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Universidad Autónoma De Madrid Facultad De Ciencias Departamento De Biología Molecular Universidad Autónoma de Madrid Facultad de Ciencias Departamento de Biología Molecular Structural biology and characterization of the human R2TP, an HSP90 co-chaperone complex PhD. Thesis Hugo Muñoz Hernández 2017 Universidad Autónoma de Madrid Facultad de Ciencias Departamento de Biología Molecular PhD. Thesis Author: Hugo Muñoz Hernández This thesis was supervised by Prof. Óscar Llorca, most of the work was realized at Centro de Investigaciones Biologicas, part of the Spanish National Research Council. The work was funded by the Spanish fellowship (FPI) of the Ministry of Economy, Industry and Competitiveness associated with the project with reference SAF2011- 22988 . Madrid, 2017 Universidad Autónoma de Madrid Facultad de Ciencias Departamento de Biología Molecular TESIS DOCTORAL Autor: Hugo Muñoz Hernández Esta tesis ha sido dirigida por el Prof. Óscar Llorca, realizada en el Centro de Investigaciones Biológicas del CSIC y financiada por el programa de Formación de Personal Investigador (FPI) del Ministerio de Economía, Industria y Competitividad asociado al proyecto con referencia SAF2011-22988. Madrid, 2017 D. ÓSCAR ANTONIO LLORCA BLANCO, Profesor investigador del Consejo Superior de Investigaciones Científicas, CERTIFICA: Que la presente Tesis Doctoral titulada “Structural bio- logy and characterization of the human R2TP, an HSP90 co-chaperone complex” ha sido realizada bajo su dirección por el Licenciado Hugo Muñoz Hernández, en el Centro de Investigaciones Biológicas para optar al grado de Doctor en Bioquímica, Biología Molecular, Bio- medicina y Biotecnología. Madrid, a 8 de Mayo de 2017. Prof. Óscar Antonio Llorca Blanco Caer es fácil, lo difícil es levantarse. I only see my goals. Ya soy amigo de la piedra de tanto tropezar. I don't believe in failure. Agradecimientos Este trabajo no hubiera sido posible sin Juan Muñoz y María Aurelia Hernández, mis padres. Los primeros y los mejores maestros que tengo. Un ejemplo a seguir en todos los aspectos de la vida. Gracias por todo, sin vosotros, seria nada. Os quiero. Por otro lado, quiero mostrar mis más sentidos agradecimientos a dos personas muy importantes. Fabrizio, te admiro. Haber estado a tu lado ha sido una experiencia muy enriquecedora, sin duda tuvimos buenos momentos y aprendimos mucho juntos. Óscar, mi más especial reconocimiento es para ti, mi mentor. Te agradezco la oportunidad que me diste de desarrollarme como investigador. No sólo eres un buen jefe, sino que también eres una maravillosa persona. Considero que eres un ejemplo a seguir. Me has enseñado mucho. Gracias por todo. Agradezco a la gente del CIB por su apoyo, dedicación y tiempo. En especial a mis compañeros: Luque, Cesar, Roberto, H. Yebenes, Carlos, Marina, R. Gomez, Teresa, Adrián, Martín, Eva, Fernando, Rafa, Begoña y a mi fiel amigo Ángel. A la gente de los grupos de C. Fernández y A. Romero. A las chicas del laboratorio de G. Rivas, con cariño a Noelia. A Carlos y a los Increíbles. Al grupo de F. Diez y en especial a Gonzalo. Doy gracias a Albert, compañero de FPI2012. A todos los organizadores de la fiesta de primavera y de las charlas pre- doctorales. Al personal de compras, seguridad, limpieza, administración, cafetería y servicio técnico del CIB que durante este tiempo ayudaron con su labor al desarrollo de esta tesis en un ambiente correcto. Quiero dar las gracias a mis compañeros microscopistas, su ayuda fue clave en el desarrollo de este trabajo. En especial a: Guy, Sasha, Jean-François, Dan, Alister, Sojnia, Rocío, Chichón, al gran David del Biogune y a la querida Jaska. Sin duda, agradezco a Iskander, Edoardo, Julia y al resto de colegas con los que he compartido los cursos y congresos. Estoy muy agradecido a la gente que me ayudo en Sussex: Laurence, Chris, Lihong, Raquel, Mariano y Alejandro. Con cariño a Mohinder, gracias. Me aportó mucho estar a tu lado en la estancia. No se como se dirá en panyabí, pero eres increíble. Gracias Maytes (incluyo a la Yayita). Supisteis ayudarme cuando las cosas no salían tan bien como hubiera querido, gracias por el apoyo y los sabios consejos que me disteis. A mis fieles amigos, Fer y Peter. Gracias por todo lo que me aportáis. Llegar o salir del trabajo sabiendo que estáis ahí es un lujo. Gracias por apoyarme tanto Fer. Os quiero hermanos. Hablando de amor, a ti Leti, cariño mio, por darme la alegría, vitalidad, bienestar, complicidad y conexión que un hombre necesita. A mis amigos de Los Molinos, por darme esos momentos de relax, sois geniales. Agradezco a mi familia chicharrera, a la madrileña, a la bachata, a la salsa y al fútbol por todo lo que habéis aportado. Por ultimo, mostrar también mis agradecimientos a todas las personas que me he encontrado en el camino académico. A mis antiguos