Molecular dynamics simulations of complexes of human dipeptidyl peptidase III with inhibitors

Dipeptidil-peptidaza III (DPP III) je o cinku ovisna peptidaza koja katalizira hidrolizu druge peptidne veze s N-kraja svojih supstrata. Enzim je široke supstratne specifičnosti, nepoznate osnovne fiziološke uloge, široke zastupljenosti među organizmima te prepoznatih uloga u procesima od interesa za razvoj lijekova. U ovom radu provedene su simulacije molekulske dinamike kompleksa ljudske DPP III s dvama inhibitorima za koje su poznati postotci inhibicije u odnosu na sintetski supstrat Arg-Arg-2-naftilamid. Za simulacije su korištene dvije konformacije enzima različite kompaktnosti kako bi se, računima slobodne energije vezanja, utvrdilo koja je povoljnija za vezanje liganda i upoznale interakcije liganda s aminokiselinskim ostatcima te cinkovim ionom u aktivnom mjestu. Dobiveni rezultati mogli bi poslužiti u daljnjem razvoju i testiranju potencijalnih inhibitora ovog enzima.Dipeptidyl peptidase III (DPP III) is a zinc dependent peptidase which catalyses hydrolysis of the second N-terminal peptide bond of its substrates. DPP III is an enzyme of broad substrate specificity and it has been found in many organisms. Although its fundamental physiological role is unknown, it has been recognised in several processes of interest for the drug development. In this thesis, molecular dynamic simulations were done for the complexes of human DPP III with two inhibitors for which the percentages of inhibition have been measured for the Arg-Arg-2-naphthylamide synthetic substrate hydrolysis. Two enzyme conformations of different compactness were used for the simulations. Subsequently, the binding free energies were calculated in order to find out which one is more favorable for ligand binding and to obtain an insight into the ligand-protein interactions. The obtained results might be useful in future development and testing of potential inhibitors of this enzyme

Studying biological assembly of ion channel complexes

Les canaux ioniques sont des complexes macromoléculaires clés exprimés dans tous les types de cellules et sont impliqués dans divers processus physiologiques, y compris la génération et la propagation de potentiels d'action. Des canaux défectueux conduisent à des maladies graves, notamment l'épilepsie, des arythmies et des syndromes douloureux, ce qui en fait une cible potentielle intéressante pour le développement de médicaments. Pour améliorer notre compréhension de ces assemblages biologiques et éventuellement trouver des traitements spécifiques pour les canalopathies, il est crucial d'étudier la structure et la fonction des canaux ioniques. L'objectif principal de cette thèse a été d'étudier ce type de détails structurels et fonctionnels pour trois canaux ioniques associés aux domaines des capteurs de douleur et des canaux potassiques voltage-dépendants en utilisant des techniques de fluorescence et d'électrophysiologie. Dans le premier projet, nous avons étudié la stœchiométrie des canaux hétéromères Kv2.1 / 6.4 (chapitre trois). La technique du décompte de sous-unités isolées (single subunit counting :ssc) permet de compter les sous-unités marquées par fluorescence d’un complexe isolé en déterminant le nombre d'événements de photoblanchiment, qui apparaissent en sauts irréversibles vers le bas sur les traces de fluorescence. Pour désigner la stœchiométrie la plus probable, nous avons utilisé des calculs de probabilités pondérées et avons constaté que les canaux Kv2.1 / 6.4 s'expriment dans un arrangement 2 : 2. Plus précisément, les études fonctionnelles des canaux concatémériques montrent que les sous-unités Kv6.4 et 2.1 doivent être disposées de manière alternée. Le deuxième projet était également basé sur des expériences de SSC et visait à déterminer l'état oligomérique du nouveau canal ionique TACAN (chapitre quatre). Nous avons trouvé une portion significative de canaux intracellulaires, ce qui a provoqué une fluorescence de fond dans les expériences de SSC traditionnelles réalisées avec les cellules mammifères. Pour améliorer le rapport du signal sur bruit de fond, nous avons effectué