Structural Biology of Long Poly-Zinc Finger Proteins

长聚锌指蛋白的结构生物学

• 批准号: RGPIN-2022-04047
• 负责人: Lavigne, Pierre
• 金额: $ 2.91万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744354
• 关键词: Structural; Biology; Long; Poly; Zinc

The C2H2 Zinc Finger (ZF) is a highly conserved protein domain involved in the sequence specific DNA recognition of transcription factors. More than 700 human genes possess at least one, with an average of 8 consecutive ZFs per gene. ZFs are separated by the conserved linker: TGEKP-(Y/F). This linker adopts an extended conformation and allows the establishment of the proper inter-ZF interface to fit in the major groove of DNA and molecular recognition. As part of our long term goal to comprehend the effect of divergences in the primary structure on the structural biology of long poly-ZF proteins, we have recently made important discoveries about the impact of non-canonical linkers on the mode of DNA binding and scanning. Briefly, we have revealed that the occurrence of phosphomimetics (D) in place of the T and of an H in place of the Y/F leads to the reversible folding of the linker into an a-helical conformation and a large-scale modulation of the inter-ZF interface. Collectively, these phenomena have a profound impact on the DNA binding affinity and mode of binding. Indeed, on the one hand, the helical conformation of the linker allows for local dissociation of ZFs on DNA and on the other hand the modulation of the inter-ZF interface allows for the recognition of bent (or coiled) conformations of DNA. Here, we will continue our long term goal by addressing: 1- how the phosphorylation of the T in the linkers by CDK1 leads to the eviction of all poly-ZF transcription factors from DNA at mitosis and 2- the impact of the evolutionary conserved and bulky Tryptophan residues in the recognition helix and at the inter-ZF interface of Krab domain poly-ZF proteins (KZNFP). KZNFPs (e.g. ZNF91) spread heterochromatin by the provoking methylation of DNA (methylation of cytosines: meC) and histones to control tissue specific gene expression and the epigenetic silencing of transposable elements such as SVAs. More specifically, we propose a series of specific objectives and training opportunities to validate the following hypotheses: 1- That the phosphorylation of the two linkers of the three ZFs of SP1 will lead to the complete loss of affinity of its cognate DNA sequence do the local dissociate of ZFs 1 and 3 due to the stabilization of the linkers in an a-helical conformation and 2- the Trp residues conserved in ZNF91 are meC readers and can stabilize methylated DNA in a bent (or coiled) conformation to promote the formation nucleosomes. The methodology will consist in established protocols in our laboratory and includes NMR-based structural and dynamical characterization of these phenomena as well as a quantitative thermodynamical characterization of these binding events. Validation of these hypotheses will significantly impact on our understanding of the structural biology of poly-ZF proteins as well as to provide a solid inter-disciplinary training environment to prepare HQPs for a career in fundamental research in all sectors of natural sciences.

C2 H2锌指（ZF）是一个高度保守的蛋白质结构域，参与转录因子的序列特异性DNA识别。超过700个人类基因拥有至少一个ZF，每个基因平均有8个连续的ZF。ZF由保守的接头TGEKP-（Y/F）分开。该接头采用延伸的构象，并允许建立适当的ZF间界面以适合DNA的大沟和分子识别。作为我们长期目标的一部分，以了解分歧的一级结构上的长poly-ZF蛋白质的结构生物学的影响，我们最近取得了重要的发现，非规范的连接器的影响的模式的DNA结合和扫描。简而言之，我们已经揭示了磷酸模拟物（D）代替T和H代替Y/F的出现导致接头可逆折叠成α-螺旋构象和ZF间界面的大规模调节。总的来说，这些现象对DNA结合亲和力和结合模式具有深远的影响。事实上，一方面，接头的螺旋构象允许DNA上ZF的局部解离，另一方面，ZF间界面的调节允许DNA的弯曲（或卷曲）构象的识别。在这里，我们将通过解决以下问题继续我们的长期目标：1-CDK 1对接头中的T的磷酸化如何导致在有丝分裂时从DNA中驱逐所有聚ZF转录因子，以及2-在识别螺旋中和在Krab结构域聚ZF蛋白（KZNFP）的ZF间界面处进化保守和庞大的色氨酸残基的影响。KZNFP（例如ZNF 91）通过激发DNA甲基化（胞嘧啶甲基化：meC）和组蛋白来扩散异染色质，以控制组织特异性基因表达和转座因子（例如SVAs）的表观遗传沉默。更具体地说，我们提出了一系列具体目标和培训机会，以验证以下假设：1-SP1的三个ZF的两个接头的磷酸化将导致其同源DNA序列的亲和力的完全丧失，这是由于接头在α-螺旋构象中的稳定化导致ZF 1和3的局部解离，2-在ZNF 91中保守的Trp残基是meC阅读器，并且可以稳定甲基化的DNA处于弯曲（或卷曲）构象以促进核小体的形成。该方法将包括在我们的实验室建立的协议，并包括基于NMR的结构和动力学表征这些现象，以及这些结合事件的定量化学表征。这些假设的验证将极大地影响我们对多聚ZF蛋白结构生物学的理解，并提供坚实的跨学科培训环境，为HQP从事自然科学各个领域的基础研究做好准备。