Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...

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Glutamate is essential for proper brain function as it is our nervous systems principal excitatory neurotransmitter, a signal that stimulates nerve cells to send messages to other cells. Glutamate activates ionotropic glutamate receptors (iGluRs), which are linked to several neurological diseases in cases when they are improperly regulated. iGluRs are transmembrane channels that allow calcium, as well as other cations, into the post synaptic neuron upon binding of glutamate or other agonists.Interestingly, iGluR homologs in plants also mediate calcium signaling upon glutamate activation and were accordingly named glutamate receptor-like channels (GLRs). Cell signaling is critical for plant survival to mediate rapid response to growth, defense, and other environmental cues. GLRs are found in all plants and vital for their health, hardiness, and adaptation for growth and survival in unfavorable conditions, such as drought, nutrient poor soil, temperature extremes, pathogens, and predators. Plant research is important with vast applications. Firstly, crops are our primary source of nutrition. In addition, plants are used as sources of drugs that we employ for treating diseases. Some examples of plant-derived neuroactive compounds include caffeine in coffee beans, nicotine in tobacco, and opium from poppy plants. In short, optimizing plant growth is beneficial to maintaining our own survival and potentially achievable by understanding GLRs role in plant health and hardiness. Despite their importance for cell signaling and implication in plant defense and regeneration, the structural basis underlying the function of these channels remains ambiguous, representing a critical barrier to our understanding of GLR function. To address this problem, I dedicated my thesis work to study the structure of GLRs and gain insight into their function. There are 20 GLRs in the model plant organism, Arabidopsis thaliana, classified into 3 different clades (AtGLR1-3). To narrow down which AtGLRs to focus our structural studies on, we investigated clade 3 representatives, as many of these GLR3s have been extensively studied in different plant species, especially crops. For example, studying AtGLR3.4 could provide useful information to how the homolog in rice, OsGLR3.4, contributes to growth and production in rice. Studying AtGLR3.4’s structure may elucidate how agonistic or antagonistic targets bind and gate the channel, potentially revealing “druggable” targets to alter plant response for defense and regeneration. Without any structural information available for GLRs, I started my studies by first focusing on their mammalian homologs, iGluRs. I first designed multiple constructs for heterologous expression and purification from cell culture (for example HEK293S GnTI- cells). Then, I optimized protein extraction and purification to obtain pure protein samples. Purified proteins were then subjected to cryo-electron microscopy (cryo-EM) which eventually allowed us to solve the structure of AtGLR3.4, the first full-length GLR structure. AtGLR3.4’s structure revealed similarities to structures of its mammalian homologs, iGluRs. In comparison to iGluRs, our GLR structure also showed tetrameric subunit assembly, with a three-layer architecture that includes the ligand binding domain (LBD) in the middle, sandwiched between the extracellular amino terminal domain (ATD) at the top and the transmembrane domain (TMD) at the bottom. In contrast to the majority of iGluR structures, however, AtGLR3.4 displayed unique symmetry and domain arrangement with the non-swapped extracellular ATD and LBD domains. We also provided further evidence supporting ligand binding promiscuity that was previously revealed in isolated LBD crystal structures from other AtGLR3s. Surprisingly, we found endogenous glutathione bound to the ATDs and demonstrated its contribution to channel activity. It is important to fill the gaps in knowledge about GLR structure to understand how these channels are activated and gated. In doing so, we will learn more about iGluRs as well as better understand plant defense and growth, which has the potential to enhance crop production for food security and our overall survival.

