Ali Bashir A, Judy Ryan M, Chowdhury Saikat, Jacobsen Nicole K, Castanzo Dominic T, Carr Kaili L, Richardson Chris D, Lander Gabriel C, Martin Andreas, Gardner Brooke M
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
bioRxiv. 2023 Sep 16:2023.09.15.557798. doi: 10.1101/2023.09.15.557798.
The heterohexameric AAA-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N-terminally bound co-factors. Here we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, . We found that although Pex1/ΔN1-Pex6 is an active ATPase , it does not support Pex1/Pex6 function at the peroxisome . An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N terminal domains of PEX1, CDC48, or NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of co-factors and substrates with Pex1/Pex6 ATPase activity.
异源六聚体AAA-ATP酶Pex1/Pex6对于过氧化物酶体的形成和维持至关重要。与其他AAA-ATP酶类似，Pex1/Pex6利用ATP水解产生的能量，通过其中心孔机械地穿入底物蛋白，从而使其展开。在相关的AAA-ATP酶马达中，底物通过与马达的N端结构域或N端结合的辅助因子结合而被招募。在这里，我们使用结构和生化技术来表征出芽酵母中Pex6的N1结构域的功能。我们发现，尽管Pex1/ΔN1-Pex6是一种活性ATP酶，但它在过氧化物酶体中不支持Pex1/Pex6的功能。分离的Pex6 N1结构域的X射线晶体结构表明，尽管序列保守性较差，但Pex6 N1结构域与PEX1、CDC48或NSF的N端结构域具有相同的折叠。将该结构与Pex1/Pex6的冷冻电镜重建、AlphaFold2预测和生化分析相结合表明，Pex6 N1介导与过氧化物酶体膜系链蛋白Pex15以及Pex1的D2 ATP酶结构域的一个延伸环的结合，该环影响Pex1/Pex6异源六聚体的稳定性。鉴于与Pex15和D2 ATP酶结构域都有直接相互作用，Pex6 N1结构域有望协调辅助因子和底物与Pex1/Pex6 ATP酶活性的结合。
