【作者】 陈泉；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2007， 博士

【摘要】 核磁共振（Nuclear Magnetic Resonance，NMR）方法是分子结构分析中不可或缺的研究手段，与X射线晶体衍射构成生物大分子三维空间结构测定的两大互补方法。随着生命科学的飞速发展，在基因组基础上研究蛋白质结构、功能，尤其是其相互作用网络，已成为后基因组时代生命科学新的前沿。分子水平蛋白质相互作用的研究对于理解许多生物学过程至关重要，在应用方面对于人类疾病分子机理的理解和药物设计有着重要的指导意义和经济价值，核磁共振方法在研究这类溶液状态下原子水平的蛋白质和其他分子相互作用及不同强度和不同时间尺度的动力学方面有着其显著的优势。本论文工作的重点是用异核多维核磁共振方法测定蛋白质-小肽配体复合物结构。我们表达纯化了人AF-6蛋白，通过NMR方法测定了AF-6蛋白PDZ结构域与Bcr蛋白C-末端小肽复合物的溶液结构，并在此基础上对两者相互作用的方式进行了探讨。我们还通过主链动力学研究发现AF-6 PDZ在配体结合前后ms-μs尺度上并无明显变化，根据相关时间推测AF-6 PDZ具有一定的二聚倾向，但并不影响结构解析。此外，我们还克隆表达纯化了人SUMO-2和SUMO-3全长蛋白，以及SUMO共价修饰体系中的酶E1（SAE1／2）和E2（Ubc9），研究了它们之间的相互作用，并尝试搭建体外共价修饰系统以获得共价修饰的复合物从而进行核磁结构解析。论文分为以下三个部分：第一章综述了各种用于研究蛋白质复合物结构的核磁共振方法，其中很多技术也适用于其他系统，如蛋白质—核酸、蛋白—糖、蛋白—脂、蛋白—药物复合物。这些技术的选择取决于以下因素：分子的大小和类型；结合平衡常数（紧结合或弱结合）；结合动力学（快交换或慢交换）；化学计量和对称性。对于不同的情况，采用不同的策略及实验方法。此部分首先介绍了研究复合物前对样品条件的优化方法：描述了在复合物的整体结构不能或不太容易确定的情况下对相互作用界面的界定：接着介绍了复合物谱峰认证的实验方法，这与标记单个蛋白质的谱峰认证完全相同；列举了X-filter NMR实验的不同类型，X-filter实验被广泛应用于选择性区分分子间或分子内的结构信息；最后介绍了如何运用这些信息进行结构计算。第二章是人AF-6蛋白PDZ结构域与Bcr蛋白C-末端小肽复合物的溶液结构及其动力学研究。首先对本工作的研究背景，即AF-6蛋白及PDZ结构域的功能和结构、Bcr蛋白的功能和结构进行了全面的介绍。AF-6基因最早是在急性淋巴性白血病人中作为ALL-1的融合配体被发现的。随后的研究表明AF-6是信号蛋白Ras和Rap1的效应蛋白，在细胞连接和信号转导中起着重要作用。AF-6可以通过其C-末端的PDZ结构域结合Bcr，而通过其N-末端的RBD结构域结合Ras，从而形成一个三元复合物，调控细胞—细胞连接处Ras介导的信号转导途径。在正常细胞中，Bcr可以磷酸化AF-6，使得AF-6通过其PDZ结构域与Bcr中的C-末端部分结合，此相互作用可以提高AF-6 RBD结构域对Ras的亲和性，通过竞争使Ras不再与Raf结合，从而下调Raf／MEK／ERK信号转导途径，使蛋白激酶级联反应失活。通过异核多维核磁共振的方法，我们获得了AF-6蛋白PDZ结构域与Bcr C-末端小肽（KRQSILFSTEV）复合物的高分辨率三维溶液结构，并对每个残基在结合过程中的作用方式进行了讨论。PDZ结构域结合前后结构变化不大，由六段β折叠和两段α螺旋组成，Bcr小肽位于PDZ结构域的αB和βB形成的疏水沟槽中，与βB形成反平行的β折叠。一般认为PDZ结构域与配基相互作用的特异性是由配体0位和-2位的残基所决定。与典型的第一类PDZ和第二类PDZ不同的是，AF-6 PDZ结构域αB：1位的残基是Gln70，与小肽-2位Thr通过范德华力作用，形成了一种非典型的结合模式。此外我们的结果表明Bcr C-末端五个残基在与AF-6 PDZ结构域的相互作用中都有贡献，而βB/βC的loop区不参与结合。我们对AF-6 PDZ的分类进行了详细的讨论，发现不论是基于配体序列还是基于PDZ序列，现有的分类方法都无法适用于AF-6 PDZ，从而需要人们对PDZ分类及现有机理进行更深入的研究。通过核磁驰豫研究，我们对结合前后的主链动力学进行了比较，推测AF-6PDZ结构域存在单体—二体平衡，但通过NMR化学位移实验证实二聚对结构没有影响。我们的工作不仅对AF-6 PDZ结构域与Bcr小肽结合的相互作用模式进行了深入的研究，也为PDZ分类学的发展和药物设计提供了分子基础。第三章简要介绍了对泛素类相关修饰蛋白质SUMO及其与酶体系相互作用的研究。SUMO是一类广泛存在，在所有真核生物中高度保守的蛋白质家族。蛋白质翻译后被SUMO修饰这一过程，称之为SUMO化，是一种重要的细胞内调控机制。细胞内蛋白质的SUMO化是涉及多个酶的多步调控的级联反应，需要活化酶E1、缀合酶E2，在多数情况下还需要连接酶E3，再加上SUMO和底物，通过顺序的酶促反应，最后SUMO羧基末端的Gly经异肽键共价连接到底物蛋白的Lys侧链ε-NH₂上，这个Lys通常位于底物蛋白保守的ΨKXE基序上（其中Ψ是疏水残基，X是任意残基）。同泛素化主要介导蛋白质进入蛋白质酶体降解不同，SUMO化在细胞内有着多方面重要的生理功能：核内外物质的运输、蛋白质定位、转录活性调控、拮抗泛素化、细胞周期行进、维持基因组的完整性等。目前的研究表明人的SUMO家族各个成员在细胞内的定位和功能既有交叠又有不同。由于SUMO-1被广泛研究，而SUMO-2／3报道不多，我们的工作重点放在对SUMO-2／3及其酶体系的相互作用研究上。首先我们成功克隆、表达、纯化了带有C末端双Gly活性位点的人SUMO-2／3全长蛋白，为了防止SUMO-2／3自身的多聚，我们突变了K11位点。然后利用MS、FPLC、CIEF等方法对蛋白的稳定性和均一性做了研究，核磁结果显示各个残基交叉峰的信号强度差异很大，表明蛋白有一定的聚合，无法进行后续的谱峰归属。此外我们用Ubc9小肽滴定SUMO-2／3，HSQC谱峰的化学位移没有任何变化，说明仅仅包含有α1的Ubc9肽段不足以与SUMO-2／3 K11R结合，空间上其他位点可能也参与了它们的相互作用。之后截去N端部分无规卷曲NMR溶液结构才得以解析，并通过Ubc9滴定得到两者相互作用的界面。我们还尝试通过体外构建共价修饰体系得到复合物结构，继而用NMR的方法对共价复合物进行结构解析，但由于E1（SAE1／2）的表达量太少而无法继续，有待进一步的研究。更多还原

【Abstract】 As the structures of more proteins and domains are solved by structural genomics projects, the future of structural biology will be oriented more toward the study of macromolecular complexes. Since so many biological processes are mediated by interactions between proteins, it is important to study them at a molecular level. The study of protein-protein interactions also has applications in a number of therapeutic areas, particularly drug design, but also in understanding the molecular basis of biological science and human diseases. Nuclear