Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India.
J Struct Biol. 2011 Oct;176(1):97-111. doi: 10.1016/j.jsb.2011.07.011. Epub 2011 Jul 26.
Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹⁵N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed β-sheet comprised of six β-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.
刚地弓形虫 ADF（TgADF）属于一种功能亚型，其特征是具有强大的 G-肌动蛋白隔离活性和低的 F-肌动蛋白切割活性。在已鉴定的 ADF/丝切蛋白中，TgADF 具有最短的长度，并且缺少一个与 F-肌动蛋白结合有关的 C 端螺旋。为了了解其特征性质，我们已经确定了 TgADF 的溶液结构，并通过 ¹⁵N 弛豫测量研究了其骨架动力学。TgADF 具有保守的 ADF/丝切蛋白折叠结构，由一个中央混合 β-片层组成，该片层由六个 β-链组成，部分被三个 α-螺旋和 C 端螺旋转角包围。TgADF 具有很高的 G-肌动蛋白隔离活性，这依赖于 G-肌动蛋白与 TgADF 的 G-肌动蛋白结合表面之间高度结构和动态优化的相互作用。通过 ITC 测量，TgADF 与兔肌肉 G-肌动蛋白的平衡解离常数为 23.81 nM，这反映了 TgADF 与 G-肌动蛋白相互作用的非常强的亲和力。TgADF 的 F-肌动蛋白结合位点部分形成，具有缩短的 F 环，该环不伸出椭圆结构，并且 C 端螺旋取代 C 端螺旋 α4。然而，它比利什曼原虫 cofilin 的 F-肌动蛋白结合位点更刚性。实验观察和结构特征不支持 PIP2 与 TgADF 的相互作用，并且 PIP2 不影响 TgADF 与 G-肌动蛋白的相互作用。总的来说，这项研究表明，G-肌动蛋白结合位点的构象灵活性增强了 TgADF 与 G-肌动蛋白的亲和力，而传统的 ADF/丝切蛋白的 F-肌动蛋白结合位点的构象刚性对于与 F-肌动蛋白的稳定结合是必要的。
