Research In Solution Structure And Function Of HBrd4 BD2 And HMog1

This PhD thesis focuses on two parts: The first part is the cloning, expression and purification of the two bromodomains in human Brd4. Their properties in solution were studied, and the solution structure of Brd4 BD2 was determined. The interactions between BDs of Brd4 and acetylated histone tails have also been studied, and their dissociation constants were measured. Backbone dynamic properties of both apo- and ligand bound- BD2 were analyzed. A ternary complex model is presented, which is composed of one molecule of diacetylated histone peptide and two molecules of Brd4 BD2. It was calculated by molecular dynamics simulation. The second part is about the expression, purification and structure determination of human Mogl. Interactions between hMogl and hRanGDP were investigated primarily. Chapter I describes the first part of our work.In the first part of Chapter I, there is a particular description of the importance of Brd4 in organisms. It is a multifunctional protein, and plays different roles through interaction with different binding partners. Brd4 regulates transcription elongation process through interaction with P-TEFb and mediator. It regulates DNA replication process and G1/S transition through interaction with RFC. It is involved in signal transduction pathway, and regulates G2/M transition by association with SPA-1. It is also involved in the life cycles of some viruses. It regulates the transcription or replication of viral genes, or facilitates their partition during mitosis. All these functions are linked to its association with acetylated chromatin. Brd4 belongs to the BET family. It reads its own histone code, and then translates it into its own regulatory processes.The protein cloning, expression, purification and other experimental processes were described in detail in the second part of Chapter I.The third part of Chapter I describes the results and discussion of the corresponding experiments. Solution structure of Brd4 BD2 demonstrates a conserved left-handed four-helix bundle. Special decorations on the long ZA loop present a unique surface on the functional hydrophobic pocket, which determines the recognition for acetylated histone tails. Both BD1 and BD2 bind to H4-AcK5 and H4-AcK12, but with subtle difference. Their bindings are rather weak. The dissociation constants were estimated at milimolar scale. BD1 and BD2 of Brd4 were both identified to be mainly monomeric in solution. No heterodimers were observed when they were mixed at equal molar. These results are different from the predictions of another group. While Brd2 BD1 and TAFII250 enhance their affinities with acetylated histone tails through formation of a homodimer or a heterodimer, and Brd4 fails to function similarly. We predict that this protein should have its own mechanism to hold onto chromosomes during mitosis and regulation of associated cellular processes. HPV E2 sets up a good model. It is possible that, in normal living cells, an unknown E2 like protein facilitates the reinforcement of Brd4 and acetyl-chromatin association. It is also possible that this unknown protein comes from the mediator complex. Analysis of backbone dynamics demonstrated that, ligand binding depressed the microsecond-millisecond conformational exchanges in some degree, while changes in picosecond-nanosecond time scale fast motions were irregular.Chapter II describes the second part of our work.Mog1 has not been investigated well up to now. The primary information comes from the yeast. We list some of its functions that have been published. One of the most important is that, Mog1 binds to Ran, and participates in the regulation of nucleocytoplasm transport. Ran plays important roles in this system. Mog1 interacts with both Ran-GTP and Ran-GDP. The interaction makes Ran release the nucleotide. The unloaded Ran still binds to Mog1. But the mechanism of this interaction in the transport system is not known. Mog1 is also involved in the SLN1-SKN7 pathway. It directly binds to some components of the pathway. In some sense, this function still links to nuclear transport. Besides, Mog1 may participate in RNA and lipid metabolism.We expressed and purified the Mog1 and Ran protein, determined the solution structure of hMog1, and investigated some properties of their interaction. We hope to find the binding interface or obtain the crystal structure of their complex. This part of work is still in process.This part of work was done cooperatively with Jianping Sun and QiHu. Plasmids of hMog1 and hRan were constructed by Dr. Jianping Sun. Solution structure of hMog1 was determined by Dr. QiHu. Expression, purification, and all the sample preparation of hMog1 were of my work. All other experiments, including protein complex crystallization, were carried out by QiHu and me.