compañeros y profesores de la UAM: Judith, Víctor, Charlie, Elena, Berta, Guille, Irene, Olga, Lorena, Mario, Lola, M. Fernández- Lobato y J. Sanz. En definitiva, con estas palabras he querido llegar al corazón de todas las personas que han participado en mi formación como científico y transmitirles el orgullo que siento de que me hayan acompañado en esta carrera de fondo. Un abrazo. Table of Contents RESUMEN EN CASTELLANO....................................................................................21 SUMMARY...................................................................................................................23 Chapter 1. INTRODUCTION.......................................................................................27 1.1. Structural studies in Molecular Biology.....................................................27 1.2. Three-dimensional Electron Microscopy for life science research..........30 1.3. HSP90 chaperone and co-chaperones complexes....................................35 1.4. The R2TP complex........................................................................................37 i ) The discovery of RTP2: composition and function.........................................37 ii ) R2TP components: RuvBL1-RuvBL2 AAA+ ATPases...................................41 iii ) R2TP components: PIH1D1.........................................................................45 iv ) R2TP components: the TPR proteins in R2TP.............................................47 1.5. Client proteins of the hR2TP-HSP90 system..............................................49 i ) R2TP in Box C/D snoRNP biogenesis...........................................................49 ii ) R2TP in PIKK signalling................................................................................50 iii ) R2TP in RNA polymerase assembly............................................................52 iv ) R2TP in telomerase assembly.....................................................................52 Chapter 2. AIM OF THIS THESIS...............................................................................57 Chapter 3. EXPERIMENTAL PROCEDURES.............................................................61 3.1. Molecular cloning and DNA preparation.....................................................61 3.2. Expression of recombinant proteins in Escherichia coli..........................61 3.3. Expression of recombinant SMGs proteins in Insect cells.......................62 3.4. Biochemical analysis of proteins................................................................63 3.5. Proteins purification.....................................................................................64 3.6. Dynamic Light scattering.............................................................................65 3.7. Sedimentation velocity assay of RPAP3-PIH1D1 complex........................66 3.8. Sedimentation equilibrium assay of RPAP3-PIH1D1 complex..................66 3.9. In vitro protein-binding experiments...........................................................66 3.10. Human R2TP reconstitution and purification using GraFix....................67 3.11. Cross-linking coupled to mass spectrometry..........................................68 3.12. Negative staining electron microscopy....................................................69 3.13. Cryo-electron microscopy.........................................................................69 3.14. Computing and images processing..........................................................70 3.15. Image processing.......................................................................................71 i ) Negative staining processing.........................................................................71 ii ) Cloud computing...........................................................................................71 Chapter 4. RESULTS..................................................................................................77 4.1. Analysis and purification of R2TP components.........................................77 i ) Human RuvBL1–RuvBL2 sample preparation...............................................77 ii ) Biochemical characterization of the RuvBL1-RuvBL2 complex.....................78 iii ) Electron microscopy of the RuvBL1-RuvBL2 complex.................................80 iv ) Human RPAP3-PIH1D1 assembly and characterization..............................83 4.2. Human R2TP reconstitution and structure.................................................86 i ) Characterization of the human R2TP assembly.............................................86 ii ) Negative stained-EM of the human R2TP complex.......................................89 iii ) Cryo-EM of the human R2TP complex.........................................................91
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