Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, 500046, India.
Sci Rep. 2021 Nov 30;11(1):23145. doi: 10.1038/s41598-021-01436-7.
STAT3, an important transcription factor constitutively activated in cancers, is bound specifically by GRIM-19 and this interaction inhibits STAT3-dependent gene expression. GRIM-19 is therefore, considered as an inhibitor of STAT3 and may be an effective anti-cancer therapeutic target. While STAT3 exists in a dimeric form in the cytoplasm and nucleus, it is mostly present in a monomeric form in the mitochondria. Although GRIM-19-binding domains of STAT3 have been identified in independent experiments, yet the identified domains are not the same, and hence, discrepancies exist. Human STAT3-GRIM-19 complex has not been crystallised yet. Dictated by fundamental biophysical principles, the binding region, interactions and effects of hotspot mutations can provide us a clue to the negative regulatory mechanisms of GRIM-19. Prompted by the very nature of STAT3 being a challenging molecule, and to understand the structural basis of binding and interactions in STAT3α-GRIM-19 complex, we performed homology modelling and ab-initio modelling with evolutionary information using I-TASSER and avant-garde AlphaFold2, respectively, to generate monomeric, and subsequently, dimeric STAT3α structures. The dimeric form of STAT3α structure was observed to potentially exist in an anti-parallel orientation of monomers. We demonstrate that during the interactions with both unphosphorylated and phosphorylated STAT3α, the NTD of GRIM-19 binds most strongly to the NTD of STAT3α, in direct contrast to the earlier works. Key arginine residues at positions 57, 58 and 68 of GRIM-19 are mainly involved in the hydrogen-bonded interactions. An intriguing feature of these arginine residues is that these display a consistent interaction pattern across unphosphorylated and phosphorylated monomers as well as unphosphorylated dimers in STAT3α-GRIM-19 complexes. MD studies verified the stability of these complexes. Analysing the binding affinity and stability through free energy changes upon mutation, we found GRIM-19 mutations Y33P and Q61L and among GRIM-19 arginines, R68P and R57M, to be one of the top-most major and minor disruptors of binding, respectively. The proportionate increase in average change in binding affinity upon mutation was inclined more towards GRIM-19 mutants, leading to the surmise that GRIM-19 may play a greater role in the complex formation. These studies propound a novel structural perspective of STAT3α-GRIM-19 binding and inhibitory mechanisms in both the monomeric and dimeric forms of STAT3α as compared to that observed from the earlier experiments, these experimental observations being inconsistent among each other.
STAT3 是一种在癌症中持续激活的重要转录因子，它特异性地与 GRIM-19 结合，这种相互作用抑制了 STAT3 依赖性基因表达。因此，GRIM-19 被认为是 STAT3 的抑制剂，可能是一种有效的抗癌治疗靶点。虽然 STAT3 以二聚体形式存在于细胞质和细胞核中，但它主要以单体形式存在于线粒体中。尽管已经在独立的实验中鉴定了 STAT3 的 GRIM-19 结合结构域，但鉴定的结构域并不相同，因此存在差异。尚未结晶人类 STAT3-GRIM-19 复合物。受基本生物物理原理的启发，热点突变的结合区域、相互作用和影响可以为我们提供 GRIM-19 负调控机制的线索。由于 STAT3 本身就是一个具有挑战性的分子，为了了解 STAT3α-GRIM-19 复合物的结合和相互作用的结构基础，我们分别使用 I-TASSER 和先进的 AlphaFold2 基于进化信息进行同源建模和从头建模，以生成单体和随后的二聚体 STAT3α 结构。观察到 STAT3α 二聚体结构可能以单体的反平行取向存在。我们证明，在与未磷酸化和磷酸化的 STAT3α 的相互作用过程中，GRIM-19 的 NTD 与 STAT3α 的 NTD 结合最强，这与早期的工作形成鲜明对比。GRIM-19 中位置 57、58 和 68 的关键精氨酸残基主要参与氢键相互作用。这些精氨酸残基的一个有趣特征是，它们在未磷酸化和磷酸化的单体以及 STAT3α-GRIM-19 复合物中的未磷酸化二聚体中表现出一致的相互作用模式。MD 研究验证了这些复合物的稳定性。通过突变时自由能变化分析结合亲和力和稳定性，我们发现 GRIM-19 突变 Y33P 和 Q61L 以及 GRIM-19 精氨酸中的 R68P 和 R57M 分别是结合的主要和次要破坏者之一。突变后结合亲和力的平均变化比例更倾向于 GRIM-19 突变体，这表明 GRIM-19 可能在复合物形成中发挥更大的作用。与早期实验观察到的相比，这些研究提出了 STAT3α-GRIM-19 结合和抑制机制的新结构视角，无论是在 STAT3α 的单体还是二聚体形式中，这些实验观察结果相互不一致。
