Structural Biophysics Laboratory, Center for Cancer Research , National Cancer Institute , Frederick , Maryland 21702-1201 , United States.
Laboratory of Cellular and Molecular Biology, Center for Cancer Research , National Cancer Institute , Bethesda , Maryland 20892 , United States.
Biochemistry. 2019 Mar 12;58(10):1423-1431. doi: 10.1021/acs.biochem.8b01323. Epub 2019 Feb 20.
Lipidated small GTP-binding proteins of the Arf family interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Here, we focus on the ADP-ribosylation factor 1 (Arf1), which interacts with numerous proteins in the Arf pathway, such as the ArfGAP ASAP1 that is highly expressed and activated in several cancer cell lines and associated with enhanced migration, invasiveness, and poor prognosis. Understanding the molecular and mechanistic details of Arf1 regulation at the membrane via structural and biophysical studies requires large quantities of fully functional protein bound to lipid bilayers. Here, we report on the production of a functional human Arf1 membrane platform on nanodiscs for biophysical studies. Large scale bacterial production of highly pure, N-myristoylated human Arf1 has been achieved, including complex isotopic labeling for nuclear magnetic resonance (NMR) studies, and the myr-Arf1 can be readily assembled in small nanoscale lipid bilayers (nanodiscs, NDs). It is determined that myr-Arf1 requires a minimum binding surface in the NDs of ∼20 lipids. Fluorescence and NMR were used to establish nucleotide exchange and ArfGAP-stimulated GTP hydrolysis at the membrane, indicating that phophoinositide stimulation of the activity of the ArfGAP ASAP1 is ≥2000-fold. Differences in nonhydrolyzable GTP analogues are observed, and GMPPCP is found to be the most stable. Combined, these observations establish a functional environment for biophysical studies of Arf1 effectors and interactions at the membrane.
Arf 家族的脂化小分子 GTP 结合蛋白与多种细胞伴侣和膜相互作用，以调节细胞内运输和细胞器结构。在这里，我们重点关注 ADP-核糖基化因子 1（Arf1），它与 Arf 途径中的许多蛋白质相互作用，如在几种癌细胞系中高度表达和激活的 ArfGAP ASAP1，与增强的迁移、侵袭和预后不良相关。通过结构和生物物理研究了解 Arf1 在膜上的分子和机制细节，需要大量与脂质双层结合的完全功能性蛋白质。在这里，我们报告了在纳米盘上生产功能性人 Arf1 膜平台用于生物物理研究。已经实现了高度纯的、N-豆蔻酰化的人 Arf1 的大规模细菌生产，包括用于核磁共振（NMR）研究的复杂同位素标记，并且可以轻松地将 myr-Arf1 组装到小纳米尺度脂质双层（纳米盘，NDs）中。确定 myr-Arf1 在 NDs 中需要至少 20 个脂质的结合表面。荧光和 NMR 用于确定在膜上进行核苷酸交换和 ArfGAP 刺激的 GTP 水解，表明磷酸肌醇对 ArfGAP ASAP1 活性的刺激作用≥2000 倍。观察到非水解 GTP 类似物的差异，并且发现 GMPPCP 最稳定。综合这些观察结果，建立了一个功能性环境，用于研究膜上 Arf1 效应物和相互作用的生物物理。
