title: The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97.
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.
authors.affiliation: From Structural and Medicinal Biochemistry and.
authors.forename: Franziska
authors.initials: F
authors.lastname: Trusch
authors.affiliation: From Structural and Medicinal Biochemistry and.
authors.forename: Anja
authors.initials: A
authors.lastname: Matena
authors.affiliation: Molecular Biology I, Centre for Medical Biotechnology (ZMB), University of Duisburg-Essen, 45117 Essen, Germany and.
authors.forename: Maja
authors.initials: M
authors.lastname: Vuk
authors.affiliation: Molecular Biology I, Centre for Medical Biotechnology (ZMB), University of Duisburg-Essen, 45117 Essen, Germany and.
authors.forename: Lisa
authors.initials: L
authors.lastname: Koerver
authors.affiliation: Molecular Biology I, Centre for Medical Biotechnology (ZMB), University of Duisburg-Essen, 45117 Essen, Germany and.
authors.forename: Helene
authors.initials: H
authors.lastname: Knævelsrud
authors.affiliation: Department of Medicine, Section of Structural Biology, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Freemont
authors.affiliation: Molecular Biology I, Centre for Medical Biotechnology (ZMB), University of Duisburg-Essen, 45117 Essen, Germany and.
authors.forename: Hemmo
authors.initials: H
authors.lastname: Meyer
authors.affiliation: From Structural and Medicinal Biochemistry and peter.bayer@uni-due.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Bayer
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D020816:Amino Acid Motifs; D000071183:Autophagy-Related Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005454:Fluorescence Polarization; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000737:chemistry; D000074405:Valosin Containing Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D051242:Caveolin 1; D018797:Cell Cycle Proteins; D000939:Epitopes; D009687:Nuclear Proteins; C533572:UBXN6 protein, human; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein
keywords: ATPase; UBXD1; endosome; nuclear magnetic resonance (NMR); p97; protein structure; protein-protein interaction