Thesis advisor: Evan R. Kantrowitz / For nearly 60 years the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and 5 X-ray structures determined in the absence and presence of a Mg2+ concentration within the physiological range. In the presence of 2 mM divalent cations (Mg2+, Ca2+, Zn2+) CTP does not significantly inhibit the enzyme while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor in vivo of ATCase is the combination of CTP, UTP and a M2+ cation and the actual allosteric activator is ATP and M2+ or ATP, GTP and M2+. The structural data reveals that two NTPs can bind to each allosteric site with a Mg2+ ion acting as a bridge between the triphosphates. Thus the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal the catalytic chains undergo essentially no alternations, however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal regions of the regulatory chains exist in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed. / Thesis (MS) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Evan R. Kantrowitz / The regulatory mechanism of allostery is exhibited by certain proteins such as Escherichia coli aspartate transcarbamoylase (ATCase), and is defined as the change in shape and activity (of enzymes) resulting from the binding of particular molecules at locations distant from the active site. This particular enzyme and the property of allostery in general have been investigated for several decades, yet the molecular mechanisms underlying allosteric regulation remain unclear. Therefore in this thesis we have attempted via several biophysical methods, along with the tools of molecular biology and biochemistry, to correlate the changes in allosteric structure with presence of the allosteric effectors and enzymatic activity. We created a double mutant version of ATCase, in which the only native cysteine residue in the catalytic chain was mutated to alanine and another alanine on a loop was mutated to cysteine, in order to lock the enzyme into the R allosteric state by disulfide bonds. This disulfide locked R state exhibited no regulation by the allosteric effectors ATP and CTP and lost all cooperativity for aspartate, and then regained those regulatory properties after the disulfide links were severed by addition of a reducing agent. This double mutant was then chemically modified by covalent attachment of a fluorescent probe. The T and R allosteric states of this fluorophore-labeled enzyme had dramatically different fluorescence emission spectra, providing a highly sensitive tool for testing the effects of the allosteric effectors on the allosteric state. The changes in the fluorescence spectra, and hence quaternary structure, matched the changes in activity after addition of ATP or CTP. This fluorophore labeled enzyme was also encapsulated within a solgel, changing the time scale of the allosteric transition from milliseconds to several hours. The fluorophore labels allowed monitoring the allosteric state within the sol-gel, and the physically trapped T and R states both showed no regulation by the allosteric effectors ATP and CTP, and no cooperativity for aspartate. The trapped T state had low-affinity for aspartate and low activity, and the trapped R state had high-affinity for aspartate and high activity. Timeresolved small-angle x-ray scattering (TR-SAXS) was used to determine the kinetics of the allosteric transition, and to monitor the structure of the enzyme in real time after the addition of substrates and allosteric effectors. These TR-SAXS studies demonstrated a correlation between the presence of the allosteric effectors, the quaternary allosteric state, and activity, suggesting like the previous studies in this thesis that the behavior of ATCase is well explained by the twostate model. However, the effector ATP appeared to destabilize the T state and CTP to destabilize the R state, suggesting a different allosteric molecular mechanism than that of the two-state model. This thesis demonstrates the validity of many of the concepts of the two-state model, while suggesting minor modifications to that elegantly simple model in order to conform with the complex structure and function of ATCase. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Over the last twenty years, fragment-based drug discovery (FBDD) has emerged as a highly successful way to provide lead compounds for subsequent optimisation into drug candidates. Initial hits usually exhibit lower potency than those identified by more traditional techniques, such as High-Throughput Screening (HTS), but the optimisation phase of FBDD is highly efficient, thus providing superior lead-like compounds. The recent application of FBDD in a variety of protein kinase campaigns has successfully led to the identification of novel binding sites and highly efficient chemical ligands. This demonstrates the utility of the FBDD strategy against well-established kinase targets, where selectivity is otherwise challenging due to significant conservation of the ATP-binding site. Protein kinase CK2 is a ubiquitously expressed and constitutively active regulator of cell growth, proliferation and apoptosis. Elevated levels of CK2 protein and activity have historically been involved in human cancer, including lung, cervical and head and neck cancer types, and its overexpression is associated with worse prognosis. A number of CK2 inhibitors are currently available displaying activity against multiple cancers in vitro and in the clinic, however the majority of these candidates target the ATP-binding site and thus display poor selectivity in kinase panel assays. Here we explore the application of FBDD towards the development of potent and selective inhibitors of the catalytic α-subunit of CK2. This project exploits a novel, conserved binding site, named the αD pocket, for the generation of allosteric inhibitor molecules. Following structure-based optimisation of a potent inhibitor series, and characterisation of a previously unreported binding mode, a fragment linking strategy between the lead αD-site fragment and a low-affinity pseudosubstrate peptide is investigated. This work validates the utility of FBDD towards the discovery of new binding modes, presents a first in class CK2α allosteric inhibitor series and provides the first X-ray crystal structure of protein kinase CK2 in complex with a ligand binding in the substrate-binding channel.

