LIMES Institute, Chemical Biology and Medicinal Chemistry Unit, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
J Am Chem Soc. 2011 Jun 1;133(21):8372-9. doi: 10.1021/ja202513s. Epub 2011 May 4.
A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.
描述了一种用于实时监测交换因子催化激活类似 ras 的小 GTP 酶的均一荧光共振能量转移 (FRET) 系统。该设计基于 GTPase 腺苷二磷酸核糖基化因子 (ARF) 与其效应蛋白 GGA3 之间的蛋白质-蛋白质相互作用所产生的超分子模板效应。当与鸟苷三磷酸 (GTP) 结合时，GTPase 被激活，而在其鸟苷二磷酸 (GDP) 结合状态下失活。这两种状态都伴随着严重的构象变化，这些变化被 GGA3 识别，GGA3 仅结合 GTPase 的“开启”状态。GDP 到 GTP 的交换，即 GTPase 激活，由鸟嘌呤核苷酸交换因子细胞溶质素-2 催化。当 GGA3 和 GTPase ARF1 用经过精心选择的 FRET 探针标记，并同时记录 ARF1 中内部色氨酸残基的荧光时，可以实时监测 GTPase 激活过程中的构象变化。我们将 FRET 系统应用于一种多重格式，允许同时识别和区分小分子抑制剂，这些抑制剂干扰细胞溶质素催化的 ARF1 激活和/或激活的 ARF1-GTP 与 GGA3 之间的相互作用。通过筛选计算机建模预测的潜在细胞溶质素抑制剂文库，我们鉴定了新的 ARF1 上细胞溶质素催化的 GDP/GTP 交换抑制剂，并在细胞增殖测定中验证了它们的增强效力。
