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[spa] Los Picornaviridae son una de las familias de virus más diversas y conocidas desde hace más tiempo. Esta familia incluye importantes agentes patógenos que afectan a humanos y a animales. Los Picornaviridae son virus pequeños, icosaédricos, de ARN de cadena sencilla de sentido positivo y causan una gran variedad de enfermedades, tales como encefalitis y poliomielitis. Se dispone de vacunas para el poliovirus, el virus de la hepatitis A y el virus de la fiebre aftosa, pero no se ha implementado ninguna profilaxis efectiva para otros picornavirus. Hasta ahora, la investigación antiviral se ha centrado en la cápside, mientras que los inhibidores dirigidos a proteínas no estructurales (como las proteasas, las helicasas y las polimerasas) están todavía por explorar. Este proyecto se centró en la caracterización estructural y bioquímica de la proteasa 3C de enterovirus B93 (EV-B93) y de los complejos de esta proteasa con varios inhibidores covalentes. El segundo objetivo fue conseguir inhibidores no covalentes de la proteasa EV-B93 3C y realizar una caracterización bioquímica, antiviral y estructural de los complejos de EV-B93 3C con estos inhibidores., [eng] Picornaviridae are among the most diverse and oldest known viral families that include many important pathogens of humans and animals. They are small, icosahedral (+)ssRNA viruses, causing a variety of diseases, such as encephalitis, and poliomyelitis. Vaccines are available for poliovirus, hepatitis A virus and foot-and mouth disease virus, but no effective prophylaxis is implemented for other picornaviruses. Thus far, anti-viral research has focused on the capsid, whereas inhibitors targeting non-structural proteins (i.e. proteases, helicases, polymerases) have remained largely unaddressed. The project was focused on structural and biochemical characterization of the enterovirus-B93 (EVB93) 3C protease alone and in complex with several covalent inhibitors. The second objective was to identify the first non-covalent potent inhibitors of the EV-B93 3C protease and their further biochemical, antiviral, and structural evaluation. This work studied the in-vitro proteolytic activity of the EV-B93 3C protease, alone and in the presence of two known covalent inhibitors - rupintrivir and compound 1, as well as three low molecular weight covalent inhibitors - NZO, NZN and DB5_60. The crystal structures of the EV-B93 3C protease alone and in complex with rupintrivir, compound 1, and NZN molecule were solved at high resolution (1.57, 1.50, 1.32, and 1.73 Å, respectively). The structures revealed that the protein adapts a chymotrypsinlike fold similarly to other picornavirus 3C proteases and possesses His-40, Glu-71 and Cys-147 as a catalytic triad. The STD NMR-based fragment screening was performed to select non-covalent binders of the EV-B93 3C protease. Validation and profiling of the most promising non-covalent hits were done using thermal shift assay (TSA), surface plasmon resonance (SPR), and proteolytic activity assay. 44 analogs of the most potent molecule were evaluated in the in-vitro proteolytic activity assay. The most active compound displayed IC50 value of 5 flM. Further chemical optimization was performed resulting in more efficient inhibitor with similar IC50 value. Selected analogs were tested in the in-vitro proteolytic assay against analogous 3C proteases from the following viruses: human rhinovirus-A49, enterovirusD68, aichivirus A, porcine sapelovirus, and equine rhinitis B virus. All compounds exhibited good inhibitory activity against three of the tested proteases. Furthermore, in a cell-based proteolytic assay and an antiviral assay the compounds did not exhibit either proteolytic or antiviral activity, which may be explained by several factors such as lack of cell permeability, low solubility and/or high toxicity. Extensive co-crystallization and soaking trials were performed to obtain crystal structures of noncovalent complexes of the EV-B93 3C protease with the most potent compounds. Regrettably, no additional electron density was identified in the proteolytic active site. Bioinformatics docking simulations suggested potential binding mode of the optimized compound. These pointed to the presumed pockets occupied by the compound that interact with the two conserved residues from the catalytic triad. Since the most potent compound is a relatively large and rigid molecule, it is unable to bind to the protease without its previous rearrangement, which is unfavorable in the crystalline state of the protein. This observation may explain the inability of the non-covalent molecules to co-crystallize with EV-B93 3C protease. The results obtained in this study may aid the design of potent, noncovalent antivirals targeting enteroviral 3C proteases.

