Abstract Charge density studies have been conducted on ten CB1 cannabinoid receptor antagonists via high resolution x-ray crystallography. Bond critical point values and various other properties derived from these studies including the electrostatic potential were analyzed in correlation to the affinity of each compound with the CB1 receptor. Correlation/anti-correlation was found between several properties and Ki. The data was also interpreted by principal component analysis with three principal components accounting for 85% of the data variation. Data mining was limit due to the low sample count and the requirements set for the inclusion of correlated/anti-correlated variables left fewer variables to analyze. The model presented is left for future interpretation.

Myostatin is a secreted growth factor of the transforming growth-factor $\beta$ (TGF$\beta$) superfamily, and a powerful negative regulator of muscle mass in vertebrates. As such, there is considerable interest in developing pharmacological agents which inhibit myostatin signalling in order to stimulate muscle growth in the context of pathological muscle wasting. Like other TGF$\beta$ family proteins, myostatin is biosynthesised as an inactive (latent) precursor protein which requires proteolytic processing to liberate the mature bioactive growth factor. To examine the molecular basis of pro-myostatin latency and the mechanism by which it is activated in the extracellular space, I have determined the crystal structure of unprocessed human pro-myostatin and studied the properties of the protein at various stages of activation. Crystallographic analysis of pro-myostatin reveals a unique domain-swapped dimeric structure, with an open V-shaped conformation distinct from the prototypical family member, TGF$\beta$1. Following cleavage of the prodomains by furin, pro-myostatin persists as a stable non-covalent complex which is resistant to the natural inhibitor follistatin and exhibits significantly weaker bioactivity than the mature growth factor. A number of distinct structural features combine to stabilise the interaction between pro and mature domains and in doing so confer latency to the pro-complex. This facilitates a controlled, step-wise process of activation in the extracellular space and contributes to a complex network of regulatory control. The results presented here provide a structural basis for understanding the effect of natural polymorphisms on myostatin function and a starting point for structure-guided development of next generation myostatin inhibitors. As a proof-of-concept, I present preliminary data on prodomain derived stapled peptides as inhibitors of myostatin signalling.

A bothropasina é uma proteína hemorrágica de 48 kDa, pertencente à classe P-III das metaloproteases, isolada a partir do veneno bruto da serpente brasileira Bothrops jararaca, e que possui os domínios adesivos desintegrina (D) e rico em cisteína (C). Neste trabalho, nós apresentamos a estrutura cristalográfica da bothropasina complexada ao inibidor POL647. O domínio catalítico , metaloprotease (M), pode ser dividido em dois subdomínios, dispostos de maneira muito similar aos descritos para essa família de metaloproteases de venenos de serpentes (em inglês \"SVMPs\"), que inclui os sítios de ligação ao zinco e ao cálcio. A cisteína livre, resíduo Cys189, está localizado em um núcleo hidrofóbico e, sendo assim, impossibilitado de fazer pontes dissulfeto ou qualquer outra interação. O domínio D não apresenta estruturas secundárias bem definidas, sendo constituído, majoritariamente, por estruturas desordenadas como \"loops\", porém estabilizados por 7 pontes dissulfeto e por dois íons cálcio. A região do motivo ECD está localizada em um \"loop\" e é estruturalmente relacionado à região RGD das desintegrinas-RGD, derivadas de SVMPs da classe P-II. O motivo ECD é estabilizado pela ponte dissulfeto Cys277-Cys310 (entre os domínios D e C), além de um íon cálcio. A cadeia lateral do Glu276 do motivo ECD está exposta ao solvente. Na bothropasina, a região hiper variada (em inglês HVR), descrita para outras P-III de SVMPs, presente no domínio C, de fato, é bastante conservada quando comparada a outros membros da classe P-III de diversas espécies. Nós propomos que esse subgrupo deva ser referido como PIII-HCR (região altamente conservada) SVMPs. Ainda é proposto que as diferenças estruturais dos domínios D, C ou DC possam estar envolvidas em uma melhor adaptação da estrutura na interação com diferentes alvos, além do reconhecimento e especificidade a um substrato para o domínio M. / Bothropasin is a 48kDa hemorrhagic P-III metalloprotease isolated from the venom of the Brazilian snake Bothrops jararaca, which has the disintegrin (D) and cysteine-rich (C) adhesive domains. We present the crystal structure of the bothropasin complexed with the inhibitor POL647. The catalytic domain, metalloprotease (M), consists of two subdomains in a very similar scaffold to the ones described for other snake venom metalloproteases (SVMPs) including the zinc and calcium binding sites. The free cysteine, residue Cys189, is in a hydrophobic core and it is not available for disulfide bonding or other interactions. The D domain does not have a defined secondary structure, but instead is composed by mostly loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and it is structurally related to the RGD region of RGD-disintegrins, which are derived from P-II SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between D and C domains) and by one calcium ion. The side chain of the Glu276 of the ECD motif is solvent exposed. In bothropasi, the HVR (hyper-variable region) described for other P-III SVMPs in the C domain in fact presents a well conserved sequence with respect to several other P-III members from different species. We propose that this subset be referred to as PIII-HCR (highly-conserved region) SVMPs. We further propose that the structural differences in the D, C or DC domains may be involved in selecting target binding which in turn could generate substrate diversity or specificity for the M domain.

