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The tubulin/FtsZ superfamily contains a large set of proteins that spans through all kingdoms of life, with αβ-tubulins being the eukaryotic representatives and FtsZ being the best studied prokaryotic homologue. It is believed that all tubulin/FtsZ-related proteins have evolved from a common ancestor, however, members from this superfamily have diverged in many aspects. αβ-tubulins polymerise into giant and hollow microtubules in the presence of GTP. Despite the size of around 25 nm wide, microtubules display sophisticated dynamics. In particular, dynamic instability, the stochastic change between fast growth and rapid shrinkage, is a hallmark of microtubules. In contrast to αβ-tubulins, FtsZ lacks the C-terminal domain of tubulins and it probably functions as single homopolymeric protofilaments, possibly through treadmilling dynamics. There is strong divergence of the biological functions in the tubulin/FtsZ superfamily. Microtubules are involved in fundamental processes such as motility, transport and chromosomal segregation, whereas FtsZ is involved in bacterial cytokinesis (bacterial cell division), and the equivalent role of FtsZ is carried out by actin-based and ESCRTIII-based systems in eukaryotes. It seems that there is a big evolutionary gap between αβ-tubulins and FtsZ, and the only properties that are conserved within the tubulin/FtsZ superfamily are fold, protofilament formation and GTPase activity. In 2002, a pair of tubulin-like genes, btuba and btubb were identified in Prosthecobacter bacteria, with higher sequence homology to eukaryotic tubulins than FtsZ or any other bacterial homologues. The crystal structures solved later revealed, again, a closer similarity to αβ-tubulins than to their prokaryotic equivalents. It has been known for a while that BtubAB form filaments in the presence of GTP, however, little knowledge has been available regarding the filament architecture. In this project, I determined the near atomic resolution structure of the in vitro BtubAB filament using cryoEM and cryoET, revealing a hollow tube that consists of four protofilaments. A closer look showed that BtubAB filaments have many conserved microtubule features including: an overall polarity, similar longitudinal contacts, M-loops in lateral interfaces, and the presence of the seam, a structural hallmark of microtubules. My study also shows that BtubC, a protein with a TPR fold, binds to the BtubAB filaments in a stoichiometric manner, similar to some MAPs on microtubules. Based on this work, I concluded that BtubAB from Prosthecobacter form bacterial ‘mini microtubules’, and my work provided interesting insight into the evolution of tubulin/FtsZ-related proteins.

Translation is the biological process that ultimately leads to the synthesis of a protein from the genetic material mRNA. Protein synthesis is essential for life as we know it, which is rooted in its extreme conservation throughout all living organisms. Translation is typically divided into four phases, the first of which, denominated translation initiation, is the most delicate step, as it entails the determining the correct start site of the produced protein. Previous structural studies allowed us to gain important insights into the position of translation Initiation Factors, and their function during the formation of the bacterial Initiation Complex and the proper positioning of the initiator tRNA on the start codon of the mRNA. Nevertheless, the limited resolution of the structures hampered gaining a pristine view of the molecular details that are essential for the correct assembly of this important step of translation. Moreover, little information is available regarding the differences underlying the initiation of translation on non-canonical start codons. Driven by recent improvements in cryo-EM, this work aims to fill these gaps and shed molecular insights into bacterial initiation of translation. This work further highlights for the first time, at molecular resolution, multiple important interactions that occurs between the 30S subunit, mRNA, initiator tRNA and initiation factors during the process of Initiation Complex formation. Supported by the structural data obtained, a new model for the order of initiation complex assembly is suggested. The model presented underlines the complexity of bacterial initiation of translation and paves the way for future experiments to gain a holistic view of this step of translation.