[spa] La presente tesis consta de dos proyectos, que detallo a continuación: - Estudio de las proteínas de la partícula regulatoria del proteosoma y del ensamblaje de la lid El sistema ubiquitina-proteosoma es la mayor ruta proteolítica citosólica en eucariotas, controla casi todos los procesos celulares básicos degradando las proteínas unidas a una cadena de ubiquitinas. El sistema se compone de una cascada de ligasas de ubiquitina que conjugan la ubiquitina a las proteínas diana y el proteosoma responsable de la degradación de dichas proteínas. El proteosoma 26S presente en eucariotas se puede dividir en dos complejos: la partícula central (20S) y la partícula regulatoria (19S), a su vez la partícula regulatoria puedo dividirse en dos subcomplejos: la base y la lid. En este proyecto se realizó el clonaje, expresión heteróloga y purificación de todas las proteínas Rpn de la lid y tres de la base (Rpn1, 2 y 10) de Saccharomyces cerevisiae en Escherichia coli. Las proteínas se expresaron individualmente y en conjunto (mediante la co-expresión y/o co-lisis). Estos experimentos resultaron en una amplia variedad de subcomplejos, desde complejos de dos proteínas hasta cinco y el reemplazo de algunas subunidades por versiones truncadas proporcionó información sobre áreas discretas de interacción entre proteínas, permitiendo crear un modelo de interacciones intermoleculares dentro del complejo de la lid. Además tres de los complejos obtenidos (Rpn5/8/9, Rpn5/8/9/11 y Rpn5/6/8/9/11) se analizaron mediante microscopia electrónica (EM) proporcionando una visión de los eventos biogénicos de la lid, ya que representan la adición consecutiva de subunidades. - Análisis estructural del dominio OBD de RepB y su unión al ADN El plásmido de amplio espectro pMV158 se replica mediante el mecanismo de círculo rodante. Su proteína de iniciación de la replicación es RepB, la cual reconoce y corta el ADN en un sitio específico. En un trabajo previo realizado en nuestro laboratorio se resolvió la estructura tridimensional de esta proteína full length, demostrando su naturaleza hexamérica. Cada monómero de RepB se compone de dos dominios: el OBD (origin-binding domain) responsable del reconocimiento del sitio de corte y el OD (oligomerization domain) que se unen entre si formando el hexámero. En este trabajo se cristalizó y resolvió mediante reemplazo molecular la estructura del OBD de RepB unido a su ADN diana, un oligonucleótido de 23bp que aloja parte de la secuencia del sitio de unión al ADN. La estructura nos permitió analizar la interacción específica proteína/ADN, la cual se establece en el surco mayor, pudiendo identificar los aminoácidos que interaccionan con áreas discretas en el ADN, siendo estos resultados comparables con aquellos descritos con anterioridad en la literatura mediante otras técnicas como mutaciones puntuales o estudios de fingerprinting. A su vez generamos un modelo que explica el modo de acción de RepB a la hora de reconocer el sitio de corte en su condición hexamérica., [eng] This PhD thesis is composed of two different projects: •Study of the proteasome regulatory particle proteins and the assembly of the lid complex. The 26S proteasome, the main protein-degradation machine in the eukaryotic cell, is composed of two discrete complexes: core and regulatory particle (RP), which can be sub-divided into the base and lid sub-complexes. By combining biochemical and structural methods, we have analyzed the interactions that hold the RP. Expressing individually or in complex the full length or truncated proteins we were able to construct a detail map of interaction within the lid complex. Three stable subcomplexes of the lid (trimeric, tetrameric and pentameric) have been solved by electron microscopy, providing a vision of lid assembly events, as they represent the consecutive addition of subunits. •Structural analysis of the RepB OBD domain and its DNA interaction. RepB initiates plasmid rolling-circle replication by binding to a triple 11-bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. This protein acts as a hexameric ring complex, where each protomer has two domains: the origin-binding domain (OBD) and the oligomerization domain (OD). The OBD shows a three-layer a–b–a sandwich fold, the active site is located at one of the faces of the bsheet and coordinates a metal ion at a short distance from the essential nucleophilic Y99. The OBD is also responsible for the recognition of the specific binding sequences. In this work we describe the structural characterization by X-ray crystallography of the OBD bound to its target DNA and we propose a model for the mechanism of action of the hexameric complex.

[eng] The herpesvirus diseases are increasing in importance as a public health problem throughout the world. Members of the human herpesvirus family are global in distribution and infect 60-95% of the world's population, both in developed and in developing countries. The terminase complex plays a key role in genome packaging and is a promising drug target for developing new antiviral compounds. The tripartite terminase 3 subunit C-terminal domain (TRM3-C) of five human herpesviruses (HSV-2, VZV, EBV, HCMV and KSHV) has been biochemically characterized, showing the need of Mn2+ cations in order to enhance the nuclease activity. The three-dimensional structure of TRM3-C HSV-2 has been determined by X-ray crystallography and showed a new position of the two Mn+2 ions in the active centre. Diverse inhibitors have been tested against the TRM3-C terminal domain of the five herpesviruses. Some of them were developed in our laboratory (BS14 and BS17) and the others are specific inhibitors of the HIV integrase. Docking models of TRM3-C have been obtained with the inhibitors that were able to inhibit the nuclease activity in a low micromolar range (BS14, BS17 and Bictegravir). Cell assays with BS14 showed that is moderately active against HCMV. Considering all the biochemical and structural data, new compounds have been designed to obtain better inhibitors against the TRM3-C target. Coronaviruses are a large family of viruses that are known to cause human illness ranging from the common cold to more severe diseases such as MERS and SARS. The emergence of SARS-CoV-2 in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19). The main protease (Mpro) is one of the best characterized drug targets among coronaviruses. Compound 1 inhibits the protease of the rhinovirus, which is homolog to the SARS-CoV-1 Mpro. Thus, could be a promising drug against coronaviruses. The three-dimensional structure of SARS-CoV-1 Mpro in complex with the inhibitor Compound 1 has been determined., [spa] Las enfermedades provocadas por herpesvirus están cobrando cada vez más importancia como problema de salud pública en todo el mundo. Los miembros de la familia del herpesvirus humano están distribuidos mundialmente e infectan al 60-95% de la población, tanto en países desarrollados como en vías de desarrollo. El complejo de la terminasa juega un papel clave en el empaquetamiento del genoma y es una diana farmacológica prometedora para el desarrollo de nuevos compuestos antivirales. Se ha caracterizado bioquímicamente el dominio C- terminal de la subunidad 3 de la terminasa tripartita (TRM3-C) de cinco herpesvirus humanos (HSV-2, VZV, EBV, HCMV y KSHV), mostrando la necesidad de cationes Mn2+ para potenciar la actividad nucleasa. La estructura tridimensional de TRM3-C HSV-2 ha sido determinada por cristalografía de rayos X, mostrando una nueva posición de los dos iones Mn+2 en el centro activo. Se han probado diversos inhibidores contra el dominio terminal TRM3-C de los cinco herpesvirus. Algunos de ellos fueron desarrollados en nuestro laboratorio (BS14 y BS17) y los demás son inhibidores específicos de la integrasa del VIH. Se han obtenido modelos de acoplamiento de TRM3-C con los inhibidores que fueron capaces de inhibir la actividad nucleasa en un rango micromolar bajo (BS14, BS17 y Bictegravir). Los ensayos celulares con BS14 mostraron que es moderadamente activo contra HCMV. Teniendo en cuenta todos los datos bioquímicos y estructurales, se han diseñado nuevos compuestos para obtener mejores inhibidores contra la diana TRM3-C. Los coronavirus son una gran familia de virus que causan enfermedades humanas que van desde el resfriado común hasta enfermedades más graves como MERS y SARS. La aparición del SARS-CoV-2 en 2019 ha desencadenado una pandemia mundial en curso de la enfermedad grave por coronavirus 2019 (COVID-19), una enfermedad similar a la neumonía. La proteasa principal (Mpro) es una de las dianas farmacológicas mejor caracterizadas entre los coronavirus. El compuesto 1 inhibe la proteasa del rinovirus, que es homóloga al SARS-CoV-1 Mpro. Por lo tanto, podría ser un fármaco prometedor contra los coronavirus. Se ha determinado la estructura tridimensional de SARS-CoV-1 Mpro en complejo con el inhibidor Compuesto 1.