magnetic resonance （NMR） spectroscopy is highly suited to investigate molecular interactions between proteins or proteins and peptides at close to physiological conditions. Such interactions may be of variable strength and sample a variety of timescales.Our work focuses on the protein-peptide complex structure determination and research by NMR spectroscopy. We expressed and purified the PDZ domain of human AF-6. The structure of this AF-6 PDZ domain complexed with the C-terminal peptide of the Bcr was determined by NMR. We explored the detailed interactions and binding mode between them. With the backbone dynamics study, we demonstrate the flexibility of AF-6 PDZ domain in free and binding form. The flexibility difference between the two forms is not obviously observed. From the correlation time estimation, we presume that the AF-6 PDZ domain might be in a monomer-dimer equilibrium in solution. However, the concentration-dependent chemical shift changes imply that dimerization neither changes the conformation nor affects the complex structure determination. Furthermore, we cloned, expressed and purified the full length of SUMO-2 and SUMO-3 protein with K11R mutation. The activating enzyme El （SAE1/2） and conjugating enzyme E2 （Ubc9） were also expressed and purified. We try to explore the interactions between them and obtain the covalent complex for NMR research.In chapter 1, NMR techniques that are being widely employed to study protein-ligand interaction are summarized. A wide variety of NMR methods can also be exploited to characterize other macromolecular complexes, such as protein-DNA, -RNA, -sugar, -lipid, -drug, etc. The technique of choice will depend upon factors including the sizes and types of molecules, equilibrium binding constant （tight vs. weak）, kinetics of binding （fast vs. slow exchange）, stoichiometry and symmetry. In this review, the important parameters used to characterize the intermolecular interaction are introduced firstly, followed by descriptions of the approaches for identifying interaction regions and how to extract valuable information related to intermolecular interaction. Finally, the NMR techniques that have been applied to map protein-ligand interaction are introduced.Chapter 2 provides the solution structure and backbone dynamics research of AF-6 PDZ domain/Bcr peptide complex. The human AF-6, a scaffold protein between cell membrane-associated proteins and actin cytoskeleton, plays an important role in special cell-cell junctions and signal transduction. It can be phosphorylated by the protein kinase Bcr, which allows efficient binding of the C-terminus of Bcr to the PDZ domain of AF-6 and consequently enhances the binding affinity of AF-6 to Ras. Formation of the AF-6, Bcr and Ras ternary complex results in down-regulation of the Ras-mediate signal transduction pathway.To better understand the molecular basis for the recognition of AF-6 PDZ domain and Bcr, we solve the solution structure of the AF-6 PDZ domain complexed with the C-terminal peptide of Bcr and explore the interactions between them in detail. It is revealed that AF-6 PDZ has a conserved fold consisting of sixβstrands flanked by two a helices and the C-terminal peptide of Bcr binds directly to the groove betweenαB andβB of the PDZ domains in an anti-parallel fashion. Compared with previously reported