Biochemical and structural investigations of the retinoblastoma protein, its binding partners, and the BRG1 protein - a subunit of human SWI/SNF remodeling complexes

Two projects were undertaken in this thesis. The first project involved functional investigations on the retinoblastoma protein (pRb) and its interaction with binding partners. The second project involved the characterization of functional domains of the human BRG1 protein including the 3D structure determination of BRG1 bromodomain. The retinoblastoma tumor suppressor protein (pRb) is a key negative regulator of cell proliferation that is frequently deregulated in human cancer. More than 130 proteins have been reported to bind to pRb directly. Many of these interactions were reported with the pRb small pocket, which is the major focus of tumorigenic mutations in pRb, and comprises the A and B cyclin - like domains (amino acids 379-578 and 641-791, respectively). However, results found in the literature are ambiguous and contradictory. We have investigated some of these interactions using purified proteins and in-vitro biophysical and biochemical methods, which included, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, isothermal titration calorimetry (ITC), the affinity chromatography pull-down assays, and gel filtration chromatography. The interactions of pRb with the following proteins were checked using this approach: MyoD, Id-2, E2F1, HDAC1, PAI2, HPV E7, SV40 large T antigen, BRG1, and gankyrin. Myogenic differentiation promoting, MyoD and inhibiting, Id-2, factors were reported to interact with the pRb pocket domain to coordinate the cell growth and differentiation during the process of myogenesis. There have been contradictory reports in the literature regarding the direct interaction between pRb and MyoD, pRb and Id-2 proteins. We probed these interactions using our multimethod in-vitro approach. Using this approach and we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, MyoD and DNA, and MyoD and Id-2. However using the same approach, we could unambiguously show that there is no direct protein-protein interaction in-vitro between the small pocket domain of pRb and the bHLH domain of MyoD and between the small pocket domain of pRb and Id-2. We propose that the indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action. Many viral oncoproteins (for example, HPV E7, E1A) are known to bind to the pRb pocket domain via a LXCXE binding motif. There are also some 20 cellular proteins that contain a LXCXE motif and have been reported to associate with the pocket domain of pRb. Using the in-vitro methods including NMR spectroscopy and isothermal calorimetry titration, I showed that LXCXE peptides of viral oncoproteins bind strongly to the pocket domain of pRb. Additionally, I showed that the LXCXE-like peptides of histone deacetylase 1 (HDAC1) bind to the same site on pRb, with however a weak (micromolar) and transient association. Systematic substitution of residues other than conserved L, C, and E show that the residues flanking the LXCXE are important for the binding. Positively charged amino acids in the XLXCXEXXX sequence signifi¬cantly weaken the interaction. Also I could show that the interactions among the peptide residues are important for orienting the LXCXE motif in a proper conformation. I discuss the ramification of the weak binding of IXCXE sequences of HDAC1 to pRb. The BRG1 protein is the central conserved ATPase of the human SWI/SNF class of the ATP dependent chromatin remodeling complex. Human BRG1 protein has an ATPase domain, an AT-hook containing region, and a bromodomain. An AT-hook motif, first described in the high-mobility-group of non-histone chromosomal proteins HMGA1/2 (formally called HMGI/Y), is a DNA binding motif. Bromodomain is an acetyl-lysine recognizing domain found in many chromatin-associated proteins. The AT-hook motif binds to the AT rich DNA sequences in the minor groove of B-DNA in a non-sequence specific manner. In this study I cloned and purified a fragment of hBRG1 which encompasses the AT hook region and the bromodomain. NMR and CD spectroscopy show that this entire recombinant domain is well-structured in solution. Functionalities of sub¬domains were checked by their interactions with N-acetylated peptides from histones. I also characterized the interaction of the AT-hook region with DNA. The ITC data shows that the primary interaction is through the AT-hook motif. The AT-hook region interacts with the linear DNA by unwinding it. These properties resemble the characteristics of the HMGA1/2 proteins and their interaction with DNA. BRG1 and BRM1 ATPase subunits of the SWI/SNF complex also contain a bromodomain at the C-terminal end of the protein. Here I report the crystal structure of human BRG1 bromodomain at a 1.5 A resolution. This is a first structure of a bromodomain from a subunit of the ATP dependent chromatin remodeling complex. The four helices in the structure are similar to the helices from other published bromodomains from histone acetyltransferase (HAT) class of proteins (GCN5, P/CAF, CBP, and double bromodomain module of TAFII250), however, a novel small anti-parallel -sheet is present in the ZA loop region (termed Z-sheet) of the BRG1 bromodomain. Sequence alignment shows that this Z-sheet is present only in hBRG1 and hBRM1 proteins and between these two it is longer in the case of hBRM1 protein. In-silico modeling shows that the N-acetyl-lysine peptide would bind in a manner similar to other bromodomain. However, we postulate that in BRG1 bromodomain the N-acetyl lysine binding groove is more open, and this would make [N-acetylated-lysine peptide]-[BRG1 bromodomain] interaction weaker. These changes in the acetyl-lysine binding site could account for the evolutionarily adapted functions of bromodomain in different proteins. Overall the results of this work should help in understanding and delineating the functions pRb and BRG1 in the important processes of cell cycle regulation and chromatin remodeling.