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<p>The intricate interplay of biomolecules acting together, rather than alone, provides insight into the most basic of cellular functions, such as cell signaling, metabolism, defense, and, ultimately, the creation of life. Inherent in each of these processes is an evolutionary tendency towards increased efficiency by means of biolgocial synergy-- the ability of individual elements of a system to produce a combined effect that is different and often greater than the sum of the effects of the parts. Modern biochemists are challenged to find model systems to characterize biological synergy.</p><p>We discuss the multicomponent, enzyme complex the cellulosome as a model system of biological synergy. Native cellulosomes comprise numerous carbohydrate-active binding proteins and enzymes designed for the efficient degradation of plant cell wall matrix polysaccharides, namely cellulose. Cellulosomes are modular enzyme complexes, comparable to enzyme "legos" that may be readily constructed into multiple geometries by synthetic design. Cellulosomal enzymes provide means to measure protein efficiency with altered complex geometry through assay of enzymatic activity as a function of geometry.</p><p>Cellulosomes are known to be highly efficient at cellulose depolymerization, and current debates on the molecular origins of this efficiency suggest two related effects provide this efficiency: i) substrate targeting, which argues that the localization of the enzyme complex at the interface of insoluble cell wall polysaccharides facilitates substrate depolymerization; and ii) proximity effects, which describe the implicit benefit for co-localizing multiple enzymes with divergent substrate preferences on the activity of the whole complex.</p><p>Substrate targeting can be traced to the activity of a single protein, the cellulosomal scaffoldin cellulose binding module CBM3a that is thought to uniquely bind highly crystalline, insoluble cellulose. We introduce methods to develop a molecular understanding of the substrate preferences for CBM3a on soluble and insoluble cellulosic substrates. Using pivaloylysis of cellulose triacetate, we obtain multiple soluble cello-oligosaccharides with increasing degree of glucose polymerization (DP) from glucose (DP1) to cellodecaose (DP10) in high yield. Using calorimetry and centrifugal titrations, cello-oligosacharides were shown to not bind Clostridial cellulolyticum CMB3a. We developed AFM cantilever functionalization protocols to immobilize CBM3a and then probe the interfacial binding between CBM3a and a cellulose nanocrystal thin film using force spectroscopy. Specific binding at the interface was demonstrated in reference to a control protein that does not bind cellulose. The results indicate that i) CBM3a specifically binds nanocrystalline cellulose and ii) specific interfacial binding may be probed by force spectroscopy with the proper introduction of controls and blocking agents.</p><p>The question of enzyme proximity effects in the cellulosome must be answered by assaying the activity of cellulosomal cellulases in response to cellulosome geometry. The kinetic characterization of cellulases requires robust and reproducible assays to quantify functional cellulase content of from recombinant enzyme preparations. To facilitate the real-time routine assay of cellulase activity, we developed a custom synthesis of a fluorogenic cellulase substrate based on the cellohexaoside of Driguez and co-workers (vide infra). Two routes to synthesize a key thiophenyl glycoside building block were presented, with the more concise route providing the disaccharide in four steps from a commercial starting material. The disaccharide building blocks were coupled by chemical activation to yield the fully protected cellohexaoside over additional six steps. Future work will include the elaboration of this compound into an underivatized FRET-paired hexasaccharide and its subsequent use in cellulase activity assays.</p><p>This dissertation also covers an experimental system for the evaluation of methonium desolvation thermodynamics. Methonium (-N+Me3, Am) is an organic cation widely distributed in biological systems. The appearance of methonium in biological transmitters and receptors seems at odds with the large unfavorable desolvation free energy reported for tetramethylammonium (TMA+), a frequently utilized surrogate of methonium. We report an experimental system that facilitates incremental internalization of methonium within the molecular cavity of cucurbit[7]uril (CB[7]).</p><p>Using a combination of experimental and computational studies we show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by a remarkably small desolvation enthalpy of just 0.5±0.3 kcal*mol-1, a value significantly less endothermic than those values suggested from gas-phase model studies (+49.3 kcal*mol-1). More surprisingly, the incremental withdrawal of methonium surface from water produces a non- monotonic response in desolvation enthalpy. A partially desolvated state exists, in which a portion of the methonium group remains exposed to solvent. This structure incurs an increased enthalpic penalty of ~3 kcal*mol-1 compared to other solvation states. We attribute this observation to the pre- encapsulation de-wetting of the methonium surface. Together, our results offer a rationale for the wide biological distribution of methonium and suggest limitations to computational estimates of binding affinities based on simple parameterization of solvent-accessible surface area.</p> / Dissertation