des expériences de SSC sur des canaux purifiés qui ont été immobilisés sur des lamelles de verre fonctionnalisées Ni-NTA. En utilisant la méthode de calcul décrite dans le premier projet, nous avons trouvé différents états oligomériques et proposons que les canaux TACAN natifs s'assemblent en tétramères qui sont instables lorsqu'ils sont solubilisés dans un détergent. Dans le dernier projet, nous avons étudié la relation structure-fonction de la sous-unité auxiliaire DPP6 pour les canaux Kv4.2 (chapitre cinq). Ici, nous avons progressivement tronqué le grand domaine extracellulaire de 700 acides aminés de DPP6 et étudié son effet sur les courants macroscopiques en utilisant la technique du cut-open voltage clamp. Nous avons constaté que les sous-unités DPP6 avec un domaine extracellulaire court ne parviennent pas à moduler les propriétés du canal aussi efficacement que la DPP6 pleine longueur. Plus précisément, la seconde moitié du domaine extracellulaire b-propeller de DPP6 est responsable d'une inactivation du canal considérablement accélérée. Sur la base de la structure cristalline du domaine extracellulaire, nous avons proposé qu'un domaine b-propeller stable et possiblement la formation de dimères DPP6 sont responsables de la déstabilisation efficace de l'état du canal ouvert.Ion channels are key macromolecular complexes expressed in all cell types and are involved in various physiological processes including the generation and propagation of action potentials. Defective channels lead to severe diseases including epilepsy, arrhythmias and pain syndromes making them an interesting potential drug target. To improve our understanding of these biological assemblies and eventually find specific treatments for channelopathies, it is crucial to study the structure and function of ion channels. The main purpose of this thesis has been to investigate such structural and functional details of three ion channel complexes from the field of pain sensors and voltage-gated potassium channels using fluorescence and electrophysiological techniques. In the first project, we studied the stoichiometry of heteromeric Kv2.1/6.4 channel complexes (chapter three). Single subunit counting (SSC) allows to directly count the number of fluorescently labeled subunits by determining the number of irreversible, step-wise photobleaching events. To determine the most probable stoichiometry, we used weighted likelihood calculations and found that Kv2.1/6.4 channels express in a 2:2 arrangement. More precisely, functional studies of concatemeric channels (performed by our collaborators) illustrate that Kv6.4 and 2.1 subunits need to be arranged in an alternating fashion. The second project was also based on SSC experiments and aimed at determining the oligomeric state of the novel ion channel TACAN (chapter four). We found a significant amount of channels in the intracellular which caused background fluorescence in traditional SSC experiments performed in cells. To improve the signal to background ratio, we performed SSC experiments on purified channels that were immobilized on Ni-NTA functionalized glass coverslips. Using the model selection method described in the first project, we found different oligomeric states and propose that native TACAN channels assemble as tetramers which are unstable when solubilized in detergent. In the last project, we investigated the structure-function relation of the auxiliary DPP6 subunit in Kv4.2 channel complexes (chapter five). Here, we progressively truncated DPP6’s 700 amino acids long extracellular domain and studied its effect on macroscopic currents using the cut-open voltage clamp technique. We found that DPP6 subunits with a short extracellular domain fail to modulate the channel properties as efficiently as the full length DPP6. More precisely, the second half of the extracellular b-propeller domain of DPP6 is responsible for drastically accelerated channel inactivation. Based on the crystal structure of the extracellular domain, we proposed that a stable b-propeller domain and possibly DPP6 dimer formation is responsible for destabilizing the open channel state efficiently

