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No description available.

The diverse functions of the MDM2 oncoprotein in growth control and tumourigenesis are managed through coordinated regulation of its discrete domains induced by both extrinsic and intrinsic stimuli. A picture of MDM2 is immerging where structurally discrete but interdependent functional domains are linked through changes in conformation. However compelling insights into how this process is carried out have been hindered by inadequate information on the structure and conformation of the full-length protein. The data presented indicates that the C-terminal RING domain of MDM2, primarily responsible of the E3 ubiquitin ligase activity of the protein, has other intriguing functions. The binding of ATP within the RING domain, triggers conformational changes of MDM2 and its main interaction partner – p53. This in effect promotes efficient binding of the p53 tumour suppressor to specific DNA promoter sequences. Moreover, results presented in this thesis demonstrate a novel role for the RING domain of MDM2 in determining the conformation and activity of its N-terminal hydrophobic cleft, the key target of anticancer drugs designed to activate the function of p53 tumour suppressor protein. Specific modulations within the RING domain, affecting Zinc coordination are synonymous with increased binding affinity of the hydrophobic pocket to the transactivation domain of p53 resulting in a gain of MDM2 transrepressor function thus leading to a decrease in p53-dependant gene expression. ThermoFluor measurements and size exclusion chromatography show that changes in the RING motif lack an effect on the overall integrity of the MDM2 protein. The intrinsic fluorescence measurements manifest that these changes generate long range conformational transitions that are transmitted through the core/central acidic domain of MDM2 resulting in allosteric regulation of the N-terminal hydrophobic pocket. Such RING generated conformational changes result in the relaxation of the hydrophobic pocket. Additionally, it is shown that the cooperation between the RING and the hydrophobic cleft in MDM2 has implications in the efficiency of binding of anticancer drugs such as Nutlin by MDM2. Cooperation between the RING and hydrophobic domain of MDM2 to regulate function demonstrates an allosteric relationship and highlights the need to study MDM2 in a native conformation. In essence the presented data demonstrates that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed towards its hydrophobic pocket.

The number of allosteric sites and active sites in phosphofructokinase from Bacillus stearothermophilus create an intricate network of communication within the enzyme. With thermodynamic linkage analysis, the overall allosteric communication can be quantified. This value, however, represents an average contribution for all the interactions involved. The recent development of a hybrid strategy has allowed for the quantification of single interactions, both heterotropic and homotropic. Focusing on the heterotropic interactions whose inhibition is entropy-driven, residues and regions within the enzyme can now be identified to further characterize each specific interaction using the hybrid strategy. Among the many components of entropy, the hybrid strategy has now allowed for the strategic placement of a reporter of side chain dynamics to identify conformational differences between the four ligand bound enzyme species of a single heterotropic interaction. In this study, a combination of these approaches was used in the methodology including constructing hybrids to isolate a single heterotropic interaction along with single tryptophan reporter. Site directed mutagenesis combined with the hybrid strategy was also implemented to directly assess the role of a single residue in the communication path of a single interaction. The region surrounding the allosteric site with the nearest active site has been implicated to be significant in transmitting the allosteric signal. In addition two single residues, T158 and D59, within this region have been identified to potentially contribute to the inhibition of this same interaction. An additional residue, G184, located outside this local region has also been identified as possibly having a significant role in the transmission of the inhibitory signal of a unique heterotropic interaction. The implications of this study have led to the initial identification of residues involved in the 22A route of allosteric communication of a single active site and allosteric site. This allosteric communication occurs to allow the enzyme to compensate for the binding of both ligands. With the location of these residues implicated to be involved in the communication of this isolated interaction, this compensation is not contained within a confined region but is however felt throughout the single subunit.

Characterization of allosteric properties of phosphofructokinase from the extreme thermophile Thermus thermophilus (TtPFK) using thermodynamic linkage analysis revealed several peculiarities. Inhibition and activation of Fru-6-P binding by the allosteric effectors phosphoenolpyruvate (PEP) and MgADP are entropically-driven in TtPFK. It is also curious that PEP binding affinity is unusually strong in TtPFK when compared to PFKs from Escherichia coli, Bacillus stearothermophilus, and Lactobacillus delbrueckii, while the magnitude of the allosteric inhibition by PEP is much smaller in TtPFK. In an effort to understand the source of weak inhibition, a putative network of residues between the allosteric site and the nearest active site was identified from the three-dimensional structures of BsPFK. Three of the residues in this network, D59, T158, and H215, are not conserved in TtPFK, and, due to their nature (N59, A158, S215), are unlikely to be involved in the same non-covalent interactions seen in BsPFK. The triple chimeric substitution N59D/A158T/S215H, results in a 2.5 kcal mol-1 increase in the coupling free energy, suggesting that the region containing these residues may be important for propagation of inhibitory response. The individual substitutions at each position resulted in an increase in the coupling free energy, and the double substitutions displayed additivity of these changes. The chimeric substitution made at N59 suggests that the polar nature of the asparagine at position 59 is key for the enhanced binding of PEP. The non-conserved R55 was found to be particularly important for the enhanced binding of PEP in TtPFK, as chimeric substitutions R55G and R55E resulted in a 3.5 kcal mol-1 and 4.5 kcal mol-1 decrease in the binding affinity for PEP, respectively. Our results also confirm the observations previously made in PFKs from E. coli and B. stearothermophilus, that the ability of the effector to bind is independent of its ability to produce allosteric response. We show that several substitutions result in a decrease in binding affinity of PEP to TtPFK, while dramatically enhancing its ability to inhibit (N59D, R55G, R55E). Similarly, some substitutions, like S215H and A158T show an enhanced inhibition by PEP, while having no effect on its binding affinity.