Conformationally constrained ligands and their flexible analogues were prepared as inhibitors of the Grb2 SH2 domain in order to study the structural and energetic effects of ligand preorganization in protein-ligand interactions. The compounds were prepared by using trans-cyclopropane-containing amino acid mimics, macrocyclization, or [alpha,alpha]-disubstituted amino acid residues. All trans-cyclopropane containing peptides were more potent than their corresponding succinate containing analogues due to an enthalpic advantage. Surprisingly, the binding of constrained peptides to the domain was entropically disfavored relative to their flexible controls. Effects of proton transfer and desolvation as being the source of the unprecedented entropic penalty for the constrained ligands relative to their respective controls were precluded, and X-ray crystallographic studies revealed that the binding conformations for the respective cyclopropane and succinate containing ligands were similar. This led us to believe that differential changes in protein dynamics may occur upon binding of the constrained and flexible ligands, which could contribute to the observed binding energetics. Two 23-membered macrocyclic ligands were slightly more potent than their corresponding linear controls. The amino acids used to link the N- and C-termini of the linear peptides to form the macrocycles were found to affect the energetics of binding. In one case, the 23-membered macrocycle was more potent than its control due to an entropic advantage, whereas the other 23-membered macrocycle was more potent than its control because it benefited from an enthalpic advantage. [alpha,alpha]-Disubstituted and [alpha]-monosubstituted residues that varied in hydrophobic character were incorporated into Grb2 SH2 domain binding tripeptides, and binding became more favorable as nonpolar surface area increased only for the set of tripeptides possessing cyclic [alpha,alpha]-disubstituted residues. The increase in affinity was due to an increasing enthalplic term, whereas the entropy of binding became less favorable. A total synthesis of (±)-lycopladine A was achieved in five steps from known compounds. The tricyclic core of the natural product was prepared utilizing a novel two-step sequence comprising a conjugate addition of a metalated picoline derivative followed by an intramolecular enolate arylation. It was demonstrated that the natural product existed in a solvent dependent equilibrium with its isomeric lactol. / text

The reaction between the tetrahedrane cluster RCCo3(CO)9{R = CHO (1), H (3)} and the redox-active diphosphine ligand 4,5-bis(diphenylphosphino)-4-cyclopenten-1,3- dione (bpcd) leads to the replacement of two CO groups and formation of RCCo3(CO)7(bpcd) {R = CHO (2), H (4)}. Clusters 2 and 4 are thermally unstable and readily transform into the new P-C bond cleavage cluster 5. All three clusters 2, 4, and 5 have been isolated and fully characterized in solution by IR and 31P NMR spectroscopy. VT 31P NMR data indicate that the bpcd ligand in RCCo3(CO)7(bpcd) is fluxional at 187 K in THF. Clusters 2, 4, and 5 have been structurally characterized by X-ray diffraction analyses.

Antibody microarrays constitute a next-generation sensing platform that has the potential to revolutionize the way that molecular detection is conducted in many scientific fields. Unfortunately, current technologies have not found mainstream use because of reliability problems that undermine trust in their results. Although several factors are involved, it is believed that undesirable protein interactions with the array surface are a fundamental source of problems where little detail about the molecular-level biophysics are known. A better understanding of antibody stability and antibody-antigen binding on the array surface is needed to improve microarray technology. Despite the availability of many laboratory methods for studying protein stability and binding, these methods either do not work when the protein is attached to a surface or they do not provide the atomistic structural information that is needed to better understand protein behavior on the surface. As a result, molecular simulation has emerged as the primary method for studying proteins on surfaces because it can provide metrics and views of atomistic structures and molecular motion. Using an advanced, coarse-grain, protein-surface model this study investigated how antibodies react to and function on different types of surfaces. Three topics were addressed: (1) the stability of individual antibodies on surfaces, (2) antibody binding to small antigens while on a surface, and (3) antibody binding to large antigens while on a surface. The results indicate that immobilizing antibodies or antibody fragments in an upright orientation on a hydrophilic surface can provide the molecules with thermal stability similar to their native aqueous stability, enhance antigen binding strength, and minimize the entropic cost of binding. Furthermore, the results indicate that it is more difficult for large antigens to approach the surface than small antigens, that multiple binding sites can aid antigen binding, and that antigen flexiblity simultaneously helps and hinders the binding process as it approaches the surface. The results provide hope that next-generation microarrays and other devices decorated with proteins can be improved through rational design.

