A novel copper(II) coordination at His186 in full-length murine prion protein

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To explore Cu(II) ion coordination by His186 in the C-terminal domain of full-length prion protein (moPrP),we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spinprobe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSLtechnique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C),moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe anda nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectrawas induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron–electrondipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distancewas within 20 Å. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1),moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 Å, 18.1 Å, 10.7 Å, and8.4 Å, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe wasnot observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2region of the C-terminal domain of moPrPC.
2010-04-09