title: Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein.
abstract: The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60-91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91-230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).
authors.affiliation: Institut für Chemie/Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany. treiber@chemie.fu-berlin.de
authors.forename: Carina
authors.initials: C
authors.lastname: Treiber
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Thompsett
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Pipkorn
authors.forename: David R
authors.initials: DR
authors.lastname: Brown
authors.forename: Gerd
authors.initials: G
authors.lastname: Multhaup
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D003035:Cobalt / Q000737:chemistry; D019286:Endopeptidase K / Q000737:chemistry; D004912:Erythrocytes / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D051379:Mice; D011328:Prions / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance
chemical_list: D008670:Metals; D011328:Prions; D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; D006639:Histidine; D019286:Endopeptidase K