Department of Biochemistry, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, United Kingdom.
J Biol Chem. 2011 Nov 4;286(44):38670-38678. doi: 10.1074/jbc.M111.274472. Epub 2011 Sep 6.
Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatory X (UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX(5)AAX(2)R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors.
必需的泛素选择性 AAA ATP 酶 p97/包含缬氨酸的蛋白（VCP）的细胞功能受决定底物特异性和命运的调节辅因子控制。大多数辅因子通过泛素调节 X（UBX）或 UBX 样结构域或线性序列基序与 p97 结合，包括迄今尚未明确的 p97/VCP 相互作用基序（VIM）。在这里，我们提出了新的最小共识序列 RX(5)AAX(2)R，作为 VIM 的通用定义，它将包括已知和假定的 p97 辅因子在内的新家族统一起来，其中包括 UBXD1 和 ZNF744/ANKZF1。我们证明这个最小的 VIM 共识序列是 p97 结合所必需的和充分的。使用 NMR 化学位移映射，我们确定了 p97 N 端结构域（N 结构域）的几个关键残基对 VIM 结合至关重要。重要的是，我们表明，酵母 VIM 包含的辅因子 Vms1 的细胞应激抗性取决于其 VIM 与酵母 p97 同源物 Cdc48 的关键 N 结构域残基之间的物理相互作用。因此，本研究中表征的 VIM-N 结构域相互作用对于 Vms1 及其可能的其他新定义的 VIM 家族辅因子成员的生理功能是必需的。
