Copper(II) and the Amyloid Beta Peptide of Alzheimer's Disease
Creators: name: Karr, Jesse William
Publisher: name: Maryland Shared Open Access Repository
Year: 2008
Subjects: Copper(II)
Amyloid Beta Peptide
Alzheimer's Disease
Electron Paramagnetic Spectroscopy
EPR
Chemistry, Inorganic (0488)
Chemistry, Biochemistry (0487)

description: Considerable effort is being directed at unraveling the molecular mechanisms of Alzheimer's disease (AD), including the participation of metal ions.  The extracellular proteinaceous plaques associated with AD are composed predominantly of the amyloid-beta (A?) peptide.  Since the A? peptide binds metal ions (Bush, A. I.; et al. Science 1994, 265, 1464), metal ions have been hypothesized to play a role in the formation and neurotoxicity of A? aggregates associated with Alzheimer's disease (Bush, A. I.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11934).  Because structure is presumed to dictate function, elucidation of the coordination environment through which transition-metal ions, especially Cu(II), bind to A? and participate in the assembly and toxicity of A? aggregates is important to understanding AD. A major controversy centers on the coordination environment of Cu(II) when bound to A?.  The research presented here focuses on the characterization of Cu(II) bound to soluble, fibrillar, and oligomeric forms of A?.  At stoichiometric Cu(II) to peptide ratios, monomeric, oligomeric, and fibrillar forms of A? produce identical EPR spectra with EPR parameters consistent with a Type 2 Cu(II) center with three nitrogen donor atoms and one oxygen donor atom in the coordination sphere of Cu(II).  Cu(II) bound in this site is mononuclear as shown by the Curie temperature dependence of the EPR signal, indicating that neither soluble nor fibrillar forms of A?40 with Cu(II) contain an antiferromagnetically exchange-coupled binuclear Cu(II) site. Low temperature EPR spectroscopy has also been used to show the N-terminus important in creating the Cu(II) binding site in native A?.  The coordination environment of Cu(II) is the same in any version of A? peptide that contain the first 16 amino acids of wild type A?.  In contrast peptides that lack one to three N-terminal amino acids of native A?, do not bind Cu(II) in the same coordination environment as the native peptide.  These results show that the native Cu(II) binding site in A? is sensitive to small changes a the N-terminus, indicating that these residues provide at least some of the ligands to Cu(II) in native A?. The three nitrogen and one oxygen-atom (3N1O) donor ligand environment of Cu(II) bound to wild type A? is accepted, but the identity of each of the ligands, in particular the oxygen-atom donor, is not.  Possible oxygen-atom donors were probed by site-specific mutagenesis.  EPR spectra of the Y10F mutant of A? showed the coordination environment for Cu(II) remains 3N1O and Cu(II) EPR spectra of this mutant are identical to the wild-type.  Mutation of glutamates-3 or 11 to glutamine or aspartate-7 to asparagine produces no effect on the EPR spectrum of Cu(II) bound to the A? peptide, indicating that these residues are not involved in Cu(II) binding.  In contrast, mutation of aspartate-1 to asparagine produces a spectrum with two components.  The spectrum of the first component is identical to that observed for wild-type peptide in the presence of Cu(II) at pH 7.2, whereas the second component has very different EPR parameters.  These data are consistent with a model in which the carboxylate of aspartate-1 creates the native binding site through hydrogen-bonding interactions rather than through direct ligation to Cu(II) in A?. Lastly, the coordination environment of bound copper as a function of Cu:A? stoichiometry and A? oligomerization states has been examined.  By EPR spectroscopy, two Cu(II) binding sites on both soluble and fibrillar A? with the same EPR parameters were spectroscopically distinguished.  The EPR parameters for the first binding site are consistent with a 3N1O or coordination environment while the EPR parameters for the second binding site are consistent with a two nitrogen, two oxygen (2N2O) coordination environment.  Consistent with other measurements, the first binding site on A? fibrils has a micromolar dissociation constant (Kd = 8 ± 2 ?M), whereas the second binding site has a millimolar dissociation constant (Kd = 1.6 ± 0.2 mM).  No change in Cu(II) coordination environment is detected by EPR spectroscopy of Cu(II) with A? (1:1 ratio) collected as a function of A? fibrillization time, which indicates that Cu(II) binding is independent of A? oligomeric state.  Transmission electron microscopy images of A? fibrils before and after Cu(II) addition are the same, showing that once incorporated, Cu(II) does not affect fibrillar structure.  On the basis of these results, a model is proposed for Cu(II) binding to A? during fibrillization that is independent of peptide oligomeric state.