Adenosine deaminase acting on tRNAs (ADAT) is a eukaryotic heterodimer, composed by ADAT2 and ADAT3, that catalyses the deamination of adenosine-to-inosine in the anticodon loop of tRNAs. ADAT proteins are essential for the cell, as the adenosine-to-inosine modification enables the proper recognition of all the codons during translation. Indeed, a single point mutation in one of the subunits of the heterodimer is causing mental retardation in some patients. However, there is no structural information of any eukaryotic deaminase, thus hampering the development of any kind of treatment. In this work, we have studied the human ADAT2-ADAT3-tRNA complex using cryo-EM and SAXS, proposing a model of the ternary complex which sheds light on the molecular insights of the deamination and paves the way for further structural studies. Also, the emergence of ADAT proteins in eukaryotes entails the enrichment of potential ADAT-substrates within tRNA pools. The only exception to this evolutionary trend is the tRNAGlyACC, which is not transcribed in humans. In this thesis, we have determined the atomic structure of the tRNAGlyACC by X-ray crystallography, which presents a typical L-shape conformation but with a cleaved anticodon loop, probably digested by a RNase. This finding supports the hypothesis of a disordered structure in the tRNAGlyACC anticodon loop nucleotides, and it may explain its absence in the human transcriptome.

[eng] The aim of this project is to structurally characterize proteins involved in bacterial signal transduction systems by applying X-ray crystallography. Bacteria use signal transduction systems to react in response to any environmental changes detected. Bacterial signal transduction is divided into two categories, one- component systems and two-component systems. Two-component systems are composed by a Response Regulator (RR) and a Histidine Kinase (HK); the Histidine Kinase auto phosphorylates an inner domain, and soon after, it phosphorylates the receiver domain on the Response Regulator, activating the output domain; usually producing a physiological effect in the cell by activating a specific gene. While in one-component systems, one protein has both, a sensory and an output domain. An example of the one-component systems would be ToxR, and an example of the two- component systems would be ComD-ComE. Bacterial transformation is a type of Horizontal Gene Transfer (HGT), which is a rapid evolutive mechanism in which entire genes can be transferred among bacterial cells. HGT is commonly deemed responsible for the appearance of antibiotic resistance, virulence factors and serotype switching. Competence for genetic transformation in Streptococcus pneumoniae is a transient physiological state whose development is coordinated by a peptide pheromone (Competence Simulating Peptide or CSP) and its receptor, which activates transcription of two downstream genes, comX and comW, and 15 other “early” genes. ComD (HK) and ComE (RR) are involved in the quorum-signaling pathway that synthesizes the CSP. They help modulate the mechanism in which bacterial transformation occurs, by allowing the inclusion of naked DNA from the environment. We have successfully formed the binary (ComE+DNA) and ternary (ComD+ComE+DNA) complexes and characterized them in the attempt of obtaining crystals to solve their 3-D structure. On the other hand, to elaborate on one-component systems, like ToxR we can discuss cholera. Cholera is caused by the causative agent Vibrio cholerae. It is estimated that there are from 1.3 to 4.0 million cases out of which up to 143,000 result in cholera deaths annually. After ingesting the V. cholerae, it travels to the small intestine colonizing it and producing the cholera toxin. ToxR is a membrane-localized transcription factor that regulates the toxT promoter. The activation of the toxT promoter triggers the virulence cascade that leads to the secretion of toxin-coregulated pilus (TCP) and the expression of cholera toxin (CTX). In recent years, our lab solved the crystal structure of the cytoplasmic domain of ToxR+20DNA, proposing molecular interactions between ToxR and the toxT promoter (Simone Pieretti’s PhD Thesis). In this study, we want to determine the crystallographic structure of three mutants of the cytoplasmic domain of ToxR bound to the toxT promoter. According to biochemical data from our collaborator (Professor Eric Krukonis, from the university of Detroit), these mutations down regulate the activation of ToxR and we aim to analyze the structural changes that these mutants suppose. Using X-ray crystallography we solved the structure of three complexes; ToxRQ78A+20DNA, ToxRS81A+20DNA and ToxRP101A+20DNA at 2.55, 2.95 and 2.95 Å resolution, respectively. We have compared the final mutant structures with the wildtype, unveiling how the structural changes result in the decrease in activation of the toxT promoter.

