Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity.
Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity.
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DOI：
10.1042/bcj20210288
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发表时间：
2021-09-17
影响因子：
4.1
通讯作者：
Chou, Tsui-Fen
中科院分区：
文献类型：
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作者：
Zhang, Xiaoyi;Gui, Lin;Li, Shan;Nandi, Purbasha;Columbres, Rod Carlo;Wong, Daniel E.;Moen, Derek R.;Lin, Henry J.;Chiu, Po-Lin;Chou, Tsui-Fen
p97蛋白是高度保守、丰富、功能多样、结构动态的含有N、D1和D2结构域的同六聚体AAA酶。含有N和D1结构域和D1-D2接头（ND 1 L）的截短的p97蛋白表现出野生型（WT）ATP酶活性的79%，而没有接头的单独ND 1结构域仅具有WT活性的2%。为了研究D1-D2接头和D1结构域之间的关系，我们产生了p97 ND 1 L突变体，并证明了这个22个残基的接头区域对D1 ATP酶活性是必需的。保守氨基酸亮氨酸464（L464）对于通过p97辅因子p37、p47和Npl 4-Ufd 1（NU）调节D1和D2 ATP酶活性是关键的。改变亮氨酸丙氨酸，脯氨酸，或谷氨酸增加的最大ATP周转率（kcat）的p47调节的ATP酶活性的突变体，但不是野生型。p37和p47增加了脯氨酸取代的接头的kcat，表明它们诱导接头构象，促进ATP水解。NU抑制WT和突变ND 1 L蛋白的D1 ATP酶活性，但激活全长p97的D2 ATP酶活性。为了进一步理解突变机制，我们使用单颗粒cryo-EM来可视化全长p97 L464 P，并揭示D1-D2接头的构象变化，导致螺旋-转角-螺旋基序的移动（543-569）。结合生物化学和结构结果，我们得出结论，接头有助于维持D1处于活性构象，并将N结构域与D1和D2 ATP酶结构域之间的通信传递到约50 nm处。
p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543–569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ~50 Å away.