分子灵活性在甲基转移酶MLL酶活调控中的作用机制探究

Molecular recognition is predominantly described by two alternative models: induced fit and conformational selection. Understanding and distinguishing the two processes is central to understand enzyme catalysis and regulation at the molecular lever. MLL (Mixed Lineage Leukemia) family of histone methyltransferases are the key players involved in epigenetic regulation. They participate in transcription activation and their malfunctions are closely associated with many human diseases including cancers. It is well known that the enzymatic activity of MLL is precisely regulated by several binding proteins, however, the mode of interaction between MLL and its regulatory proteins is still controversial and the precise mechanism for its activation is not fully resolved. In this proposal, we aim to understand the mode of action describing MLL’s activation using a combination of genetic selection, biochemical, biophysical, and structural biology techniques. We will obtain thermodynamically more stabilized mutants of the catalytic domain of MLL, and from these mutants screen for the ones that are locked in a single conformation using single molecular techniques. By comparing the methyltransferase activity of the mutants and their abilities to interact with MLL’s regulatory proteins, we hope to discover the relationship between stability, structural flexibility, and catalytic activity of the MLL proteins and the mode of action of the interaction between MLL and its regulatory proteins. Our research will not only provide some evidence for the newly established conformational selection model but also shed some light on novel drug design considering MLL as the target.

分子识别中的诱导契合和构象选择模型是目前流行的两种模型，指导着酶催化及调控领域的研究。MLL（Mixed Lineage Leukemia）家族蛋白是一类参与表观遗传调控、与癌症密切相关的组蛋白甲基转移酶，其活性受到若干结合蛋白的精确调控，但MLL和调控蛋白的相互作用究竟遵循哪种分子识别模型至今还存在争议，其催化活性调控的机制还未完全解析。本课题我们拟综合运用生物化学、生物物理、遗传筛选、结构生物学等手段，从提升MLL催化结构域的稳定性着手，分离鉴定具有单一构象的突变体，利用单分子技术考察其结构动态性与催化活性之间的联系，揭示这一类重要酶家族的活化模型，进一步加深对这类酶的性质及其调节规律的理解，希望所得的规律能够被用于指导以MLL为靶点的新型药物研发。同时，本研究将为较为新兴的构象选择模型提供新的实验证据，也为阐释蛋白质的稳定性、结构灵活性及生物活性的相互关系提供一定的帮助。

组蛋白H3K4甲基化是激活基因转录的表观遗传标记，该过程由MLL家族组蛋白H3K4甲基转移酶MLL1-4，SET1A和SET1B催化，其活性依赖于与其他保守蛋白的相互作用。然而，调控MLL活性的机制却一直存在争议。首先，我们利用双底物酶反应动力学等生物化学手段，系统揭示了MLL3和MLL4的SET结构域采用截然不同的催化机制：MLL3遵循快速平衡随机顺序双-双反应机制；MLL4则采用稳态有序顺序双-双反应机制，其中辅因子SAM的结合将MLL4稳定在允许H3结合的活性构象，使其进一步结合H3并催化甲基转移反应，为进一步阐明其在正常细胞中的精确活性调控和异常细胞中的病理机制提供了坚实的分子基础。其次，我们开发了一种体内蛋白质稳定性监测工具——CysGA探针，并利用此工具对MLL3SET进行了定向进化和筛选，获得了11个稳定性增强的MLL3SET突变体，为富集处于不同天然构象的MLLSET结构域奠定了基础。最后，成功建立起了综合利用smFRET、分子动力学模拟等技术考察MLL3SET构象动态性和结构灵活性的方法，区分了MLL3SET的不同构象，并获得了催化活性提升3.28倍的自激活突变体I4801T。同时，分子动力学模拟结果表明其SET-I结构域的灵活性下降。这些证据表明MLL家族蛋白通过SET-I结构域的灵活性严格调控其甲基转移酶活性。这一系列工作将为以MLL家族蛋白及其调控蛋白亚基为靶点的小分子药物设计提供新方向。