A new method to determine the structure of the metal environment in metalloproteins: investigation of the prion protein octapeptide repeat Cu2+ complex

Abstract

Since high-intensity synchrotron radiation is available, "extended X-ray absorption fine structure" spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu(2+) complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.

New Insights into Metal Interactions with the Prion Protein: EXAFS Analysis and Structure Calculations of Copper Binding to a Single Octarepeat from the Prion Protein

“…In their initial study, EXAFS data is collected on the Cu(II)OR species at pH 7.0, as opposed to pH 7.5 herein, with a k -range of ~3-11 Å −1 . 37 The lower pH likely means there are additional contributions from N2O2 equatorial Cu 2+ coordination, in addition to the expected N3O1 primary coordination environment (excluding consideration of apical solvation). 32 As the Cu K-edge near edge spectrum is not shown it is not possible to determine whether photoreduction contributes to their Cu(II) EXAFS data.…”

“…35 Several groups have performed EXAFS characterizations of Cu 2+ binding to PrP, or related peptide fragments, however, despite the wealth of published data in this area to-date much of the previous EXAFS work in this area has not been rigorously analyzed or reported transparently. 36,37,38,39 …”

Environmental Effects on a Prion's Helix II Domain: Copper(II) and Membrane Interactions with PrP180–193 and Its Analogues

“…[6] Although there is agreement on the biological relevance of this metal binding region, the attribution of the primary location of the copper binding site and the structure of copper(ii) complexes with the octapeptide ligand still remain a matter of debate, especially since contradictory results have been reported. [7][8][9] Preferential copper(ii) coordination to an unstructured region of PrP encompassed between residues 90 and 115 has recently been suggested [9] by studying the competitive effect of the octarepeat domain on Cu 2 + binding to the PrP91-115 fragment. Furthermore, the coordination of a single copper(ii) ion to both His96 and His111 influences the structuring of this amyloidogenic region and induces a b-sheet-like conformation.…”

The Role of Metals in β -Amyloid Peptide Aggregation: X-Ray Spectroscopy and Numerical Simulations

“…From the experimental point of view, this review will be focused on the XAS technique, as the latter displays a number of very interesting features when it is employed in investigating biological systems, and especially in the study of metal-proteins [24,[30][31][32][33][34]. Perhaps the most important of them is that XAS can be used for samples in any state of aggregation.…”