MOLECULAR MODEL OF AN ACTIN FILAMENT CAPPED BY SEVERING PROTEIN, GELSOLIN

明胶蛋白是一种调节肌动蛋白的6个结构域蛋白(82 KDa) 通过切断、覆盖和使细丝成核来组装。我们有 G2-6，a修饰的F-肌动蛋白的电子冷冻显微镜观察 缺乏高亲和力单体结合的明胶蛋白缺失突变体 有效切断所需的域。电子 钙离子修饰G2-6的F-肌动蛋白的冷冻显微镜观察 EGTA，一种表现出正常切断活动的情况，显示 细丝的戏剧性扭曲似乎与 切断活动。G2-6：F-肌动蛋白的三维重建 是在允许切断的条件下获得的，但价格较低 效率。结构表明，明胶连接了两个 当它结合细丝时，纵向缔合的单体。这个 F-肌动蛋白结合部位在3亚区轴向居中，径向居中 在上游肌动蛋白单体的亚区1和3之间。这两个 装饰细丝的变形外观和位置 G2-6密度表明，为了使G4结构域与肌动蛋白和 切断细丝，明胶蛋白和肌动蛋白都会经历 构象变化。

Gelsolin is a six domain protein (82 kDa) which regulates actin assembly by severing, capping, and nucleating filaments. We have performed electron cryomicroscopy of F-actin decorated with G2-6, a gelsolin deletion mutant which lacks the high affinity monomer binding domain that is required for efficient severing. Electron cryomicrographs of F-actin decorated with G2-6 in calcium, but not in EGTA, a condition that exhibits normal severing activity, show dramatic distortions of filaments which appear to correlate with severing activity. A three-dimensional reconstruction of G2-6:F-actin was obtained under conditions which permit severing, but at a lower efficiency. The structure shows that gelsolin bridges two longitudinally-associated monomers when it binds the filament. The F-actin binding site is centered axially at subdomain 3 and radially between subdomains 1 and 3 of the upper actin monomer. Both the distorted appearance of the decorated filaments and the location of the G2-6 density suggest that in order for domain G4 to bind actin and sever the filament, both gelsolin and actin undergo large conformational changes.