Complex I (NADH:ubiquinone oxidoreductase), located in the mitochondrial inner membrane, is a major electron entry point to the respiratory chain. It couples the energy released from electron transfer (from NADH to ubiquinone) to the concomitant pumping of protons across the membrane, to generate an electrochemical proton motive force. Mammalian complex I is composed of 45 subunits, 14 of which comprise its simpler bacterial homologues. It is encoded by both the mitochondrial and nuclear genomes, and pathological mutations in both sets of subunits result in severe neuromuscular disorders such as Leigh syndrome. Several structures of mammalian complex I from various organisms have been determined, but the limited resolutions of the structures, which typically refer to poorly characterised enzyme states, has hampered detailed analyses of mechanistic features. The first part of this thesis describes development of a method for purifying complex I from the genetically amenable and medically relevant model organism Mus musculus (mouse), in a pure, stable and active state. The enzyme from mouse heart mitochondria was then comprehensively characterised, to ensure the presence of all the expected subunits and co-factors, and to define its kinetic properties. The second part of this thesis describes structural studies by single particle electron cryomicroscopy (cryo-EM) on the purified mouse enzyme in two distinct states, the 'active' and 'de-active' states. The active state was determined to 3.3 Å resolution, the highest resolution structure of a eukaryotic complex I so far. Subsequently, comparison of the two mouse structures, together with previously determined mammalian and bacterial structures, revealed variations in key structural elements in the membrane domain, which may be crucial for the catalytic mechanism. Moreover, in the high-resolution active mouse complex I structure a nucleotide co-factor was observed bound to the nucleoside kinase subunit NDUFA10. Finally, complex I from the Ndufs4 knockout mouse model, which recapitulates the effects of a human mutation that causes Leigh syndrome, was purified and subjected to kinetic and proteomic analyses. Following cross-linking and preliminary structural studies, it was concluded that the detrimental effects of deleting NDUFS4 are due to lack of stability of the mature complex.

La détermination de la structure des protéines à une résolution atomique est cruciale pour la compréhension de leur fonction cellulaire. Actuellement, la cristallographie aux rayons X est la méthode la plus efficace pour la détermination à haute résolution de la structure de protéines monomériques allant 40 et 100 kDa. Par contre, elle est limitée par la croissance de cristaux de bonne qualité, qui est problématique pour nombreuses cibles. La cryo-microscopie électronique (cryoME) permet la détermination structurale à résolution quasi-atomique de larges structures protéiques, de préférence symétrique et en solution. Cependant, les images de cryoME sont très bruitées, car une faible dose d'électrons est appliquée de manière à limiter les dommages d'irradiation. En moyennant des dizaines d'images correspondant à la même orientation moléculaire, le rapport signal sur bruit est amélioré. La combinaison des images moyennées de plusieurs orientations permet l'obtention d'une carte de densité électronique 3D de la molécule d'intérêt. Si la taille et la symétrie de la molécule diminuent, l'analyse cryoME devient de moins en moins précise, il est alors impossible d'analyser des protéines monomériques de taille inférieure à 100 kDa. Le but de ce travail a été de développer une nouvelle approche pour réduire cette limite de poids moléculaire. Elle consiste à fusionner la protéine d'intérêt (cible) à une matrice homo-oligomérique, générant une particule symétrique et de taille importante adaptée à l'analyse par cryoME. Dans cette thèse, nous avons cherché à tester et démontrer la faisabilité de cette approche de symétrisation en utilisant des protéines cibles de structure connue.Pour mettre en place notre étude pilote, nous avons choisi différentes combinaisons de cibles et de matrices connectées par des peptides de liaison (linker) de longueur différentes. Nous avons caractérisé les fusions exprimées en bactéries par microscopie électronique après coloration négative et par plusieurs techniques biophysiques. Grace à ces techniques, nous avons trouvé que la meilleure combinaison est la fusion entre la protéine matrice glutamine synthétase (GS), un 12-mer de symétrie D6 et la cible maltose binding protein (Mbp), connectées par un linker contenant trois alanines, que nous avons appelée « Mag ». En jouant sur la longueur du linker nous avons ensuite sélectionné la fusion la plus compacte pour l'analyse cryoME: MagΔ5. Nous avons obtenu la carte cryoME à 10 Å de MagΔ5, qui présente une bonne corrélation avec les modèles atomiques de