In this thesis, I focus on studies of the mechanism of inactivation in KcsA. Allosteric coupling between the pH gate and the selectivity filter in the protein is hypothesized to be the cause of inactivation. Allosteric coupling refers to changes at one site of a protein due to perturbations at a remote site. In chapter 3, I measured the potassium affinities at the selectivity filter at neutral and low pH, which corresponds to the closed and open conformation at the pH gate. The results show a three order of magnitude shift in the potassium affinity. This is direct evidence that the pH gate and the selectivity filter are coupled, in support of the activation-coupled inactivation hypothesis. The allosteric coupling factor, defined as the ratio of the affinities, can be used as a benchmark to study other factors in the allosteric process, such as the membrane and specific residues. Because of the potential deleterious effect of the acidic pH on the protein and membrane, we studied a mutant E118A&H25R, in which the pH gate is mutated to be open. Thus we were able to measure the K+ affinity change in the open and closed conformation at the pH gate at neutral pH. The results confirmed that the opening of the pH gate results in an energetic stabilization of the collapsed (K+-unbound) state, and shifts the K+ affinity towards looser binding. In chapter 4, I tested the important role of residue F103 in mediating allosteric coupling, as suggested by electrophysiology and crystallography studies. I mutated this residue and measured the allosteric coupling factor on the mutant. The affinity at low pH is much tighter than wild-type and the coupling factor is significantly reduced. From the spectra, I observe local structural changes on I100 and T74 as a result of F103A mutation, implying the interaction among F103, I100 and T74 to mediate the allosteric coupling. F103 is distant from the pH gate and the selectivity filter; its effect on the coupling and inactivation behaviors confirms that inactivation involves coupling between the pH gate and the selectivity filter. In chapter 5, I developed a method to probe those allosteric participants, such as F103 in KcsA by NMR measurements. I tested this method on KcsA, dissecting KcsA into various functional compartments. Various allosteric participants T75Cg T74Cg I100 were identified. The importance of residue T74 for the coupling was confirmed by electrophysiology and NMR thermodynamics characterization. In chapter 6, we applied SSNMR to probe the structural and magnetic properties of superatom clusters.