Although the tremendous catalytic power of enzymes is widely recognized, their exact mechanisms of action are still a source of debate. In order to elucidate the origin of their power, it is necessary to look at individual residues and atoms, and establish their contribution to ligand binding, activation, and reaction. Given the present limitations of experimental techniques, only computational tools allow for such detailed analysis. During my PhD studies I have applied a variety of computational methods, reviewed in Chapter 2, to the study of two enzymes: DfrB dihydrofolate reductase (DHFR) and methyltetrahydrofolate: corrinoid/iron-sulfur protein methyltransferase (MeTr). ¶ The DfrB enzyme has intrigued microbiologists since it was discovered thirty years ago, because of its simple structure, enzymatic inefficiency, and its insensitivity to trimethoprim. This bacterial enzyme shows neither structural nor sequence similarity with its chromosomal counterpart, despite both catalysing the reduction of dihydrofolate (DHF) using NADPH as a cofactor. As numerous attempts to obtain experimental structures of an enzyme ternary complex have been unsuccessful, I combined docking studies and molecular dynamics simulations to produce a reliable model of the reactive DfrB•DHF•NADPH complex. These results, combined with published empirical data, showed that multiple binding modes of the ligands are possible within DfrB. ¶ Comprehensive sequence and structural analysis provided further insight into the DfrB family. The presence of the dfrB genes within integrons and their level of sequence conservation suggest that they are old structures that had been diverging well before the introduction of trimethoprim. Each monomer of the tetrameric active enzyme presents an SH3-fold domain; this is a eukaryotic auxiliary domain never found before as the sole domain of a protein, let alone as the catalytic one. Overall, DfrB DHFR seems to be a poorly adapted catalyst, a ‘minimalistic’ enzyme that promotes the reaction by facilitating the approach of the ligands rather than by using specific catalytic residues. ¶ MeTr initiates the Wood-Ljungdahl pathway of anaerobic CO2 fixation. It catalyses the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH3THF) to the cobalt centre of a corrinoid/iron-sulfur protein. For the reaction to occur, the N5 position of CH3THF is expected to be activated by protonation. As experimental studies have led to conflicting suggestions, computational approaches were used to address the activation mechanism. ¶ Initially, I tested the accuracy of quantum mechanical (QM) methods to predict protonation positions and pKas of pterin, folate, and their analogues. Then, different protonation states of CH3THF and active-site aspartic residues were analysed. Fragment QM calculations suggested that the pKa of N5 in CH3THF is likely to increase upon protein binding. Further, ONIOM calculations which accounted for the complete protein structure indicated that active-site aspartic residues are likely to be protonated before the ligand. Finally, solvation and binding free energies of several protonated forms of CH3THF were compared using the thermodynamic integration approach. Taken together, these preliminary results suggest that further work with particular emphasis on the protonation state of active-site aspartic residues is needed in order to elucidate the protonation and activation mechanism of CH3THF within MeTr.

<p>Computational protein design aims at identifying protein mutations and conformations with desired target properties (such as increased protein stability, switch of substrate specificity, or novel function) from a vast combinatorial space of candidate solutions. The development of algorithms to efficiently and accurately solve problems in protein design has thus posed significant computational and modeling challenges. Despite the inherent hardness of protein design, a number of computational techniques have been previously developed and applied to a wide range of protein design problems. In many cases, however, the available computational protein design techniques are deficient both in computational power and modeling accuracy. Typical simplifying modeling assumptions for computational protein design are the rigidity of the protein backbone and the discretization of the protein side-chain conformations. Here, we present the derivation, proofs of correctness and complexity, implementation, and application of novel algorithms for computational protein design that, unlike previous approaches, have provably-accurate guarantees even when backbone or continuous side-chain flexibility are incorporated into the model. We also describe novel divide-and-conquer and dynamic programming algorithms for improved computational efficiency that are shown to result in speed-ups of up to several orders of magnitude as compared to previously-available techniques. Our novel algorithms are further incorporated as part of K*, a provably-accurate ensemble-based algorithm for protein-ligand binding prediction and protein design. The application of our suite of protein design algorithms to a variety of problems, including enzyme redesign and small-molecule inhibitor design, is described. Experimental validation, performed by our collaborators, of a set of our computational predictions confirms the feasibility and usefulness of our novel algorithms for computational protein design.</p> / Dissertation