Biophysics of Macromolecular Complexes

CTCF和染色质结构。 组蛋白在细胞核内将基因组DNA包装并浓缩成染色质。 蛋白质如CCCTC结合因子（CTCF）通过施加拓扑约束、介导远程基因组相互作用和参与转录事件，通过被动和主动机制帮助指导染色质高阶组织。 CTCF是一种高度保守的DNA结合蛋白，只存在于双侧性动物中。 哺乳动物形式由11个锌指DNA结合结构域组成，两侧是保守的N-末端和C-末端尾，占蛋白质的约57%。 虽然锌指特异性识别DNA基序并与RNA相互作用，但N-和C-末端结构域的作用在很大程度上仍然未知。 这些末端与粘着蛋白相互作用，并帮助稳定描绘拓扑相关的染色质结构域的复合物。 但是，这不可能是他们唯一的作用。 我们已经表明，这些N-和C-末端在溶液中本质上是无序的，许多核和DNA结合蛋白共有的属性。 目前的工作重点是确定结合到CTCF的N-和C-末端的蛋白质伴侣，并研究形成的复合物，以了解CTCF如何调节真核细胞核内的高阶基因组组织。 人类CTCF具有直系同源物CTCFL，主要与精子发生和一些癌症类型相关。 虽然CTCF和CTCFL具有高度保守的11个锌指DNA结合结构域，并识别相同的DNA基序，但它们的N-和C-末端显著不同，这表明这些蛋白质的不同作用来自它们的末端。 类似地，虽然在两侧性动物中和在进化过程中具有核心锌指DNA结合结构域是保守的，但CTCF在门中可能具有不同的末端。 我们有兴趣从选定的物种中表征CTCF的蛋白质伴侣，以进一步剖析蛋白质在组织基因组中所起的多重作用。 具有生物学意义的大分子组装体。 我们利用流体动力学方法，特别是沉降速度和沉降平衡分析离心，以表征关键的生物组件，确定它们的形状和化学计量，并测量它们的相互作用亲和力。 与John Louis（LCP-NIDDK）合作，我们正在研究SARS-CoV-2主要蛋白酶（MPro）的成熟和治疗性共价抑制剂的作用。 MPro的成熟形式作为二聚体起作用，并且对于病毒复制和繁殖是必不可少的。 MPro作为单体形成，是多蛋白链的一部分，自动加工导致其释放和随后的二聚化。 MPro单体的结构研究表明，它采用了天然样折叠，具有未缠绕的含氧阴离子环构象（E），定义了催化非活性状态。 酶促分析表明，这种大部分无活性构象与活性次要构象（E*）处于平衡状态。 分别被批准用于治疗猫和人冠状病毒感染的共价抑制剂GC 373和Nirmatrelvir（NMV）与MPro单体结合。 沉降研究表明，这些抑制剂，特别是NMV，促进MPro的弱二聚化，这需要N-末端残基的存在。 对复合物的结构研究表明，E构象转化为活性形式（E*）类似于成熟的野生型二聚体所观察到的。 N-末端的有组织结构和蛋白质的改善的构象稳定性使NMV复合物易于二聚化。 所观察到的在N-末端的微型前体的自动加工表明，前体中的N-末端切割位点序列的结合稳定了E* 构象，导致N-末端的释放并使MPro倾向于成熟为二聚体。 这些观察结果可能为设计和鉴定更有效的蛋白酶抑制剂提供有价值的工具。

CTCF and chromatin structure. Histone proteins package and condense genomic DNA into chromatin within the cell nucleus. Proteins such as the CCCTC binding factor (CTCF) help direct chromatin higher-order organization through passive and active mechanisms by imposing topological constraints, mediating long-range genomic interactions, and participating in transcriptional events. CTCF is a highly conserved DNA-binding protein found exclusively in bilaterians. The mammalian form consists of an eleven zinc-finger DNA binding domain, flanked by conserved N-terminal and C-terminal tails constituting about 57 percent of the protein. While the zinc fingers specifically recognize DNA motifs and interact with RNA, the roles of the N- and C-terminal domains remain unknown for the most part. These termini interact with cohesin and help stabilize the complexes delineating topologically associated chromatin domains. However, it is unlikely that this is their sole role. We have shown that these N- and C-termini are intrinsically disordered in solution, a property shared by many nuclear and DNA-binding proteins. Current work focuses on identifying protein partners that bind to the N- and C-termini of CTCF and a study of the complexes formed to understand how CTCF regulates higher-order genome organization within the eukaryotic nucleus. Human CTCF has an ortholog CTCFL, primarily associated with spermatogenesis and some cancer types. While CTCF and CTCFL have highly conserved eleven zinc-finger DNA binding domains and recognize identical DNA motifs, they differ significantly in their N- and C-termini suggesting that the diverse roles for these proteins arise from their termini. Similarly, while conserved among bilaterians and across evolution with a core zinc-finger DNA binding domain, CTCF may have divergent termini across phyla. We are interested in characterizing protein partners for CTCF from select species to dissect further the multiple roles that the protein plays in organizing the genome. Macromolecular assemblies of biological interest. We utilize hydrodynamic methods, particularly sedimentation velocity and sedimentation equilibrium analytical centrifugation, to characterize critical biological assemblies, determine their shape and stoichiometry, and measure their interaction affinities. In collaboration with John Louis (LCP-NIDDK), we are studying the maturation of the SARS-CoV-2 main protease (MPro) and the effects of therapeutic covalent inhibitors. The mature form of MPro functions as a dimer and is indispensable for viral replication and propagation. MPro is formed as a monomer, part of a polyprotein chain, and auto-processing leads to its release and subsequent dimerization. Structural studies of the MPro monomer reveal that it adopts a native-like fold with an unwound oxyanion loop conformation (E), defining the catalytically inactive state. Enzymatic assays indicate that this majority inactive conformer is in equilibrium with an active minor conformer (E*). The covalent inhibitors, GC373 and nirmatrelvir (NMV), approved for treating feline and human coronavirus infections, respectively, bind to the MPro monomer. Sedimentation studies indicate that these inhibitors, particularly NMV, promote the weak dimerization of MPro and that this requires the presence of the N-terminal residues. Structural studies on the complex demonstrate the conversion of the E conformation to an active form (E*) resembling that observed for the mature wild-type dimer. The N-terminus's organized structure and the protein's improved conformational stability predispose the NMV complex to dimerize. The observed auto-processing of a mini-precursor at the N-terminus indicates that binding of the N-terminal cleavage site sequence in the precursor stabilizes the E* conformation, leading to the liberation of the N-terminus and predisposing MPro for maturation as a dimer. These observations may provide a valuable tool for designing and identifying more effective protease inhibitors.