Division of Medical Genetics, Department of Pediatrics, Harbor-UCLA Medical Center and The Lundquist Institute, Torrance, California 90502, U.S.A.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, U.S.A.
Biochem J. 2021 Sep 17;478(17):3185-3204. doi: 10.1042/BCJ20210288.
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.
p97 蛋白是一种高度保守、丰富、功能多样、结构动态的同六聚体 AAA 酶，包含 N、D1 和 D2 结构域。一种包含 N 和 D1 结构域以及 D1-D2 接头（ND1L）的截短 p97 蛋白表现出 79%的野生型（WT）ATP 酶活性，而单独的 D1 结构域没有接头仅有 2%的 WT 活性。为了研究 D1-D2 接头与 D1 结构域之间的关系，我们生成了 p97 ND1L 突变体，并证明这个 22 个残基的接头区域对于 D1 ATP 酶活性是必需的。保守的氨基酸亮氨酸 464（L464）对于 p97 辅助因子 p37、p47 和 Npl4-Ufd1（NU）调节 D1 和 D2 ATP 酶活性是至关重要的。将亮氨酸突变为丙氨酸、脯氨酸或谷氨酸会增加这些突变体 p47 调节的 ATP 酶活性的最大速率（kcat），但对 WT 没有影响。p37 和 p47 增加了脯氨酸取代接头的 kcat，表明它们诱导了有利于 ATP 水解的接头构象。NU 抑制 WT 和突变 ND1L 蛋白的 D1 ATP 酶活性，但激活全长 p97 的 D2 ATP 酶活性。为了进一步了解突变体机制，我们使用单颗粒冷冻电镜可视化全长 p97L464P，并揭示了 D1-D2 接头的构象变化，导致螺旋-转角-螺旋模体（543-569）的运动。综合生物化学和结构结果，我们得出结论，接头有助于将 D1 维持在有能力的构象中，并将通讯从 N 结构域传递到 D1 和 D2 ATP 酶结构域，这两个结构域之间的距离约为 50 Å。