[eng] The Escherichia coli infecting T7 bacteriophage shares a common dsDNA packaging mechanism with other bacteriophages of the Caudovirales order, Herpesviruses and Adenoviruses. The packaging machinery comprises the portal protein and the terminase complex. The portal protein is a channel located at a unique portal vertex that provides a conduit for DNA translocation, while the terminase complex recognizes a long concatemer of DNA, performs the nuclease catalytic activity and hydrolyses ATP. Available structural information about portal proteins describes them as oligomeric rings with an axial channel. High quality samples suitable for structural characterization of the portal protein of T7 bacteriophage were obtained and characterized. Both X-ray crystallography and cryo-electron microscopy (cryo-EM) data were collected, and an initial model built on the 5.8Å cryo-EM map was used to phase the crystallographic data, which allowed the building of a model of a tridecameric particle at 2.8Å resolution. The T7 portal particle is 170Å tall and 110Å wide toroidal protein with a central channel that ranges from 23Å to 95Å in diameter. Four domains have been identified in the structure: the wing, the stem, the clip and the crown. The a10-tunnel loop valve is proposed to play an important functional role. During packaging, it may adapt while DNA is translocated and rotated, and once the genome has been packed the side chain of tunnel loop residue Arg368 may be able to seal the channel and stabilize the DNA inside the capsid before tail assembly. Interestingly, these mechanisms would not only imply the flexibility of a loop region, but also the kink of the longer helix of the portal structure, a10.

[eng] Cholera is an acute diarrheal infection caused by the bacterium Vibrio cholerae. An estimated 2.9 million of cases and about 100000 cholera deaths occur annually all over the world. Upon ingestion of the V. cholerae, the bacterium travels to small intestine where it colonizes and produces the cholera toxin. Cholera toxin raises intracellular cyclic AMP and leads to chloride secretion and the subsequent secretory diarrhoea. Cholera toxin production is regulated through the master virulence regulator ToxT. Trascriptional activation of toxT is activated by membrane-localized ToxR, in association with another membrane protein named TcpP. Both ToxR and TcpP work as a two-component regulatory system merged in single proteins: they receive an external signal through its periplasmic C-terminal domain and bind to the toxT promoter by their cytoplasmic N-terminal domains. This project thesis aims at characterizing part of the system by studying ToxR-DNA complexes, since two ToxR molecules are supposed to bind the promoter to recruit TcpP and hence the RNA polymerase for transcription activation. Using X-ray crystallography, we have solved the structure of three complexes of the ToxR DNA binding domain with 20-bp, 40-bp and 25-bp oligonucleotides at 2.0 A, 2.6 A and 3.2 A resolution, respectively. According to the three structures, ToxR is able to bind to an extensive region of the toxT promoter that goes from the position -97 to the position -45. Considering an integrated model of the three structures, there are four ToxR molecules binding the toxT promoter: two molecules bind the DNA in tandem, one molecule binds the ToxR degenerate box and the last one is binding what is supposed to be the TcpP binding site. The structure determination of the three complexes is important to define with more accuracy the ToxR binding site in the toxT promoter. This site is characterized by eleven bases with a high A-T rich region sequence followed by a CATA/CATG/TGTA box, where the last two bases perform direct and specific contacts with the protein. In the three structures, ToxR shows a winged helix-turn-helix (w-HTH) fold. The wing interacts with the minor groove in an A-T rich region sequence while the recognition helix enters in the major groove at the region with a sequence corresponding to a CATA box. We have compared ToxR with the other w-HTH family proteins and we have found a new structural element, the secondary wing, which displays interactions with the DNA. We have analyzed the protein-DNA contacts in the three structures, and also the protein-protein interactions in the ToxR-DNA40 structure, thus validating the data published on ToxR defective mutations. Finally, we put forward a model of toxT promoter activation at molecular level, based on our crystal structures and on what is known in literature and from our collaborators. We propose that ToxR acts as co-activator in the first steps of toxT transcription activation at different levels. First, it would capture the DNA and hold it close to the cytoplasmic membrane, since both ToxR and TcpP are membrane proteins. Second, it would play a key-role in relieving H-NS from the toxT promoter: H-NS binds the DNA and transcription is repressed, but ToxR is able to replace it in a region that goes from the position -97 to the position -45. Third, ToxR would not be recruiting the RNA polymerase directly, but creating the suitable conditions for the action of TcpP. ToxR recruits TcpP probably through a hand-holding mechanism since one of the ToxR binding site is very close to the TcpP's binding site., [spa] El cólera es una infección diarreica aguda causada por la bacteria Vibrio cholerae. La producción de la toxina colérica se controla a través del regulador maestro de virulencia ToxT, cuya activación se lleva a cabo por las proteínas de membrana ToxR y TcpP. Este proyecto de tesis tiene como objetivo el estudio de los complejos formados por ToxR junto con el ADN, dado que se conoce que ToxR se une al promotor toxT para reclutar TcpP y consecuentemente la ARN polimerasa, produciendo la activación de la transcripción. Mediante cristalografía de rayos X hemos resuelto la estructura de tres complejos del dominio de unión a ADN de ToxR con oligonucleótidos de 20, 40 y 25 pares de bases a resoluciones de 2.0 A, 2.6 A y 3.2 A, respectivamente. De acuerdo con las tres estructuras, ToxR es capaz de unirse a una amplia región del promotor toxT que se expande desde la posición -97 hasta la posición -45. Teniendo en cuenta el modelo integrado de las tres estructuras, cuatro moléculas de ToxR se unen al promotor toxT en tándem e invertidas. En las tres estructuras, ToxR muestra un tipo de plegamiento winged helix-turn-helix (w-HTH). El ala (wing) interactúa con el surco menor del ADN, mientras que la hélice de reconocimiento penetra en el surco mayor. Comparando ToxR con el resto de proteínas de la familia w-HTH, hemos encontrado un nuevo elemento estructural, el ala secundaria (secondary wing), que interacciona con el ADN. La determinación de la estructura de los tres complejos es importante para definir con mayor precisión el sitio de unión de ToxR en el promotor toxT. Este sitio se caracteriza por once bases con una secuencia rica en A-T seguida de una caja CATA/CATG/TGTA, donde las dos últimas bases contactan directamente y específicamente con la proteína. Proponemos que ToxR actuaría como co-activador de la transcripción de toxT a diferentes niveles: (i) podría ser responsable de capturar el ADN y mantenerlo cerca de la membrana citoplasmática, (ii) podría jugar un papel clave en el desplazamiento de H-NS, (iii) podría reclutar TcpP y estabilizar su interacción con el promotor.