Six new crystal structures were determined using a Rigaku Mercurcy 375/MCCD(XtaLab mini) diffractometer. The structure of a molybdenum cofactor was solved resulting in an R1 (R1 = Σ ||Fo| - |Fc|| / Σ |Fo|) of 3.61% despite the presence of a disordered DMSO molecule. New Tetrakis(N-phenylacetamidato) Dirhodium(II) complexes were synthesized and characterized. Two 2,2-cis-[Rh2(NPhCOCH3)4]•(C3H4N2)x where x= 1 or 2 were successfully crystallized and solved with R1 values below 5%. Additional studies were conducted via NMR to observe formation of both products. Three potential catalysts were synthesized starting with 3,1-[Rh2(NPhCOCH3)4]. The resulting compounds were a mono adduct 3,1-[Rh2(NPhCOCH3)4]•(C3H4N2), and two dimer of dimers complexes with amine bridges 3,1-[Rh2(NPhCOCH3)4]2•(C8H6N2) and 3,1-[Rh2(NPhCOCH3)4]2•(C10H8N2). All three complexes were crystallized and solved with R1 values less than 10%. Additional NMR studies were conducted to elucidate solid and solution phase structures and to determine the possibility of additional amine bonds forming.

For structure-based drug discovery, structural information of a target protein is necessary. NMR, or X-ray crystallography can provide necessary information on active site configuration that can lead a successful virtual screening campaign into identifying binders that may then be optimized into potent inhibitors. However, many challenges exist in the structure-based drug discovery cycle. For instance, structure determination of a protein of interest can many times be a daunting task. In addition, complex structure determination, which can allow essential characterization of protein-ligand interactions, is also challenging and many times impossible. Virtual screening heavily relies on such structural information, but hit-to-lead optimization schemes do as well. Furthermore, inherent protein characteristics such as conformational flexibility only add to the complexities in using structural information to identifying and optimizing inhibitors. In the scope of the work presented here, a structure-based drug discovery approach against three different protein targets is described. Each is presented with it's own set of challenges, but each has successfully led to the identification of new ligands. The drug discovery project against CXCL12 will first be described. CXCL12 is a small chemokine (~10KDa) that binds to the CXCR4 receptor promoting chemotaxis of lymphocytes but also metastasis of cancer cells. This interaction is further supported by sulfated tyrosines on CXCR4 that bind specific sites on the CXCL12 surface. The CXCL12-CXCR4 signaling axis has been a major focus of drug discovery, but efforts are mainly focused on CXCR4, since CXCL12 is a small protein lacking surface characteristics that are thought to be druggable. Yet, through a combination of rigid, flexible, and ensemble docking in virtual screening studies, we have successfully identified compounds that bind each of the three sulfotyrosine recognition sites on CXCL12, which normally bind the sulfated tyrosines on CXCR4 (sY7, sY12, and sY21). Furthermore, we have led a hit-to-lead approach in optimizing compounds against the sY21-binding site, aided by trivial information gained through crystallographic complex structure determination of CXCL12 bound by such a compound. We aim to eventually link compounds against different sites together and greatly improve potency. Next, the drug discovery project against P. aeruginosa LpxA will be described. In Gram-negative bacteria, the first step of lipid A biosynthesis is catalyzed by UDP-N-acetylglucosamine acyltrasferase (LpxA) through the transfer of a R-3-hydroxyacyl chain from the acyl carrier protein (ACP) to the 3'-hydroxyl group of UDP-GlcNAc. Acyl chain length selectivity varies between species of bacteria, but is highly specific and conserved within certain species. In E. coli and L. interrogans for example, LpxA is highly selective for longer R-3-hydroxyacil chains (C14 and C12 respectively), while in P. aeruginosa the enzyme is highly selective for