Three residues, Phe190, Leu193 and Val302, which have been proposed to define the S₁ site of prolidase of <i>Lactococcus lactis</i> NRRL B-1821 (<i>L. lactis</i> prolidase), may limit the size and polarity of specific substrates accepted by this enzyme (Yang, S. I., and Tanaka, T. 2008. Characterization of recombinant prolidase from <i>L. lactis</i> changes in substrate specificity by metal cations, and allosteric behavior of the peptidase. FEBS J. 275, 271-280). These residues form a hydrophobic pocket to determine the substrate specificity of <i>L. lactis</i> prolidase towards hydrophobic peptides, such as Leu-Pro and Phe-Pro, while little activity was observed for anionic Asp-Pro and Glu-Pro. It is hypothesized that the substrate specificity of <i>L. lactis</i> prolidase would be changed if these residues are substituted with hydrophilic amino acid residues individually or in combinations by site-directed mutagenesis (SDM). In addition to the changes in substrate specificity, other characteristics of wild type prolidase, such as allosteric behaviour and substrate inhibition may receive influences by the mutations (Yang & Tanaka, 2008). To test this hypothesis, mutations were conducted on these three residues at the S₁ site. Mutated <i>L. lactis</i> prolidases were subsequently analyzed in order to examine the roles of these residues in the substrate specificity, allosteric behaviour, pH dependency, thermal dependency and metal dependency of prolidase. The results showed the significant changes in these kinetic characteristics of single mutants, such as L193E, L193R, V302D and V302K and double mutants, L193E/V302D and L193R/V302D. Leu193 was suggested to be a key residue for substrate binding. The mutants L193R, V302D, L193R/V302D and L193E/V302D lost their allosteric behaviour, and the substrate inhibition of the wild type was no longer observed in V302D and L193E/V302D. The results indicated Val302 to be more important for these properties than other S₁ site residues. Moreover, together with the observations in molecular modelling of the mutants, it was proposed that interactions of Asp302 with Arg293 and His296 caused the loss of allosteric behaviour and substrate inhibition in the V302D mutant. The investigations on the pH dependency suggested that His296 acted as proton acceptor in <i>L. lactis</i> prolidase's catalysis. It was expected that the electrostatic microenvironment surrounding His296 was influenced by the charged mutated residues and side chains of dipeptide substrates, thus the protonation of His296 was affected. It was suggested that the introduced positive charge would stabilize the deprotonated form of His296 thus to maintain the activities of the mutants in more acidic condition compared to wild type prolidase. The study of thermal dependency revealed that all non-allosteric prolidases had higher optimum temperatures, suggesting that the loss of allosteric behaviour resulted in more rigid structures in these prolidases.

Three residues, Phe190, Leu193 and Val302, which have been proposed to define the S₁ site of prolidase of <i>Lactococcus lactis</i> NRRL B-1821 (<i>L. lactis</i> prolidase), may limit the size and polarity of specific substrates accepted by this enzyme (Yang, S. I., and Tanaka, T. 2008. Characterization of recombinant prolidase from <i>L. lactis</i> changes in substrate specificity by metal cations, and allosteric behavior of the peptidase. FEBS J. 275, 271-280). These residues form a hydrophobic pocket to determine the substrate specificity of <i>L. lactis</i> prolidase towards hydrophobic peptides, such as Leu-Pro and Phe-Pro, while little activity was observed for anionic Asp-Pro and Glu-Pro. It is hypothesized that the substrate specificity of <i>L. lactis</i> prolidase would be changed if these residues are substituted with hydrophilic amino acid residues individually or in combinations by site-directed mutagenesis (SDM). In addition to the changes in substrate specificity, other characteristics of wild type prolidase, such as allosteric behaviour and substrate inhibition may receive influences by the mutations (Yang & Tanaka, 2008). To test this hypothesis, mutations were conducted on these three residues at the S₁ site. Mutated <i>L. lactis</i> prolidases were subsequently analyzed in order to examine the roles of these residues in the substrate specificity, allosteric behaviour, pH dependency, thermal dependency and metal dependency of prolidase. The results showed the significant changes in these kinetic characteristics of single mutants, such as L193E, L193R, V302D and V302K and double mutants, L193E/V302D and L193R/V302D. Leu193 was suggested to be a key residue for substrate binding. The mutants L193R, V302D, L193R/V302D and L193E/V302D lost their allosteric behaviour, and the substrate inhibition of the wild type was no longer observed in V302D and L193E/V302D. The results indicated Val302 to be more important for these properties than other S₁ site residues. Moreover, together with the observations in molecular modelling of the mutants, it was proposed that interactions of Asp302 with Arg293 and His296 caused the loss of allosteric behaviour and substrate inhibition in the V302D mutant. The investigations on the pH dependency suggested that His296 acted as proton acceptor in <i>L. lactis</i> prolidase's catalysis. It was expected that the electrostatic microenvironment surrounding His296 was influenced by the charged mutated residues and side chains of dipeptide substrates, thus the protonation of His296 was affected. It was suggested that the introduced positive charge would stabilize the deprotonated form of His296 thus to maintain the activities of the mutants in more acidic condition compared to wild type prolidase. The study of thermal dependency revealed that all non-allosteric prolidases had higher optimum temperatures, suggesting that the loss of allosteric behaviour resulted in more rigid structures in these prolidases.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

