From the School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT Leeds, West Yorkshire, United Kingdom,
From the School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT Leeds, West Yorkshire, United Kingdom.
J Biol Chem. 2014 Jun 6;289(23):16399-408. doi: 10.1074/jbc.M113.541250. Epub 2014 May 2.
The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.
液泡型 ATP 酶（V-ATPase）是一种 1MDa 的跨膜质子泵，通过 ATP 驱动的旋转机制运行。对于真核细胞的内稳态至关重要，它在骨重塑和肿瘤侵袭性中发挥核心作用，使其成为一个重要的治疗靶点。它在节肢动物生理学中的重要性也使其成为一种有前途的杀虫剂靶点。设计针对 V-ATPase 的先导化合物的主要挑战是其普遍存在性，因此任何治疗剂都必须能够针对特定的同工酶。在这里，我们已经表征了豌豆白蛋白 1b（PA1b）与 V-ATPase 的结合位点，PA1b 是一种小的半胱氨酸结蛋白，对昆虫 V-ATPase 具有极高的选择性抑制作用。电子显微镜显示，PA1b 的结合发生在膜域 c 环的一系列等效位点上。在 Mg·ATP 的存在下，PA1b 定位到一个远离亚基 a 的单一位点，预测该位点是其他抑制剂的结合界面。光亲和标记研究表明亚基 c 和 e 的放射性标记。此外，象鼻虫对 PA1b 的抗性与 bafilomycin 抗性有关，这是由亚基 c 的突变引起的。这些数据表明存在一个结合位点，亚基 c 和 e 都参与其中，并且抑制作用涉及将 c 环转子锁定在静态的亚基 e 上，而不是亚基 a 上。这对于理解 V-ATPase 的机制以及具有治疗或杀虫剂潜力的抑制剂具有重要意义。它还为复合物中亚基 e 的位置提供了第一个证据。