Structural insights into coronavirus binding to host aminopeptidase N and interaction dynamics

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-11-2014Coronaviruses (CoV) are large enveloped RNA viruses of animals and humans associated mostly with enteric and respiratory diseases. These viruses were earlier considered more of a veterinary interest and were associated to humans with mild flu. The last decade brought outbreaks with high mortality rates caused by CoV transmission from animals to man. This remarkable cross-species transmission potential is related to CoV adaptation to a variety of cell surface molecules for entry into host cells. The CoV particles bear exposed spike (S) proteins in their envelope that attach to specific cell entry receptors, which determines CoV host cell range and tropism. CoV can recognize diverse entry receptors, but they preferentially use membrane bound ectoenzymes. A subset of CoV recognizes the cell surface aminopeptidase N (APN), a membrane-bound metalloprotease. APN (CD13) is a “moonlighting” ectoenzyme linked to multiple functions such as angiogenesis, cell-cell adhesion and tumorogenesis. It cleaves neutral amino acid side chains from the N-terminus of oligopeptides, it is distributed in wide variety of tissues its expression is dysregulated in tumors. APN is an important target for cancer therapies and anti-inflammatory drug design, as well as a major CoV entry receptor. This Thesis presents an extensive structural study on mammalian APN ectodomains as well as its function as CoV receptor. APN crystal structures revealed ectodomain architecture, dimerization and motions important for peptide hydrolysis and CoV recognition. Allosteric inhibition of APN catalysis can be mediated by the suppression of APN movements. Moreover, the crystal structure of a porcine CoV spike fragment bound to the pig APN (pAPN) ectodomain uncovers how CoV bind to its APN receptor. A protruding receptor-binding edge in the S penetrates in small APN cavities, a receptor-binding mode distinct from other CoV-receptor interactions. CoV specifically bind to the open APN conformation. Structure-guided studies identified key virus and receptor motifs at the CoV-APN binding interface and they show that the receptor-binding region is a major antigenic determinant in CoV binding to APN. CoV neutralizing antibodies target key receptor-binding residues, showing that they prevent CoV binding to the APN receptor and infection. The Thesis provides a compelling view on CoV cell entry and neutralization, as well as important structural insights to understand the multifunctional APN protein.Los coronavirus (CoV) son virus ARN con envuelta responsables de enfermedades entéricas y respiratorias en animales y humanos. Estos virus fueron inicialmente considerados de interés veterinario y se asociaron con resfriados en humanos. La última década trajo infecciones de CoV con altas tasas de mortalidad, causadas por la transmisión de CoV animales a humanos. Este potencial de transmisión transversal entre especies está relacionado con la adaptación CoV al uso de diferentes moléculas de la superficie celular para la entrada en la célula huésped. Las partículas CoV contienen espículas (S) expuestas en la envuelta que adhieren las particulas virales a receptores para su penetración en la célula, que determina el tipo de células permisivas para CoV y su tropismo. Los CoV pueden reconocer diversos receptores para la entrada, pero preferentemente utilizan ectoenzimas. Un grupo de CoV reconoce la aminopeptidasa N (APN), una metaloproteasa unida a la membrana celular. APN (CD13) es una ectoenzima que se ha relacionado múltiples funciones, tales como la angiogénesis, la adhesión célula-célula y la tumorogénesis. Esta proteína hidroliza aminoácidos N-terminales neutros en oligopéptidos, se encuentra en gran variedad de tejidos y su expresión aumenta en tumores. APN es una diana importante de terapias contra el cáncer y de fármacos anti-inflamatorios, así como un receptor para la entrada CoV en la célula. Esta tesis presenta un extenso estudio estructural sobre los ectodominios de APN de mamíferos, así como sobre su función como receptor de CoV. Las estructuras cristalográficas de la APN revelaron la arquitectura modular de su ectodominio, dimerización y los movimientos del mismo, importantes para la catálisis y el reconocimiento de CoV. Se muestra la posible inhibición alostérica de la actividad catalítics de la APN mediante la supresión de los cambios conformacionales de la APN. Asimismo, la estructura cristalográfica de un fragmento de la espícula de un CoV porcino unido al ectodominio de la APN de cerdo, han identificado cómo los CoV se unen a su receptor. Una región expuesta en la proteína S del CoV penetra en pequeñas cavidades de la APN; este modo de unión al receptor es distinto de otras interacciones CoV-receptor descritas. Los CoV se unen específicamente a la conformación abierta de la APN. Estudios funcionales identificaron motivos clave para la unión del virus y receptor y muestran que la región de unión al receptor es un determinante antigénico en CoV, reconocido por anticuerpos neutralizantes, que inhiben la unión de los CoV a la APN y la infección. La Tesis proporciona una visión general sobre la entrada de CoV en el huésped y su neutralización, así como importantes puntos de vista estructurales para comprender las múltiples funciones de la AP

Molecular, Genetic, and Biochemical Analysis of Phage Lambda Lysis

Spanins are required for outer membrane disruption, the final step in phage lysis of Gram-negative hosts. Recently spanins were shown to be fusogenic and it was proposed that spanins function by fusing the inner and outer membrane. This work uses a genetic approach to probe the functional domains of the λ spanins Rz and Rz1 by selecting for lysis-defective alleles. Our selection showed single missense mutations clustered within subdomains essential to other membrane fusion systems, including coiled-coil domains and a proline-rich region. Surprisingly, most products of lysis defective alleles were normal for accumulation and complex formation. This suggests that a majority of the mutations blocked function at a downstream step, e.g. membrane fusion. To gain insight into spanin function, we selected for spontaneous suppressors that restored plaque formation to lysis defective alleles. Strikingly, regardless of the site of inactivating mutation, the second site rescuing mutations clustered within a coiledcoil domain near the cytoplasmic membrane. These changes encoded polar insertions into the hydrophobic core and were not allele specific. Furthermore, suppressor mutants were defective for Rz accumulation and exhibited a defect in lysis morphology. Instead of identifying point-to-point contacts, a global suppression pattern was indicated. This suggests that destabilization of the membrane-proximal segment of the Rz coiled-coil can rescue function for lysis defective alleles of Rz or Rz1 at cost of normal saltatory function. Lastly, it is not known how λ causes lysis from the poles of E. coli. To address this, we use time-lapse microscopy to monitor the activity and subcellular localization of lysis proteins in the seconds prior to lysis. Results exclude the endolysin and spanin and indicate that the holin, which initiates lysis, also controls the site of lysis

Insights into Protein-Ligand Molecular Recognition: Thermodynamic, Kinetic and Structural Characterization of Inhibitor Binding to Aldose Reductase and Carbonic Anhydrase II

Two pathological relevant proteins, human aldose reductase and human carbonic anhydrase, were used as model proteins to get insights into the process of molecular recognition. The thermodynamics and kinetics of the formation process of protein ligand complex formation were studied