Suri Charu, Joshi Harish C, Naik Pradeep Kumar
Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, 173234, Himachal Pradesh, India.
Proteins. 2015 May;83(5):827-43. doi: 10.1002/prot.24773. Epub 2015 Feb 28.
The initiation of microtubule assembly within cells is guided by a cone shaped multi-protein complex, γ-tubulin ring complex (γTuRC) containing γ-tubulin and atleast five other γ-tubulin-complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ-tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/β-tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ-tubulin and GCP4 through computational techniques. We simulated two complexes of γ-tubulin-GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of -158.82 and -170.19 kcal/mol for dimer1 and -79.53 and -101.50 kcal/mol for dimer2 using MM-PBSA and MM-GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ-tubulin. From the results of the free-energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ-tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ-tubulin-GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ-tubulin-GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo-noscapine followed by noscapine and amino-noscapine. This offers a novel chemical scaffold for γ-tubulin binding drugs near γ-tubulin-GCP4 interface.
细胞内微管组装的起始由一种锥形多蛋白复合物——γ-微管蛋白环复合物（γTuRC）引导，γTuRC包含γ-微管蛋白以及至少其他五种γ-微管蛋白复合物蛋白（GCPs），即GCP2、GCP3、GCP4、GCP5和GCP6。γTuRC的边缘是一圈γ-微管蛋白分子，它通过其纵向界面之一与GCP2、GCP3或GCP4相互作用，并通过其他界面与为微管晶格形成而招募的α/β-微管蛋白二聚体相互作用。然而，在缺乏γTuRC亚基功能重组晶体结构的情况下，这些相互作用尚未得到很好的理解。在这项研究中，我们通过计算技术阐明了γ-微管蛋白与GCP4之间的原子相互作用。我们对γ-微管蛋白-GCP4复合物的两种复合物（我们称为二聚体1和二聚体2）进行了25纳秒的模拟，以获得稳定的复合物，并分别使用MM-PBSA和MM-GBSA方法计算出二聚体1的结合自由能的系综平均值为-158.82和-170.19千卡/摩尔，二聚体2的为-79.53和-101.50千卡/摩尔。这些非常有利的结合自由能值表明GCP4与γ-微管蛋白之间存在非常强的相互作用。从自由能分解结果和计算丙氨酸扫描计算中，我们确定了γ-微管蛋白与GCP4相互作用的关键氨基酸，即热点。此外，为了寻找可能在γ-微管蛋白-GCP4复合物界面相互作用的化学先导物；我们发现了一类基于植物生物碱那可丁的化合物，与先前报道的化合物相比，它们在靠近γ-微管蛋白-GCP4界面的一个腔中以高亲和力结合。在整个模拟过程中，所有那可丁类化合物都表现出稳定的相互作用，然而，观察到溴化那可丁的相互作用最强烈，其次是那可丁和氨基那可丁。这为在γ-微管蛋白-GCP4界面附近的γ-微管蛋白结合药物提供了一种新的化学支架。
