Altered Domain Structure of the Prion Protein Caused by Cu2+ Binding and Functionally Relevant Mutations: Analysis by Cross-Linking, MS/MS, and NMR

Alex J McDonald; Deborah R Leon; Kathleen A Markham; Bei Wu; Christian F Heckendorf; Kevin Schilling; Hollis D Showalter; Philip C Andrews; Mark E McComb; M Jake Pushie; Catherine E Costello; Glenn L Millhauser; David A Harris

doi:10.1016/j.str.2019.03.008

Altered Domain Structure of the Prion Protein Caused by Cu²⁺ Binding and Functionally Relevant Mutations: Analysis by Cross-Linking, MS/MS, and NMR

Structure. 2019 Jun 4;27(6):907-922.e5. doi: 10.1016/j.str.2019.03.008. Epub 2019 Apr 4.

Affiliations

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 (PrP^C) serves as precursor to the infectious isoform (PrP^Sc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu²⁺ ions. PrP^C 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 PrP^C. 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.

Keywords: NMR; copper; cross-linking; ion channel; mass spectrometry; molecular dynamics; mutation; patch clamp; prion; protein domain.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Cell Line
Copper / chemistry*
Copper / metabolism
Cross-Linking Reagents / chemistry
Cross-Linking Reagents / metabolism
Humans
Magnetic Resonance Spectroscopy / methods
Mice
Molecular Dynamics Simulation*
Mutation*
Prion Proteins / chemistry*
Prion Proteins / genetics*
Prion Proteins / metabolism
Protein Binding
Protein Domains*
Protein Isoforms / chemistry
Protein Isoforms / genetics
Protein Isoforms / metabolism
Tandem Mass Spectrometry / methods

Substances

Cross-Linking Reagents
Prion Proteins
Protein Isoforms
Copper