The activation of Protein Kinase A (PKA) and of Hyperpolarization-activated and Cyclic Nucleotide-modulated channels (HCN) is controlled by cAMP through cAMP binding domains (CBDs), which serve as cAMP-dependent conformational switches to regulate downstream signaling pathways. The binding of the cAMP allosteric effector removes the auto-inhibition imposed by linkers that are adjacent to the CBDs of PKA and HCN. However, our understanding of how cAMP binding to the structured CBD controls the adjacent inhibitory linkers is currently limited. Herein, we investigate through NMR spectroscopy the interactions between the CBDs of HCN and PKA and the respective adjacent linkers. Chapters 2 and 3 of this thesis focus on the linkers N-terminal to PKA CBD-A and CBD-B, respectively, while Chapter 4 centers on the linker N-terminal to the HCN CBD. We show that in the case of PKA the linker N-terminal to CBD-A is flexible, but is coupled to the CBD-A through state active selective interactions. In the case of the CBD-B of PKA the state selective interactions with the linker N-terminal to it are to a large extent lost and replaced by state-selective inter-CBD interactions, which in turn control the conformational ensemble accessible to the inter-domain linker. Unlike PKA, in the case of HCN, the primary mechanism of cAMP-dependent linker control is through the state-selective destabilization of the structured tetrameric N-terminal linker. Overall, this thesis reports three distinct mechanisms through which linkers in HCN and PKA serve not only as simple covalent threads, but also as integral parts of the allosteric networks underlying auto-inhibition and cAMP dependent activation. / Thesis / Doctor of Philosophy (PhD) / Cyclic adenosine monophosphate or cAMP is a second messenger that is produced by cells to control the internal cellular metabolism in response to external stimuli. The goal of this thesis is to elucidate the structural and dynamical changes that translate the cAMP signal into a specific biological response necessary for the survival of the cell. We used Nuclear Magnetic Resonance (NMR) Spectroscopy to investigate how, under physiological solution conditions, the cAMP interacts with and modifies the cAMP-dependent protein kinase A (PKA) and the hyperpolarization-activated and cyclic nucleotide-gated channels (HCN). Knowledge of both structure and dynamics on both proteins is required in order to fully understand at a molecular level how cAMP works in the human heart. The elucidation of the structural and dynamical changes associated with cAMP-binding is expected to help define general rules applicable to the design of drugs for cardiovascular disorders.

Allosteric regulation of proteins by reversible ligand binding is essential for regulation of fundamental biological processes. The mechanism by which a binding event alters the function of a distant site in a protein is only poorly understood. In this thesis, I use the Tetracycline Repressor (TetR) as a model system to study ligand induced allostery. The transcription of genes encoding the resistance to the antibiotic, tetracycline (Tc), is repressed by TetR, which is a homodimeric alpha-helical protein possessing a small N-terminal DNA binding domain (DNB domain) and a larger C-terminal tetracycline binding and dimerization domain (TBD domain). Based on previous structural and thermodynamic studies, the DNB domains are thought to exist in two stable, distinct conformations. One conformation is able to bind the Tc resistance operator sequence (tetO) with high affinity, while the other, which is induced by Tc binding, binds very weakly. While most previous studies on TetR have focused on the effects of Tc binding on the DNB domain conformation, here I have investigated the role of the DNB domain in modulating Tc binding. By introducing destabilizing mutations into the DNB domain I ascertained that the conformation and stability of the DNB domain plays an important role in determining Tc binding affinity. I also discovered that in the absence of ligand, the DNB domain exists in an unstable and flexible state with respect to the TBD domain. However, Tc binding to the TBD domain stabilizes the DNB domain, causing it to fold cooperatively with the TBD domain. I have discovered that the behavior of previously isolated non-inducible mutants is caused by the inability of Tc to stabilize the DNB domain in these mutants. Furthermore, reverse TetR mutants, which bind DNA better in the presence of Tc have an unfolded DNB domain that is only partially stabilized by Tc binding. My work suggests a new comprehensive, Tc induced stabilization and domain cooperativity model that can describe the mechanism of allostery in TetR and previously unexplainable mutants. A practical outcome of this research is the creation of a Tc induced folding switch that can be exploited to control the in vivo degradation of a protein of interest.

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.