The Octarepeat Domain of the Prion Protein Binds Cu(II) with Three Distinct Coordination Modes at pH 7.4

The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, three distinct coordination modes are identified. Each mode is fully characterized to reveal a series of copper-dependent octarepeat domain structures. Multiple His coordination is clearly identified at low copper stoichiometry. In addition, EPR detected copper−copper interactions at full occupancy suggest that the octarepeat domain partially collapses, perhaps stabilizing this specific binding mode and facilitating cooperative copper uptake. This work provides the first complete characterization of all dominant copper coordination modes at pH 7.4

Aspects of Structure and Bonding in Copper - Amino Acid Complexes Revealed by Single-Crystal EPR/ENDOR Spectroscopy and Density Functional Calculations

This work deduces from a series of well-defined copper-doped amino acid crystals, relationships between structural features of the copper complexes, and ligand-bound proton hyperfine parameters. These were established by combining results from electron paramagnetic resonance (EPR)/electron−nuclear double resonance (ENDOR) studies, crystallography, and were further assessed by quantum mechanical (QM) calculations. A detailed evaluation of previous studies on Cu2+ doped into α-glycine, triglycine sulfate, α-glycylglycine, and l-alanine crystals reveal correlations between geometric features of the copper sites and proton hyperfine couplings from amino-bound and carbon-bound hydrogens. Experimental variations in proton isotropic hyperfine coupling values (aiso) could be fit to cosine-square dependences on dihedral angles, namely, for Cα-bound hydrogens, aiso = −1.09 + 8.21 cos2 θ MHz, and for amino hydrogens, aiso = −6.16 + 4.15 cos2 φ MHz. For the Cα hydrogens, this dependency suggests a hyperconjugative-like mechanism for transfer of spin density into the hydrogen 1s orbital. In the course of this work, it was also necessary to reanalyze the ENDOR measurements from Cu2+-doped α-glycine because the initial study determined the 14N coupling parameters without holding its nuclear quadrupole tensor traceless. This new treatment of the data was needed to correctly align the 14N hyperfine tensor principal directions in the molecular complex. To provide a theoretical basis for the coupling variations, QM calculations performed at the DFT level were used to compute the proton hyperfine tensors in the four crystal complexes as well as in a geometry-optimized Cu2+(glycine)2 model. These theoretical calculations confirmed systematic changes in couplings with dihedral angles but greatly overestimated the experimental geometric sensitivity to the amino hydrogen isotropic coupling

Electron Spin−Echo Envelope Modulation Study of Multicrystalline Cu2+-Insulin: Effects of Cd2+ on the Nuclear Quadrupole Interaction of the Cu2+-Coordinated Imidazole Remote Nitrogen

A comparison of electron spin−echo envelope modulation (ESEEM) spectra from multi-crystalline Cu2+-insulin with and without additional Cd2+ show a dramatic change in the quadrupole coupling parameters of the remote nitrogens of the two histidine imidazoles that ligate to copper. Without Cd2+, the quadrupole parameters are like those observed in blue copper proteins and in copper substituted lactoferrin. With Cd2+ soaked into the Cu2+-insulin crystals, the quadrupole parameters are similar to those found in galactose oxidase. Theoretical simulations of ESEEM spectra guided by structure modeling suggest that these changes originate from differences in the hydrogen bonding environments of the imidazole remote nitrogen. In addition, a compilation of results from previous ESEEM studies of copper proteins reveals that the asymmetry parameter, η, may be an indicator of type of hydrogen bond the imidazole remote nitrogen makes. When η ≥ 0.9, the nitrogen hydrogen bonds to water, whereas when η \u3c 0.9, the nitrogen hydrogen bonds to the protein