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.
Highlights
Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis
P97 and ubiquitin regulatory X domain-containing protein 1 (UBXD1) are recruited to CAV1-containing endosomes, which can be stimulated by CAV1 overexpression [14, 21, 22]
CAV1-HA was detected by immunofluorescence with HA antibodies, and colocalization of CAV1-HA and UBXD1-GFP was visualized by epifluorescence microscopy (Fig. 1C)
Summary
Cell-based Experiments—The human CAV1-HA and UBXD1-GFP constructs were described elsewhere [14, 21, 22]. pEGFP-UBXD1–5xPUB-GFP was generated by QuikChange mutagenesis, resulting in the following codon changes: N184A, K193A, Y194A, K198A, and N201A in the PUB domain as described elsewhere [20]. pEGFP-UBXD1-RL-GFP was mutated to code for changes R62A/L63A in the VIM as defined by Stapf et al [23]. Cell-based Experiments—The human CAV1-HA and UBXD1-GFP constructs were described elsewhere [14, 21, 22]. Plasmid Constructs for Recombinant Protein Expression— Different UBXD1 constructs (amino acids 1–133, 32–133, and 1– 80) and the D1D2 domain of p97 (amino acids 200 – 806) were cloned into a modified pET41 vector (Invitrogen) as described elsewhere [24] with an N-terminal GST tag and a PreScission protease cleavage site. The UBXD1 constructs were separated from the GST tag by size exclusion chromatography (Superdex 75 26/600 column, GE Healthcare) combined with a glutathione column in phosphate buffer (50 mM KPi, 150 mM KCl, pH 6.5). The measurements were done in phosphate buffer (50 mM KPi, 0.05% Tween, pH 7.2) at 25 °C on a Cary Eclipse fluorescence spectrometer (Agilent Technologies, Waldbronn, Germany). After a 2-h incubation at 37 °C, each sample was mixed with SDS loading buffer and loaded for SDS-PAGE
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