structure, the complex exhibits a special binding mode of PDZ/peptide. The interaction mode does not adapt to the existing classification rules that have been put forward, whether based on the ligand or on the PDZ domain specificity. The unique Gln⁷⁰ at aB:l of AF-6 PDZ domain determines the distinct binding mode of PDZ/peptide. In addition, the backbone dynamic analysis shows the flexibility of AF-6 PDZ domain/Bcr peptide complex, which is similar with the ligand-free form. From the correlation time estimation, we presume that the AF-6 PDZ domain might be in a monomer-dimer equilibrium in solution. However, the concentration-dependent chemical shift changes imply that dimerization neither changes the conformation nor affects the complex structure determination. Our work not only characterizes the structural details of AF-6 PDZ/Bcr peptide complex interaction, but also provides the potential target for future drug design and therapy.In chapter 3, a brief review of SUMO modification, including the family classification, biochemical reaction process and biological functions was provided first. SUMO is ubiquitiously expressed and highly conserved in all eukaryotes. Post-translational modification with SUMO, termed sumoylation, has emerged as an important cellular regulatory mechanism. SUMO modification of cellular proteins is a multi-step cascade reaction which many enzymes are involved in. The pathway requires the activating enzyme El and conjugating enzyme E2, and in most cases, requires a ligating enzyme E3, leading to the conjugation between SUMO and a substrate protein. The linkage between them is an isopeptide bond between the C-terminal glycine of SUMO and theε-amino group of a lysine residue in the substrate. This lysine is frequently found at a conservedψFKXE motif, whereψis a hydrophobic amino acid residue and X is any residue. Unlike ubiquitin which mostly tags proteins for degradation, SUMO has many important cellular functions, such as nucleocytoplasmic trafficking, protein localization, transcription regulation, signal transduction, antagonizing ubiquitination, cell cycle and genome integrity. The present studies suggest that SUMO family members are both overlapped and distinct in cellular localization and functions. Because SUMO-1 was broadly reported before, we concentrated our research on the interaction between SUMO-2/3 and enzymes in the covalent modification pathway.Firstly, we cloned, expressed and purified the full length SUMO-2/3 with the di-glycine motif at the C-terminal tail. To avoild polymerization, we mutate the site Lysl1 to Arg. MS, FPLC and CIEF methods were used to study the stability and uniformity. However, the singal intensity of the amino acids was distinct in the HSQC spectrum, indicating aggregation exist in the full length SUMO solution. Furthermore, we studied the interaction between SUMO-2/3 and the peptide of the conjugating enzyme Ubc9 using NMR chemical shift mapping. The result indicated that only the peptide including al of Ubc9 can not bind to SUMO-2/3 K11R. The interaction needs other spatial sites. The truncated SUMO was finally used to do the structure determination and interaction reserch.We also tried to obtain the covalent complex for NMR structural determination. However, the work can’t proceed with the low expression of SAE1/2, which needs further research.更多还原