NR ARCW
AU Treiber,C.; Simons,A.; Pipkorn,R.; Thompsett,A.; Brown,D.R.; Multhaup,G.
TI Metal ion binding of PrP
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Vortrag V-13
PT Konferenz-Vortrag
AB
Whereas the physiological function of PrP is yet to be fully determined the ability of PrP to bind Cu(II) in vivo suggests a role in Cu homeostasis. Dissociation constants for Cu(II) binding have been reported to vary between micro-, nano- and micromolar. Comparative binding studies were performed to determine affinities of PrP to metal-ions (Cu, Mn, Co) by Surface Plasmon Resonance (SPR) analysis. Based on a method which we had initially worked out for the analysis of the Cu binding domain of APP (Simons et al., Biochemistry 41, 9310-20, 2002), we could analyze PrP binding kinetics of Cu(II) binding on Cu(II)-loaded NTA-dextran affinity sensors by assaying the formation of a ternary complex of NTA-Cu(II)-PrP on the chip surface. Recombinant bacterial PrP and four different synthetic peptides covering PrP amino acid residues 92-114 (including the metal ion chelating histidines 96 and 111), 60-91 (covering the metal ion octarepeat region), 86-107 (including the metal ion chelating histidine 96) and 100-121 (including the metal ion chelating histidine 111) were used to measure the kinetics of binding to the Cu-charged NTA surface.
SPR results revealed that PrP specifically bound to Cu, Mn or Co charged NTA-surfaces. A peptide covering residues 86-107 specifically bound to Cu-charged surfaces and residues 100-121 were identified for Co binding whereas none of the peptides was able to bind to Mn-NTA. Full-length PrP and PrP lacking the octapeptide region bound to Cu-, Mn- and Co-NTA surfaces. Both forms of recombinant PrP had a similar affinity to Cu and Mn but the affinity to Co differed between these forms by one magnitude and was lower for the truncated protein.
Taken together, (1) there are at least two independent binding sites for Cu within PrP, one in the octarepeat region and a second site provided by histidine 96; (2) since none of the peptides could bind to Mn but both recombinant proteins did, a novel conformational binding site for Mn is proposed for PrP with an affinity that is modulated by the octapeptide region; (3) Histidine 111 is important for Co binding and the octarepeat region enhances the affinity for Co by one magnitude.
Thus, SPR results for binding of PrP to Cu were in fully agreement with the literature thus validating SPR results obtained for PrP binding to Mn and Co, for mapping of metal ion binding sites and the binding kinetics.
AD Carina Treiber, ZMBH-Heidelberg; Andreas Simons, Gerd Multhaup, Freie Universität Berlin, Institut für Chemie/Biochemie; Rüdiger Pipkorn, DKFZ-Heidelberg; Andrew Thompsett, David R. Brown, University of Bath, Department of Biology and Biochemistry
SP englisch
PO Deutschland
OR Tagungsband