Protein Complex Structure Research By NMR Spectroscopy

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.