Mbp et GS. Plus particulièrement, le site catalytique et quelques hélices α sont identifiables. Ces résultats sont confirmés par l'étude cristallographique que nous avons conduite sur MagΔ5. L'ensemble de ce travail souligne que la présence d'une grande interface d'interactions cible-matrice stabilise la fusion et améliore la résolution en cryoME. Pour la symétrisation d'une cible inconnue, nous envisageons la même procédure expérimentale que celle développée pour MagΔ5. La matrice et le linker les plus adaptés devront être identifiés en utilisant les mêmes méthodes biophysiques.En conclusion, ce travail établit la preuve de concept que la méthode de symétrisation des protéines permet la détermination de la structure de protéines de poids moléculaire inférieur à 100 kDa par cryoME. Cette méthode a le potentiel d'être un nouvel outil prometteur, qui faciliterait l'analyse de cibles résistantes à l'analyse structurale conventionnelle. / Structural determination of proteins at atomic level resolution is crucial for unravelling their function. X-ray crystallography has successfully been used to determine macromolecular structures with sizes ranging from kDa to MDa, and currently remains the most efficient method for the high-resolution structure determination of monomeric proteins within the 40-100 kDa range. However, this method is limited by the ability to grow well diffracting crystals, which is problematic for several targets, such as membrane proteins. Single particle cryo electron microscopy (cryoEM) allows near atomic (3-4Å) resolution structural determination of large, preferably symmetric, assemblies in solution. Biological molecules scatter electrons weakly and, to avoid radiation damage, only low electron doses can be used during imaging. Consequently, raw cryoEM images are extremely noisy. However, averaging many molecular images aligned in the same orientation permits one to increase the signal-to-noise ratio, ultimately allowing the achievement of a 3D density map of the molecule of interest. Nevertheless, as the molecular size and degree of symmetry decrease, the individual images loose adequate features for accurate alignment. Currently, cryoEM analysis is practically impossible for monomeric proteins below ~100 kDa in mass. We propose to circumvent this obstacle by fusing such monomeric target proteins to a homo-oligomeric protein (template), thereby generating a self-assembling particle whose large size and symmetry should facilitate cryoEM analysis. In the present thesis we seek to test and demonstrate the feasibility of this ‘protein symmetrization' approach and to evaluate its usefulness for protein structure determination. To set up the pilot study we combined selected targets of known structure with two templates: Glutamine Synthetase (GS), a 12-mer with D6 symmetry and a helical N-terminus, and the E2 subunit of the pyruvate dehydrogenase complex, a 60-mer with icosahedral symmetry and an unstructured N-terminus. After recombinant production in E.coli we identified by negative stain EM a promising dodecameric chimera for structural analysis, comprising maltose binding protein (Mbp) connected to GS by a tri-alanine linker (denoted “Mag”). In order to optimize sample homogeneity we produced a panel of Mag deletion constructs by sequentially truncating the 17 residues between the Mbp and GS domains. A combination of biophysical techniques (thermal shift assay, dynamic light scattering, size exclusion chromatography) and negative stain EM allowed us to select the best candidate for cryoEM analysis, MagΔ5. By enforcing D6 symmetry we obtained a cryoEM map with a resolution of 10Å (FSC 0.5 criterion). The density of the symmetrized 40 kDa Mbp presents shape and features corresponding to the known atomic structure. In particular, the catalytic pocket and specific α-helical elements are distinguishable. The cryoEM map is additionally validated by a 7Å crystal structure of the MagΔ5 oligomer. The presence of a continuous helical connection between target (Mbp) and template (GS) likely contributed to the conformational homogeneity of MagΔ5. Moreover, comparing MagΔ5 with other chimeras studied in this work suggests that a large buried surface area and favorable interactions between the target and template limit the flexibility of the chimera and improve its resolution by cryoEM. For the symmetrization of a target of unknown structure, we envisage proceeding by a trial and error approach by fusing it to a panel of templates with helical termini and different surface properties, and subsequently selecting the best ones using biophysical assays. In conclusion, the present work establishes the proof-of-concept that protein symmetrization can be used for the structure determination of monomeric proteins below 100 kDa by cryoEM, thereby providing a promising new tool for analyzing targets resistant to conventional structural analysis.