MobM relaxase from the promiscuous antibiotic resistance plasmid pMV158 is a prototype of the Mob_Pre/MOBV family of relaxases, the major family of relaxases found in Staphylococcus aureus. Staphylococcal infections cause the highest number of lethal cases among antibiotic-resistant bacterial infections. Relaxases initiate the conjugative DNA transfer, a major route for the antibiotic resistance acquisition in bacteria, by nicking their substrate DNA through formation of a covalent DNA-relaxase adduct and terminate the transfer in the recipient cells by rejoining ends of the linearized plasmid. MobM forms a DNA-histidine adduct, unique to MOBV relaxases, instead of a DNA-tyrosine adduct, thus representing a distinct category of relaxases with specialization towards the transfer of short mobile genetic elements in Gram-positive pathogenic bacteria. MobM overall fold resembles the fold of other structurally characterized relaxases, although some important structural differences are present. Molecular basis for the MobM processing of plasmid origin of transfer and active site mechanism are described herein., La relaxasa MobM del promiscuo plásmido de resistencia a antibióticos pMV158 es un prototipo de la familia Mob_Pre/MOBV de relaxasas, la mayor familia de relaxasas se encuentran en Staphylococcu aureus. Las infecciones por estafilococos causan el mayor número de casos mortales entre las infecciones bacterianas resistentes a los antibióticos. Relaxases iniciar la transferencia conjugativa de ADN, una ruta el más frecuente para la adquisición de resistencia a antibióticos por bacterias, por mellar su ADN sustrato mediante la formación de un aducto covalente de ADN-relaxasa y terminan la transferencia en las células receptoras por reincorporarse extremos del plásmido linealizado. MobM forma un aducto de ADN-histidina, único para MOBV relaxases, en lugar de un aducto de ADN-tirosina, lo que representa una categoría distinta de relaxases con especialización hacia la transferencia de elementos genéticos móviles cortos en bacterias patógenas Gram-positivas. MobM estructura general se asemeja a la de otras veces relaxases caracterizan estructuralmente, aunque algunas diferencias estructurales importantes están presentes. Base molecular para el procesamiento de origen del plásmido por MobM y mecanismo de sitio activo se describe en esta thesis.

La incidència d’infeccions gonocòciques i pneumocòciques roman alta en països en vies de desenvolupament i està augmentant a moltes parts del món. La necessitat de tractaments per a l’individu i per al control de la malaltia a nivel comunitàri és punyent però escollir el tractament adequat ha esdevingut complex com a conseqüència de la capacitat de Neisseria gonorrhoeae i Streptococcus pneumoniae de desenvolupar resistència envers antibiòtics. Aquesta tesi de doctorat investiga mecanismes relacionats amb la transferència genética horitzontal i amb l’arrest del creixement a N. gonorrhoeae and S. pneumoniae. La primera part de la tesi està dedicada a l’estudi de la transformació natural a N. gonorrhoeae i descriu els procediments que han portat a l’expressió, purificació i caracterització bioquímica de ComE, ComA i DprA, tres proteïnes que tenen un paper en la presa i el processament de DNA ambiental. La segona part de la tesi descriu la caracterització estructural mitjançant cristal·lografia de rajos X de RelBE2 de S. pneumoniae, un sistema toxina-antitoxina cromosòmic de tipus II que s’ha relacionat amb la moderació de la traducció i amb l’arrest del creixement en condicions d’escassetat de nutrients, i proposa un model per a la seva regulació., The incidence of gonococcal and pneumococcal infections remains high in developing countries and is increasing in many parts of the world. The need not only for treatment of the individual but also for control of the diseases at a community level is acute but the selection of appropriate treatments has become a complicated issue by the ability of Neisseria gonorrhoeae and Streptococcus pneumoniae to develop resistance to antibiotics. This PhD thesis investigates mechanisms related to horizontal gene transfer and growth arrest in N. gonorrhoeae and S. pneumoniae. The first part of the thesis is devoted to the study of natural transformation in N. gonorrhoeae and describes the procedures that have lead to the expression, purification and biochemical characterization of ComE, ComA and DprA, three proteins involved in the uptake and processing of environmental DNA. The second part of the thesis describes the structural characterization by X-ray crystallography of S. pneumoniae RelBE2, a chromosomally-encoded type II toxin-antitoxin system that has been linked to translation moderation and growth arrest under starvation conditions, and proposes a model for its regulation.