R-3-hydroxydecanoyl, a 10-hydrocarbon long acyl chain. Three P. aeruginosa LpxA crystal structures will be described here for the first time; the apo form, the complex with its substrate UDP-GlcNAc, and the complex with its product UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc. A comparison between the APO form and complexes identifies key residues that position UDP-GlcNAc appropriately for catalysis, and supports the role of His121 in generating the nucleophile by interacting with the UDP-GlcNAc 3'-hydroxyl group. Furthermore, the product-complex structure supports the role of Met169 as the "hydrocarbon ruler", providing structural information on how P. aeruginosa LpxA is granted its exceptional selectivity for the 10-hydrocarbon long acyl chain. Structural information of the active site was subsequently used in designing virtual screening experiments that led to the identification of two ligands, confirmed by X-ray crystallography screening to bind to the active site. We aim to continue application of X-ray crystallography into screening compound binding, and to also use a hit-to-lead approach in compound optimization. Finally, the drug discovery project against the Tiam1 PDZ domain will be described. Tiam1 (T-cell lymphoma invasion and metastasis gene 1) is a GEF (guanine exchange factor) protein that activates Rac1 and initiates tumor formation. Tiam1 is regulated through its PDZ domain, which binds to syndecan1. We have successfully applied a virtual screening strategy to an existing crystallographic structure of the Tiam1 PDZ domain complexed to a syndecan1 peptide and identified four ligands that bind to the PDZ domain with low affinities. These compounds provide a starting point for future hit-to-lead optimization strategies.

La régulation de la transcription est un processus dynamique faisant intervenir le recrutement de complexes protéiques impliqués dans le remodelage de la chromatine. Parmi eux, mon travail s’est focalisé sur le complexe NuRD (Nucleosome Remodeling and histone Deacetylation) et sa sous-unité de liaison à l’ADN CpG MBD3. Pour cela une approche de biologie structurale intégrative combinant la préparation biochimique, la caractérisation biophysique et l’étude structurale par cryo-EM et cristallographie aux rayons-X a été mise en place. Les caractérisations biophysiques de MBD3 ont permis de mettre en évidence son interaction avec un ADN non-modifié CpG et des cristaux diffractant jusqu’à 3.9 Å ont été obtenu. De plus la région désordonnée en aval du domaine de liaison a été identifiée et son impact dans la formation de complexe caractérisé. Des cristaux pour les différentes constructions en complexe avec l’ADN ont été obtenus et sont actuellement optimisés. Enfin l’optimisation de la purification et la préparation du complexe, ont permis la visualisation du complexe NuRD et mettent en avant pour la première fois une organisation en domaines du complexe. / Transcription regulation of chromatin is a very dynamic process regulated through the recruitment of chromatin-remodeling complexes. My work focuses on NuRD for Nucleosome remodeling and histones deacetylation complex a 1 MDa multi-subunit protein complex and its subunit MBD3 a CpG-binding protein and more precisely on an integrated biology approach of this molecular assembly and its interaction with DNA. It combines biochemical preparation, biophysical characterization, single particle cryo-eletron microscopy and x-ray crystallography. Biophysical analysis show that MBD domain of MBD3 interacts with unmodified CpG DNA, a crystal diffracting up to 3.9 Å were obtained. Moreover a C-terminal intrinsically disordered region of MBD3 were identified and despite is inherent disorder seems to increase the binding affinity of MBD3 for DNA. Crystals were obtained for both constructs in complex with DNA and are currently optimized.Cryo-EM study of NuRD complex allows us to develop and optimized purification and grids preparation for the visualization of the complex. The present results reveal a domain organization of the complex never identify before.

