend both on His position within the peptide sequence and on the adjacent residues. We have used a series of protected oligopeptides having His at the C- or the N-terminus, inducing different binding modes to amide nitrogens around the His residue, either towards the N- or C-terminus. His imidazole acts as an anchoring site for Cu(2+) and then binding to ionized amide nitrogens follows. When it is directed towards the C-terminus the formation of a less stable seven-membered chelate ring with a {N(im), N(-)} binding mode occurs. When coordination goes towards the N-terminus the thermodynamically more stable six-membered chelate ring is formed. NMR data suggest that both the coordination modes are possible for the model peptides; however, the thermodynamic measurements show that they only slightly differ in energy and the influence of the adjacent amino acid residues can address the coordination toward the C- or the N-terminus.

Impact of ring size on the copper(II) coordination abilities of cyclic tetrapeptides

“…In the CD spectrum (Fig. 3) the charge transfer contribution around the 290 nm seems also to support an increasing contribution from the Cu-N transitions [21][22][23] starting from CuH À1 L species. Notably, large CD perturbation is observed in concomitance with the transition from CuH À1 L to the CuH À2 L species, while this latter show similar CD behavior to the CuH À3 L and CuH À4 L species.…”

“…The CD features that are observed around 290 nm should be assigned to amide to copper(II) charge transfers [21][22][23]. In turn, features assignable to His to Cu(II) CT should fall in the 330-350 nm range [23].…”

Cross‐Talk Between the Octarepeat Domain and the Fifth Binding Site of Prion Protein Driven by the Interaction of Copper(II) with the N‐terminus

“…[67,70,72,73,75,76,[88][89][90][91][92][93][94][95][96] Although there is no general consensus, most of the results obtained so far indicate His111 as the preferential, if not exclusive, copper(II) binding site. [67,72,73,75,76,90,91,95,96] Our systematic studies carried out on small peptide fragments of PrP led to the same conclusions, and the high copper(II) binding affinities of the His96, [97] His111, [98] and even His187 [99] residues have unambiguously been demonstrated. A comparison of the thermodynamic stabilities of the histidinecontaining fragments revealed that the peptides that encompass the amino acid sequences outside the octarepeat domain are even more efficient chelators of copper(II) than the single octarepeat fragments.…”

“…At pH 7.4 three metal-coordination modes have been found so far within the tetraoctarepeat domain: [81] Two copper(II) binding sites, centred around His96 and His111, have also been found for the amyloidogenic PrP region, [67,69,78] with His111 having a higher affinity for copper(II). [72,73,75,90,91,108] (N im ,N À ,N À ) is the main metal-binding mode experienced by Cu 2+ , although there is no general agreement on the contribution of additional donor atoms. [72,74,89,90,[93][94][95][96][97][98][109][110][111] Structural rearrangement of the N-terminus caused by copper(II) binding has also been studied but conflicting results have been reported for peptides that contain different domains.…”

“…[72,73,75,90,91,108] (N im ,N À ,N À ) is the main metal-binding mode experienced by Cu 2+ , although there is no general agreement on the contribution of additional donor atoms. [72,74,89,90,[93][94][95][96][97][98][109][110][111] Structural rearrangement of the N-terminus caused by copper(II) binding has also been studied but conflicting results have been reported for peptides that contain different domains. [112,113] The majority of studies with PrP peptide fragments allowed the assessment of the copper(II) coordination modes, but the results represented a simplified model of the copper-binding chemistry and did not consider important aspects of the metal-PrP interactions because protein folding and interregion interactions were not accounted for.…”