El genoma mitocondrial humano es una molécula circular de doble cadena localizada en la matriz mitocondrial. La transcripción del DNAmt se inicia a partir de tres promotores localizados en el D-loop, uno para la cadena ligera (L) y dos para la cadena pesada (H1 y H2). Las unidades de transcripción que se inician en H2 y en L producen una especie policistrónica gigante que cubre casi toda la cadena. En cambio, la transcripción iniciada en H1 se termina en el extremo 3’ del rRNA 16S. El papel principal de esta atenuación se debe al factor de terminación de la transcripción, mTERF. mTERF, es una proteína codificada en núcleo, cuya forma madura tiene 342 aminoácidos. Se ha descrito que se une a varios lugares estratégicos del DNA mitocondrial, donde controla procesos como la terminación de la transcripción o la pausa de la maquinaria de replicación mitocondrial. De estos lugares, el que presenta mayor afinidad es la secuencia de 28 pares de bases (DNA28pb) en el gen del tRNALeu(UUR) mitocondrial. Ensayos in vitro han demostrado que la unión de mTERF a esta región provoca la terminación de la transcripción del DNA mitocondrial en ambos sentidos. En este trabajo se estudia, desde un punto de vista estructural y mediante técnicas de cristalografía de proteínas, el complejo de mTERF con el DNA que contiene la secuencia de terminación. Durante la producción de mTERF recombinante, la proteólisis espontánea de la forma entera de la proteína (Arg56-Ala399) dio lugar a una variante corta, mTERF-ΔN (Arg99-Ala399) que mantenía la unión al DNA. Se determinó la estructura tridimensional del complejo con DNA de mTERF-ΔN a 2.4 Å de resolución. La disposición del DNA en el eje z del cristal provocó que los cristales de mTERF-ΔN en complejo con un DNA29pb, con un parámetro c de la celda unidad demasiado pequeño para albergarlo, presentaran graves problemas de anisotropía y desorden, dando lugar a mapas de densidad electrónica muy distorsionados. Se realizaron entonces ensayos de cristalización con oligonucleótidos de menor longitud, pudiéndose trazar y refinar el DNA y la proteína completa con los cristales de mTERF-ΔN en complejo con el DNA12pb. La estructura del fragmento ΔN se usó para resolver la estructura del complejo de mTERF entera con el DNA15pb a 3.1 Å de resolución. En estos cristales, la proteína interacciona mediante los subdominio N-terminal y C-terminal con dos moléculas de DNA simétricas que no interaccionan entre ellas y cuya posición relativa viene dada por un ángulo de ~36º. mTERF se une a la largo del surco mayor del DNA, estableciendo contactos con los fosfatos mediante 19 residuos y con bases nitrogenadas mediante 5 residuos. La estructura cristalográfica de mTERF consiste en nueve repeticiones estructurales, de TERF-I a IX, flanqueadas por un segmento N-terminal y una hélice α C-terminal. Cada motivo TERF está formado por unos 35 aminoácidos que se estructuran en tres hélices α (H1, H2 y H3) formando una superhélice trigonal levógira con un núcleo hidrofóbico central. La combinación de la conectividad en superhélice levógira con la propagación en tándem del motivo para formar un solenoide es única de las proteínas de la familia MTERF. En base a la estructura cristalográfica se generaron modelos para estudiar los complejos en solución mediante la técnica de SAXS. La representación gráfica del error asociado al ajuste entre la curva teórica de dispersión para cada modelo y la curva medida experimentalmente, presenta un mínimo, indicando que la unión de mTERF al DNA28pb se da preferentemente sobre una zona del DNA. Los ensayos de unión y especificidad de mTERF y las formas truncadas de ésta con el DNA28pb mediante geles nativos de retardo muestran que mTERF-ΔC y ΔN-mTERF-ΔC no unen DNA y que mTERF y mTERF-ΔN lo unen específicamente., The human mitochondrial genome is a closed circular molecule located within the mitochondrial matrix. mtDNA transcription is initiated from tree promoters in the D-loop, one for L-strand (L) and two for the H-strand (H1 and H2). Transcription from H2 and L proceeds around the mtDNA circle, creating a polycistronic RNA. On the other hand, transcription initiated in H1 ends at the 3’end of rRNA 16S. The main role of this attenuation is due to the mitochondrial transcription termination factor mTERF. mTERF, is a nucleus-encoded protein whose mature form has 342 amino acids. It has been described that mTERF binds to several strategic mtDNA sites, where it controls processes such as transcription termination or pausing of the replication machinery. Among them, the 28bp sequence from the tRNALeuUUR gene shows the highest affinity of binding to mTERF. In vitro assays have shown that binding of mTERF to this site promotes termination of transcription bidirectionally. We study, from a structural point of view, the mTERF complex with the oligonucleotide containing the termination sequence. During recombinant mTERF production, spontaneous proteolysis of the full-length protein (Arg56–Ala399) yielded a shorter variant (Arg99–Ala399; mTERF-ΔN) yet DNA-binding form. The three-dimensional struc¬ture of mTERF-ΔN was determined from SeMet-derivatized crystals at 2.4Å resolution. In the first mTERF-ΔN-DNA crystals, the arrangement in the z-axis of the large 29bpDNA molecule, compared to the c parameter of the unit cell, caused severe problems of anisotropy and disorder, which resulted in low quality electron density maps. Then crystallization assays were performed with shorter oligonucleotides, being able to trace and refine the complex structure with mTERF-ΔN-12bpDNA crystals. The structure of ΔN fragment was used to solve the full-length mTERF structure at 3.1Å resolution in complex with 15bpDNA. In these crystals, two crystallographically related identical oligonucleotides related by ~36° are bound by two distinct regions of a single protein moiety, the N-terminal and C-terminal subdomains. mTERF wraps round the DNA along the major groove through 19 nonspecific interactions with both dsDNA backbone phosphates and 5 interactions with DNA nitrogen bases. mTERF consists of nine structural repeats, TERF-I–TERF-IX flanked by an N-terminal extended segment and a C-terminal α-helix C. Within each repeat, roughly 35 residues fold into three α-helices (H1, H2 and H3) that are arranged in a left-handed triangular superhelix around a central hydrophobic core. The combination of left-handed helical connec¬tivity with tandem propagation is unique to mTERF. Based on the crystallographic structure, we constructed models to study the complex in solution by small-angle X-ray scattering (SAXS). The graphical representation of agreement of each of the models to the experimental SAXS curve presents a minimum, indicating the specificity of the binding between mTERF and the 28bpDNA. The 28bpDNA binding affinity and specificity of mTERF and truncated forms, assayed by native gels, shows that mTERF-ΔC and ΔN-mTERF-ΔC do not bind DNA while mTERF and mTERF-ΔN bind it specifically.

