Alex J McDonald1, Deborah R Leon2, Kathleen A Markham3, Bei Wu1, Christian F Heckendorf2, Kevin Schilling3, Hollis D Showalter4, Philip C Andrews5, Mark E McComb2, M Jake Pushie6, Catherine E Costello7, Glenn L Millhauser8, David A Harris9. 1. Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. 2. Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA; Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA 02118, USA. 3. Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA. 4. Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA. 5. Department of Biological Chemistry, Department of Chemistry, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109, USA. 6. Department of Surgery, Division of Neurosurgery, College of Medicine, University of Saskatchewan, Saskatoon, Canada. 7. Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA; Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA 02118, USA. Electronic address: cecmsms@bu.edu. 8. Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA. Electronic address: glennm@ucsc.edu. 9. Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. Electronic address: daharris@bu.edu.
Abstract
The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define. Here, we employ a combination of chemical cross-linking, mass spectrometry, NMR, molecular dynamics simulations, and functional assays to identify residue-level contacts between the N- and C-terminal domains of PrPC. We also determine how these interdomain contacts are altered by binding of Cu2+ ions and by functionally relevant mutations. Our results provide a structural basis for interpreting both the normal and toxic activities of PrP.
The cellular isoform of the n class="Species">prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define. Here, we employ a combination of chemical cross-linking, mass spectrometry, NMR, molecular dynamics simulations, and functional assays to identify residue-level contacts between the N- and C-terminal domains of PrPC. We also determine how these interdomain contacts are altered by binding of Cu2+ions and by functionally relevant mutations. Our results provide a structural basis for interpreting both the normal and toxic activities of PrP.
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