Prolidase has potential applications in cheese debittering, organophosphate detoxification and as an enzyme replacement therapy in prolidase-deficient patients. Recombinant Lactococcus lactis prolidases and their catalytic properties have previously been characterized in Dr. Tanaka's research group. Unlike other prolidases, L. lactis prolidase shows allosteric behaviour, metal-dependent substrate specificity and substrate inhibition. The current project focuses on elucidating the three-dimensional structure of L. lactis prolidase using X-ray crystallography. Hexagonal plate-like crystals of wild-type L. lactis prolidase were grown by the hanging drop vapour diffusion method, allowing the crystals to grow to about 50 µm in their longest dimension. The crystallization cocktail in which they grew contained 0.08 M sodium cacodylate (pH 6.5), 0.16 M calcium acetate, 14 % PEG 8000 and 18 % glycerol. Crystal diffraction data was collected at a wavelength of 0.9795 Å on beamline 08ID-1 of the Canadian Macromolecular Crystallography Facility at the Canadian Light Source and was processed using X-ray Detector Software. The crystals belonged to space group C2 and estimated to contain three molecules in an asymmetric unit. The electron density map of this structure was solved by the molecular replacement method and the structure model was refined against 2.25 Å resolution data. Molecule A forms a dimer with molecule B, while molecule C forms a dimer with molecule C', which is located in the neighbouring crystal asymmetric unit. The electron density of molecule A was well-defined and complete. Therefore, all the 362 amino acid residues of L. lactis prolidase were fitted. The other two molecules were incomplete and less defined. Only 360 and 352 residues could be fitted in molecules B and C, respectively. Molecule C, the worst of the three, compromised the overall quality of the refined structure. However, the functional interpretation of the structure was not compromised since the well-defined molecules form a dimer with each other and the biologically-functional form of L. lactis prolidase is a homodimer. The final Rwork and Rfree are 22.39 and 27.77, respectively. Comparison with other known prolidases revealed that Asp 36 and His 38 are unique to L. lactis prolidase. These residues have been shown to be involved in the allosteric behaviour and substrate inhibition of this enzyme, respectively. Therefore, this crystal structure further supports their suggested contribution in L. lactis prolidase's unique catalytic properties.

Accumulating evidence suggests a prominent role of the arrestin-dependent signaling pathway in triggering most of the deleterious side effects observed using δ-OR targeting drugs. Numerous small molecules targeting the δ-OR receptors have been developed but their pharmacological properties, including their functional selectivity, have been poorly characterized. The absence of functionally selective opioid drugs, and the lack of knowledge of the pharmacological profile and signaling properties of the δ-OR receptor, limits its therapeutic exploitation. The development of functionally selective modulator toward the canonical G protein pathway could importantly increase the therapeutic potential of this receptor while decreasing its deleterious effects. An approach to fine-tune the functional selectivity of a GPCR is by using allosteric modulators. These allosteric modulators would reduce problems associated with drugs targeting the orthosteric site by not chronically activating the receptor. The overall goal of the proposed research is to study the molecular mechanism by which sodium-channel inhibitors allosterically regulates the delta opioid receptor (δ-OR) signaling and functional selectivity. Additionally, the signaling features of the δ-OR signal transduction triggered by biased receptor activation have been investigated. A combination of approaches, including functional studies, molecular modeling and mutagenesis, were used to study the general mechanism underlying the activation and tuning of the δ-OR signal transduction behavior. Thus, this work suggests the druggability of the allosteric sodium pocket by using sodium channel inhibitors. The current research represent discovery of two different allosteric profiles for the β-arrestin recruitment and one allosteric profile for the G-protein pathway at activated DOR and would serve as scaffold for further refinement of modulators with the desired pharmacological profile.

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