PSD95 is the canonical member of the Membrane Associated Guanylate Kinase class of scaffold proteins. PSD95 is a five-domain major scaffolding protein abundant in the postsynaptic density (PSD) of the neuronal excitatory synapse. Within PSD95 three PDZ domains modulate protein-protein interactions by selectively binding to short peptide motifs of target proteins. Under the direction of the multivalent PDZ domain interactions, the interacting proteins tend to cluster at the PSD, a phenomenon that is critical for synaptic signalling regulation. Earlier studies have shown that the N-terminal PDZ domains of PSD95 are obligatory for the clustering to occur. This thesis focuses on the strong inwardly rectifying potassium channel, Kir2.1 as the PSD95 binding partner. Kir2.1 is known to maintain membrane resting potential and control cell excitability. Previous studies have reported that Kir2.1 clustered into ordered tetrad complexes upon association with PSD95.This study investigates the detailed clustering mechanisms of Kir2.1 by PDZ domains. To achieve this, components that are involved in the formation of a complex namely PSD95 sub-domains comprising single PDZ and the tandem N terminal PDZ double domain (PDZ1-2), and Kir2.1 cytoplasmic domains(Kir2.1NC) are studied in detail via different structural and biophysical approaches; 1) PDZ1-2 is examined in apo- and bound ligand form with a Kir2.1 Cterminal peptide in crystal and solution via X-ray crystallography and small angle X-ray scattering; 2) the tandem and the single PDZ domain interaction with ligand are measured thermodynamically via isothermal calorimetry (ITC); 3) the complex of full length PSD95 with Kir2.1NC is analyzed with electron microscopy (EM). The protein components are produced in high quality by protein expression and multiple-step protein purification techniques. PDZ1-2 crystallographic structures were solved at 2.02A and 2.19A in theapo- and the liganded forms respectively. The solution state analysis showed domain separation and structural extension of the tandem domain when incorporated with the ligand. The ITC experiment revealed PDZ1-2 to have greater affinity towards the peptide ligand relative to the single PDZ domains. These combinatorial outcomes lead to the conclusion that PSD95 clusters Kir2.1 by adopting an enhanced binding interaction which is associated with increased PDZ1-2 inter-domain separation. The preliminary analysis of PSD95-Kir2.1NC complex with cryo-EM showed the establishment of a tetrad and led to a reconstruction at 40A resolution. The work in obtaining a higher resolution complex structure is promising with further data collection required to allow the employment of more sophisticated model reconstruction processes.

Semester project deals with the processing of data from TEM and their analysis (to- mography, single particle analysis). The main aim of this work is to determine the 3D structure of the studied enzyme. As a test sample with low symmetry is used restriction endonuclease EcoR124I.

<p dir="ltr">The CRISPR-Cas systems, originally evolved as bacterial and archaeal adaptive immune systems against viral infections, have been ingeniously repurposed for genome editing. The ongoing evolutionary competition between bacteria and phages has given rise to the diversification of CRISPR-Cas systems, which can be broadly classified into two classes and six types. Among these, the versatile CRISPR type V family stands out as a promising source for discovering new CRISPR-Cas effectors to expand the genome editing toolbox. However, before proceeding to genome editing applications, it is imperative to get a comprehensive understanding of the mechanisms underlying how Cas effectors function as programmable RNA-guided nucleases. Structural studies play a pivotal role in elucidating these mechanisms, providing a clear picture of processes such as DNA recognition and cleavage.</p><p><br></p><p dir="ltr">In the first part of this thesis, we embarked on determining the cryo-EM structures of an extraordinarily small type V-F CRISPR-Cas effector, Cas12f. Our findings unveiled that Cas12f functions as an asymmetric dimer. Through structural analysis and mutagenesis experiments, we elucidated the mechanisms of PAM recognition and substrate cleavage by Cas12f. Furthermore, we provided insights into the activation mechanism of Cas12f by monitoring its conformational changes before and after the crRNA-target DNA heteroduplex formation. Our results contribute to our understanding of the type V Cas effector nucleases and hold promise for possible applications of genome editing.