Es presenta el model atòmic a 4.5 Å de DnaB, la principal helicasa replicativa bacteriana, d'Aquifex aeolicus. És un anell hexamèric de 100 Å d'amplada i 80 Å d'alçada amb dues capes de simetria diferenciada, la dels dominis N-terminals en C3 i la dels C-terminals propera a C6. El diàmetre central és de 25 Å al llarg d'ambdues capes, principal diferència amb les estructures prèvies, on era 25 Å més estret a la capa N-terminal. L'estretament s'origina pel trencament d'una de les dues superfícies d'interacció entre monòmers N-terminals, cosa que augmenta la flexibilitat del subdomini implicat. Només l'ssDNA pot atravessar l'anell, quan a les estructures prèvies hi podia passar tant ssDNA com dsDNA. L'estructura aquí presentada és més propera a la conformació funcional de DnaB durant la realització de l'activitat helicasa, mentre que les anteriors correspondrien a la forma inactiva o a la conformació capaç de translocar-se sobre dsDNA., DnaB is the main replicative helicase in bacteria. An atomic model for the DnaB from Aquifex aeolicus at a 4.5 Å resolution is presented. It´s a ring-shaped homohexamer (100 Å width and 80 Å hight) with two simmetry layers, a C3 N-terminal layer and an almost C6 C-terminal one. The diameter of the central channel is 25 Å along both layers, being the main diference with the previously solved structures, which were 25 Å smaller along the N-terminal layer. This is due to one of the previous interacting surphaces being lost in the current structure, thus enabling a higher felxibility of the subdomain involved. Only ssDNA can pass trhough the ring, while both ssDNA and dsDNA could in the previous structures. So, the present structure is closer to the functional conformation, while the previous ones would correspond to the inactive form or the conformation that is only able to translocate along dsDNA.

La conjugación bacteriana es uno de los mecanismos de transferencia horizontal genética más importante y la causa principal de la rápida adquisición de resistencia a los antibióticos en las bacterias. La capacidad de las bacterias para conjugarse fue descubierta en 1946 en cultivos de E. coli y, años más tarde, en bacterias Gram-positivas. El proceso de conjugación comienza con el corte de un enlace fosfo-diester específico por medio de una relaxasa que juega un papel clave en la iniciación de dicha transferencia.En el plásmido R388 de E. coli, TrwC es la transferasa que cataliza la etapa inicial y final de la transferencia conjugativa del ADN. La estructura cristalina del dominio N-terminal de la relaxasa TrwC en complejo con un oligonucleótico de 27 bases que contiene la horquilla de reconocimiento y el enlace fosfo-diester a cortar ha sido determinada por difracción de rayos X. Asimismo, se han resuelto las estructuras de una serie de complejos ternarios proteína-ADN-metal con diferentes cationes divalentes ocupando el sitio activo. Un análisis sistemático, mediante difracción anómala, permitió determinar sin ambigüedad la naturaleza del metal localizado en el sitio activo, encontrándose que Zn2+, Ni2+ y Cu2+ se unían a la tríada de histidinas. La comparación de las estructuras de los diferentes complejos sugiere la existencia de dos caminos que permiten la salida de la cadena de ADN del centro activo y que probablemente son utilizados en diferentes etapas de la reacción de proceso de conjugación. La información estructural permitió proponer (i) un mecanismo enzimático en el que el ión metálico polariza el enlace fosfo-diester a cortar facilitando el ataque por parte de la tirosina catalítica, y (ii) la serie de eventos que ocurren durante el procesamiento del ADN a transferir y que explican la función biológica de la relaxasa.Asimismo se ha estudiado y caracterizado por difracción de rayos X el dominio N-terminal de la relaxasa MobM del plásmido pMV158 de Streptococcus agalactiae en complejo con un oligonucleótido de 26 bases que mimetiza la secuencia del oriT justo hasta el sitio de corte (nic). El plegamiento global de la proteína muestra un núcleo central formado por cinco hebras betas anti-paralelas flanqueadas por dos pares de hélices alfa. Este plegamiento es estructuralmente homólogo al observado en la relaxasas de bacterias Gram-negativas, pese a la baja homología de secuencia existente entre dichas proteínas. La cadena de ADN se une a MobM mediante numerosas interacciones electrostáticas alojándose a lo largo de la amplia cavidad que la superficie de la proteína presenta. Los contactos intermoleculares se agrupan en cuatro regiones; los dos más importantes involucran dos giros betas consecutivos de la cadena proteica que penetra en el surco menor del ADN y un largo lazo flexible que rodea el extremo de simple cadena del ADN., Bacterial conjugation is one of the most important horizontal gene transfer mechanisms and the main cause of the rapidly spread of the antibiotic resistance in bacteria. The ability of bacteria to conjugate was discovered in 1946 in E. coli cultures and years later in Gram-positive bacteria. The conjugative process begins with the cleavage of a specific phosphodiester bond through a relaxase protein, playing a key role in the initiation of the transfer.TrwC is the DNA strand transferase that catalyzes the initial and final stages of conjugative DNA transfer in the plasmid R388 from E. coli. We have solved the crystal structure of the N-terminal relaxase domain of TrwC in complex with a 27 base-long DNA oligonucleotide that contains both the recognition hairpin and the scissile phosphate. In addition, a series of ternary structures of protein-DNA complexes with different divalent cations at the active site have been solved. Systematic anomalous difference analysis allowed us to determine unambiguously the nature of the metal bound. Zn2+, Ni2+ and Cu2+ were found to bind the histidine-triad metal binding site. Comparison of the structures of the different complexes suggests two pathways for the DNA to exit the active pocket. They are probably used at different steps of the conjugative DNA-processing reaction. The structural information allows us to propose (i) an enzyme mechanism where the scissile phosphate is polarized by the metal ion facilitating the nucleophilic attack of the catalytic tyrosine, and (ii) a probable sequence of events during conjugative DNA processing that explains the biological function of the relaxase.We also investigated the N-terminal relaxase domain of the protein MobM from plasmid pMV158 from Streptococcus agalactiae, and herein we report the X-ray crystal structure of the complex between this domain and a 26-mer oligonucleotide that mimics the oriT sequence just upstream the nic site. The overall fold of the protein shows a core domain comprising five anti-parallel beta-strands flanked by pairs of alfa-helices. This fold resembles that of Gram-negative relaxases in spite of the very low sequence homology. The DNA molecule binds to MobM through a number of electrostatic interactions following a large crevice over the surface of the protein. Intermolecular contacts are clustered in four main regions; the two most important ones involve a bulge formed by two consecutive beta-turns, and a long unstructured loop that wraps around the single stranded DNA part.

