The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97

J Biol Chem. 2015 Dec 4;290(49):29414-27. doi: 10.1074/jbc.M115.680686. Epub 2015 Oct 16.

Abstract

Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors.

Keywords: ATPase; UBXD1; endosome; nuclear magnetic resonance (NMR); p97; protein structure; protein-protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Motifs
  • Autophagy-Related Proteins
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism*
  • Caveolin 1 / metabolism
  • Cell Cycle Proteins / metabolism*
  • Cell Line
  • Circular Dichroism
  • Endosomes / metabolism
  • Epitopes / chemistry
  • Fluorescence Polarization
  • Green Fluorescent Proteins / metabolism
  • Homeostasis
  • Humans
  • Lysosomes / metabolism
  • Magnetic Resonance Spectroscopy
  • Mutation
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Ubiquitin / chemistry
  • Valosin Containing Protein

Substances

  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Autophagy-Related Proteins
  • Carrier Proteins
  • Caveolin 1
  • Cell Cycle Proteins
  • Epitopes
  • Nuclear Proteins
  • UBXN6 protein, human
  • Ubiquitin
  • Green Fluorescent Proteins
  • Adenosine Triphosphatases
  • VCP protein, human
  • Valosin Containing Protein

Associated data

  • PDB/1E32