</p><p><br></p><p dir="ltr">In the second part, we focused on study of CRISPR-associated transposons (CASTs). Specifically, we delved into Cas12k, a component of the type V-K CRISPR-Cas system, which is a naturally inactivated nuclease but is interestingly associated with transposons and is capable for guiding transposition. We determined the structure of Cas12k in complex with the guide RNA and target DNA. Our studies revealed target site recognition mechanism and the structural features of Cas12k critical for downstream CIRPSR-guided DNA transposition.</p><p><br></p><p dir="ltr">Lastly, we directed our attention towards the ancestor of CRISPR type V systems, TnpB, which serves as a minimal programmable RNA-guided DNA nuclease originating from the IS200/IS605-like transposon family. To reveal the molecular mechanisms of substrate recognition and cleavage, multiple approaches including artificial dimers was introduced to obtained the cryo-EM structure of <i>Isdra2</i> TnpB-gRNA-target DNA ternary complex. Furthermore, our exploration extended to the investigation of newly emerged TnpB variants. Among these variants, one was identified as a naturally occurring transcription repressor. We attained the cryo-EM structure of this variant at 3.12 Å and currently working on understanding its mechanism.</p>

<p>The β-barrel assembly machinery (BAM) is responsible for the biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria. These OMPs have a membrane-embedded domain consisting of a β-barrel fold which can vary from 8 to 36 β-strands, with each serving an important role in the cell such as nutrient uptake and virulence. BAM was first identified nearly two decades ago, but only recently has the molecular structure of the full complex been reported. Together with many years of functional characterization, we have a significantly clearer depiction of BAM's structure, the intra-complex interactions, conformational changes that BAM may undergo during OMP biogenesis, and the role chaperones may play. But still, despite advances over the past two decades, the mechanism for BAM-mediated OMP biogenesis has remained elusive. Over the years, several theories have been proposed that have varying degrees of support from the literature, but none has of yet been conclusive enough to be widely accepted as the sole mechanism. Here we present our recent work on the structures of BAM in its near native environment, characterized by cryo-EM, and study its interaction with OMP substrates. Specifically, we focused on the role of BAM-mediated EspP biogenesis, and structurally characterized crosslinked intermediates to atomic resolution, allowing for a more complete understanding of BAM-mediated OMP biogenesis. We also characterized BAM-mediated OmpT and OmpA biogenesis, which further supports a BamA-budding model for OMP biogenesis. Given its essential role in Gram-negative bacteria, BAM is an attractive target for antibiotics, and we contributed to characterizing a novel antibiotic designed against BAM called darobactin, which binds to the lateral gate of BAM, thereby disrupting OMP biogenesis and leading to programmed bacterial lysis.</p>

Transcriptional deregulation has emerged as one of the leading causes in various human diseases. More than fifty percent of cancers arise due to frequent mutations in genes regulating transcription. Higher-order assembly via protein-protein interactions is one common mechanism of transcriptional regulation. Despite their critical role in regulating gene transcription and therapeutic relevance, detailed mechanistic understanding of these assemblies remains scarce. The primary focus of this thesis is to uncover important structural principles underlying the assembly and stability of multi-domain protein assemblies by characterizing components of the IFNβ enhanceosome and the CtBP-mediated repression complex. Using a combination of biochemical and structural analyses, I showed that the transcriptional activator C-terminal binding protein 2 (CtBP2) is a tetramer by solving the 3.6Å cryoEM structure of CtBP2. I highlighted the types of interactions that stabilize the homo-tetramer and showed the relevance of the tetramer in CtBP2 transcriptional activity. Second, I used an integrative approach to investigate the structural features leading to activation of interferon regulator factor 3 (IRF3) and its interaction with DNA. Although this work mostly focused on components of the CtBP2-mediated complex and IFNβ enhanceosome, the principles described here can be applied to other complexes. Therefore, these studies provide an overall understanding on how other macromolecular complexes regulate gene transcription. Furthermore, our structural-based analyses will provide a basis for designing drugs that can regulate gene transcription in cancer and immunological disorders.