Trusch Franziska, Matena Anja, Vuk Maja, Koerver Lisa, Knævelsrud Helene, Freemont Paul S, Meyer Hemmo, Bayer Peter
From Structural and Medicinal Biochemistry and.
Molecular Biology I, Centre for Medical Biotechnology (ZMB), University of Duisburg-Essen, 45117 Essen, Germany and.
J Biol Chem. 2015 Dec 4;290(49):29414-27. doi: 10.1074/jbc.M115.680686. Epub 2015 Oct 16.
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.
含缬酪肽蛋白/p97是一种由ATP驱动的蛋白分选酶，它与不同的蛋白辅因子协同作用，以控制细胞稳态的各个方面。调节性N结构域与两个ATP酶结构域（D1和D2）中的第一个之间的界面处发生的突变会使ATP酶活性失调，并导致一种多系统退行性疾病，即与骨Paget病和额颞叶痴呆/肌萎缩侧索硬化相关的包涵体肌病。有趣的是，这些突变仅影响p97介导的部分途径，这与辅因子相互作用失衡相关，并且在小窝蛋白-1的内溶酶体分选过程中，最显著受损的是含泛素调节X结构域蛋白1（UBXD1）辅因子的结合。然而，迄今为止，这些突变如何影响p97与辅因子的相互作用尚不清楚。在基于细胞的内体定位研究中，我们确定了UBXD1的N端区域（UBXD1-N）的关键作用。使用核磁共振（NMR）和圆二色光谱（CD）的生物物理研究表明，UBXD1-N可归类为内在无序蛋白。NMR滴定实验证实了含缬酪肽蛋白/p97的相互作用基序，并确定UBXD1-N的螺旋1和螺旋2处的第二个结合位点为p97的结合界面。在反向滴定实验中，我们在p97 N结构域上确定了两个相距较远的表位，其中包括与疾病相关的残基，并且通过荧光各向异性证实了UBXD1-N与p97的D1D2桶之间存在额外的相互作用。在功能上，UBXD1-N与p97的结合导致ATP酶活性降低，并提供部分抗蛋白酶解保护。这些发现表明，UBXD1-N插入到p97-ND1界面中，从而调节p97结构域之间的结构域间通讯及其与疾病发病机制相关的活性。我们提出，以UBXD1-N为特征的多价结合模式是定义p97辅因子子集的更普遍原则。