En aquest treball es descriu la determinació estructural mitjançant cristal·lografia de raigs X de la proteïna p4 del bacteriòfag phi29 en la seva forma lliure i en complex amb DNA.La proteïna p4 és essencial en el procés infectiu del bacteriòfag, ja que regula la transcripció dels seus gens, establint-se dues fases transcripcionals clarament diferenciades. Això permet separar en el temps la síntesi de proteïnes implicades en la replicació del DNA fàgic i la de les proteïnes estructurals que formen la partícula del bacteriòfag. Existeixen 3 llocs funcionals de unió de p4 en el genoma del bacteriòfag phi29, que es localitzen en una regió intergènica de 219 pb on es troben els 3 promotors més importants: el A2b, el A2c i el A3.En absència de la proteïna p4, els promotors A2b i A2c són transcripcionalment actius, mentre que el promotor A3, degut a l'absència de una caixa -35 en la seva seqüència és incapaç d'unir l'RNA polimerasa i es manté inactiu.En el moment en que la proteïna p4 és sintetitzada, aquesta s'uneix als seus llocs d'unió i produeix diferents efectes depenent del promotor que considerem. En el promotor A2b, el lloc d'unió de la proteïna p4 solapa amb la seva caixa -35 i impedeix la unió de l'RNA polimerasa inhibint-se la transcripció. En el promotor A2c, la proteïna p4 afavoreix la unió de l'RNA polimerasa però aquesta no pot realitzar la seva funció perque es produeix una sobreestabilització del complex i l'RNA polimerasa és incapaç d'alliberar-se del promotor. En el promotor A3, la proteïna p4 recluta l'RNA polimerasa permetent l'inici de la transcripció.La proteïna p4 va cristal·litzar en dos grups espacials, C2221 i P212121 en presència i absència d'un compost de platí, respectivament. L'obtenció de fases es va realitzar pel mètode MAD, recollint 3 conjunts de dades a diferent longitud d'ona de manera que les intensitats fossin diferents en les reflexions simètriques per l'efecte del platí com a dispersor anòmal. El model de la proteïna es va afinar fins a 3 Å en el grup espacial C2221 i aquest va servir per poder fer una cerca de reemplaçament molecular en el cristall P212121. En aquest grup espacial es va poder afinar el model fins a 2 Å.El complex format per la proteïna p4 i el DNA va cristal·litzar en el grup espacial P2, apareixent un complex per unitat asimètrica. El fasejat es va realitzar pel mètode del reemplaçament molecular utilitzant com a model de cerca un dímer de la proteïna p4 i un fragment ideal de DNA en conformació B.La proteïna p4 és una proteïna de 125 aminoàcids que s'uneix al DNA en forma de dímer. La unió de la proteïna al DNA provoca una corvatura de 70º. La unió es realitza de manera específica en la regió N-terminal de la proteïna i de manera inespecífica en la regió de dimerització. Els contactes realitzats amb l'esquelet de fosfat del DNA tenen com a objectiu fixar el solc menor de la molècula de DNA en una conformació comprimida., This work describes the structural determination by X-ray crystallography of the phi29 bacteriophage protein p4, both bound and unbound to DNA.Protein p4 is essential for the bateriophage infective step, as it regulates its genes transcription, thus establishing two clearly differentiated transcriptional phases. This allows temporal separation of the phage protein synthesis: the proteins involved in phage DNA replication are synthesized first and the structural proteins that form the bacteriophage particle later. There are three p4 functional binding sites in phi29 genome, which are located in a 219bp long intergenic region where the three most important promoters (A2b, A2c and A3) are located.In p4 absence, A2b and A2c promoters are active, while A3 promoter, due to not having a -35 box in its sequence, is unable of binding RNA polymerase and remains inactive.When p4 protein is synthesized, it binds to its binding sites, thus producing different effects depending on the promoter. In A2b promoter, protein p4 binding site encloses the promoter -35 box, thus preventing RNA polymerase binding and transcription consequently. In A2c promoter, although protein p4 enhances RNA polymerase binding, the enzyme is unable to perform its function because the complex is overstabilized and so RNA polymerase cannot escape from the promoter. In A3 promotor, protein p4 recruits RNA polymerase, thus allowing transcription initiation.Protein p4 was crystallized in two different space groups, C2221 in presence of a platinum compost and P212121 in its absence. Phases were obtained by MAD, collecting 3 data sets at different wavelengths to have differences in the symmetric reflections intensities due to the effect as an anomal dispersor of the platinium. The protein model was refined up to 3 Å in the C2221 space group, and this model was used to perform a molecular replacement in the P212121 crystal. In this former space group, the model could be refined up to 2 Å .The complex formed by protein p4 and DNA was crystallized in P2 space group, with one complex per asymmetric unit. Molecular replacement was used for phase determination, taking a protein p4 dimer and an ideal DNA fragment in B conformation as a searching model.P4 is a 125 aminoacid long protein that binds DNA as a dimer. P4 binding to the DNA induces a 70º curvature on it. This binding is specific at the n-terminal region and unspecific at the dimerization area. The contacts with the phosphate backbone of the DNA fix the minor groove of the DNA molecule in a narrowed conformation.