<p>Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation. </p><p>Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation.</p><p>We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils.</p><p>The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases.</p>

<p>Cytochrome <i>cd</i>₁ nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the <i>d</i>₁ haem iron of the enzyme.</p><p>Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions.</p><p>Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction.</p><p>Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty.</p><p>Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.</p>

<p>Microorganisms that degrade lignocellulose play an important role in maintaining the global carbon cycle. Under cellulolytic conditions, the fungus <i>Phanerochaete chrysosporium</i> produces an extracellular flavocytochrome, cellobiose dehydrogenase (CDH), with a proposed role in lignocellulose degradation. CDH consists of 755 amino acids including a C-terminal flavodehydrogenase linked by a peptide hinge to an N-terminal <i>b</i>-type cytochrome. The enzyme catalyses the oxidation of cellobiose to cellobiono-1,5-lactone, followed by transfer of electrons to an electron acceptor, either directly by the flavodehydrogenase domain, or via the cytochrome domain. This thesis presents a structural study on the individual domains of <i>P. chrysosporium</i> cellobiose dehydrogenase.</p><p>The crystal structure of the cytochrome was determined at 1.9 Å resolution. It folds as a β-sandwich with the topology of the antibody Fab V(H) domain, and the haem iron is ligated by Met65 and His163. This is only the second example of a <i>b</i>-type cytochrome with this ligation. The haem propionates are surface exposed to facilitate interdomain electron transfer.</p><p>The structure of a cytochrome Met65His mutant was determined at 1.9 Å resolution. In the mutant, the iron is ligated by the histidyl δ and ε nitrogens, rather than the usual N-ε/N-εligation. This is the first example of a <i>bis</i>-His N-ε/N-δ coordinated protoporphyrin IX iron. The structure of the flavoprotein domain was determined at 1.5 Å resolution. It is partitioned into an FAD-binding subdomain of α/β-type and a substrate-binding subdomain consisting of a seven-stranded β-sheet and six α-helices. Furthermore, the structure of the flavoprotein with the inhibitor cellobiono-1,5-lactam at 1.8 Å resolution lends support to a hydride-transfer mechanism for the reductive-half reaction of CDH although a radical mechanism cannot be excluded.</p>

<p>Binding proteins are present both in gram-negative and gram-positive bacteria. They are the recognition components of the ABC transport systems that transport different nutrients into the cell, and are in some cases also involved in chemotaxis. In gram-negative bacteria, they are present in the periplasm between the inner and the porous outer membrane. Here, these highly specific proteins can bind to a certain ligand such as ions, sugars and amino acids. The protein-ligand complex can then interact with permeases bound to the inner membrane that transport the nutrient into the cell. Gram-positive bacteria lack an outer membrane and the binding protein must therefore be anchored to the cell membrane.</p><p>In this thesis different aspects of three members of the super-family of the periplasmic binding proteins have been studied. In the case of the allose-binding protein (ALBP) from <i>E. coli</i> we focused on the movement of the protein when ligand is bound and released. This protein was also compared with the ribose-binding protein (RBP) which belongs to the same structural cluster and from which both open and closed structures are available. The leucine-binding protein (LBP) from <i>E. coli</i> was studied with regards to the structural basis of its specificity for different ligands as well as its conformational changes. The leucine-isoleucine-valine protein has 80% sequence identity with LBP but still exhibits a different preference for ligands. The structure of the maltose-binding protein (MBP) was obtained from a gram-positive thermoacidophile, <i>A. acidocaldarius. </i>Here, our goal was to study acid-stability of proteins. Since little is known about this and structures of the mesophilic counterpart in <i>E. coli</i> are available, as well as structures from two hyperthermophiles, we had an opportunity to study differences in their structural properties that could explain their differing stabilities.</p>