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Estudio Estructural Y Funcional De Un Inhibidor Proteínico Monodominio

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Estudio Estructural Y Funcional De Un Inhibidor Proteínico Monodominio Portada: Fotografía de un as, una moneda de bronce utilizada durante la Segunda República Romana (siglo segundo antes de Cristo), la cual muestra la imagen del dios Jano (fotografía tomada en el Museu Nacional d'Art de Catalunya, Barcelona; www.mnac.cat). Sobre la moneda se muestra el modelo de la estructura del complejo ternario subtilisina- sermetstatina-esnapalisina (en naranja, verde y amarillo, respectivamente) obtenido en la presente tesis doctoral. UNIVERSITAT AUTÒNOMA DE BARCELONA Institut de Biotecnologia i Biomedicina CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS Institut de Biologia Molecular de Barcelona Departament de Biologia Estructural Proteolysis Laboratory Estudio estructural y funcional de un inhibidor proteínico monodominio de doble faz, sermetstatina, en complejo con dos peptidasas de diferente clase, subtilisina y esnapalisina Sergio Trillo Muyo Barcelona, Mayo 2013 UNIVERSITAT AUTÒNOMA DE BARCELONA Institut de Biotecnologia i Biomedicina Programa de Doctorado en Estructura y Función de Proteínas 2008/2009 Programa con mención de calidad otorgado por el Ministerio de Educación y Ciencia, enmarcado en las directrices establecidas por el Real Decreto 778/98 Estudio estructural y funcional de un inhibidor proteínico monodominio de doble faz, sermetstatina, en complejo con dos peptidasas de diferente clase, subtilisina y esnapalisina Memoria presentada por Sergio Trillo Muyo para optar al título de doctor por la Universitat Autònoma de Barcelona Sergio Trillo Muyo Directores de Tesis: Tutor: Prof. F.Xavier Gomis Rüth Dr. Joan López Arolas Prof. Xavier Daura Ribera A mis padres A mi tía AGRADECIMIENTOS La portada de esta memoria de tesis se ilustra con una imagen del dios romano Jano, el dios de las puertas, de las transiciones, de los comienzos y los finales. Precisamente, después de todos estos años, de tanto trabajo y de tantas horas, me encuentro aquí, en el final de una etapa y el inicio de otra. La representación de este dios, con dos caras mirando hacia ambos lados de su perfil, me recuerda que en los momentos de transición hay que saber mirar atrás y adelante, al pasado y al futuro. Así que sin apartar la vista del futuro, de los nuevos proyectos y oportunidades, es también hora de mirar atrás, de no dejar olvidadas las cosas que me han llevado hasta aquí. Mirando al pasado veo momentos, algunos buenos y otros no tanto, que de un modo u otro me han marcado en este periodo de crecimiento científico y personal que es la realización de una tesis. Pero, más importantes que esos momentos, son las personas con quienes los compartí, y este es el momento agradecerles el haber estado ahí. Quiero empezar agradeciendo a Xavier Gomis la oportunidad que me dio de realizar esta tesis. Sin él, sin su tiempo, su apoyo, su guía, su motivación y sus ideas, este trabajo no hubiese sido posible. También quiero agradecer especialmente a Joan L. Arolas haber codirigido esta tesis, por todo su trabajo, sus ideas y por todos los conocimientos que me ha transmitido. Tengo que dar las gracias a todos los miembros del laboratorio Cri3 con los que he tenido la suerte de trabajar durante estos años y compartir el día a día. De todos ellos debo mencionar a Goretti Mallorquí, por guiar mis primeros pasos, por motivarme como lo hizo, porque sin ella esta etapa que ahora acaba no habría empezado. Gracias también a Tibisay Guevara, por haberme acompañado durante toda mi estancia en el laboratorio, por todo su trabajo y todo su apoyo. Además, debo dar las gracias a los miembros de los otros Cri con los que he coincidido, especialmente a Isabel Usón por haberme acogido temporalmente en su laboratorio. Gracias también a Anna Mañosas, por su apoyo incondicional, por todas las transiciones que hemos vivido y viviremos juntos. Finalmente, quiero dar las gracias a mi familia. A mis padres, por haber educado a dos hijos como lo han hecho, por todo su sacrificio. A mi hermano, por estar siempre ahí. A mi tía, que siempre tendré presente. Índice ÍNDICE I Índice Índice .............................................................................................................................. I Figuras ................................................................................................................................. VI Tablas ................................................................................................................................ VIII Abreviaturas .................................................................................................................. IX Nomenclatura .............................................................................................................. XV Prefacio ....................................................................................................................... XIX 1. Introducción ............................................................................................................... 1 1.1. Bacterias del suelo ........................................................................................................ 3 1.1.1. Género Streptomyces ............................................................................................. 5 1.2. Peptidasas ..................................................................................................................... 8 1.2.1. Clasificación ............................................................................................................ 9 1.2.1.1. Base de datos EC .......................................................................................... 10 1.2.1.2. Base de datos MEROPS ................................................................................ 11 1.2.2. Metalopeptidasas ................................................................................................ 11 1.2.2.1. Metzinquinas ................................................................................................ 15 1.2.2.1.1. Esnapalisina ....................................................................................... 18 1.2.2.1.2. Regulación por zimogenicidad en metzinquinas............................... 20 1.2.3. Serín peptidasas ................................................................................................... 26 1.2.3.1. Clan PA ......................................................................................................... 29 1.2.3.2. Clan SB .......................................................................................................... 31 1.3. Inhibidores proteínicos de peptidasas ........................................................................ 33 1.3.1. Mecanismo estándar de inhibición ...................................................................... 38 1.3.2. Inhibidores proteínicos de metalopeptidasas ..................................................... 40 1.3.3. Inhibidores proteínicos de serín peptidasas ........................................................ 43 1.3.3.1. Inhibidor de subtilisina de Streptomyces ..................................................... 45 1.3.3.2. Inhibidor de peptidasa neutra de Streptomyces caespitosus ...................... 49 2. Objetivos .................................................................................................................. 51 3. Materiales y métodos ................................................................................................ 55 3.1. Producción y purificación de sermetstatina ............................................................... 57 3.2. Obtención de esnapalisina y proesnapalisina. Análisis del prodominio ..................... 58 3.2.1. Análisis bioinformático ........................................................................................ 59 3.2.2. Producción y purificación de proesnapalisina ..................................................... 59 3.2.3. Producción y purificación de esnapalisina ........................................................... 60 III Índice 3.2.4. Estudios de activación in-vitro de proesnapalisina .............................................. 61 3.2.5. Cristalización de proesnapalisina E164A .............................................................. 62 3.2.6. Estudios de actividad ........................................................................................... 62 3.3. Análisis de proteólisis y su inhibición por sermetstatina ............................................ 63 3.4. Formación y purificación de complejos....................................................................... 64 3.4.1. Subtilisina-sermetstatina ..................................................................................... 64 3.4.2. Esnapalisina-sermetstatina .................................................................................. 64 3.4.3. Subtilisina-sermetstatina-esnapalisina ................................................................ 65 3.5. Cristalización y colección de datos de difracción de rayos X ...................................... 65 3.6. Resolución de estructuras y refinamiento .................................................................. 67 3.7. Experimentos de estabilidad de sermetstatina. Puentes disulfuro ............................ 68 3.8. Miscelánea .................................................................................................................. 68 4. Resultados y discusión .............................................................................................. 69 4.1. Producción y purificación de sermetstatina ...............................................................
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