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Universidade De Lisboa Faculdade De Medicina Veterinária UNIVERSIDADE DE LISBOA FACULDADE DE MEDICINA VETERINÁRIA DIVERGENT CELLULOSOME ARCHITECTURE IN RUMEN BACTERIA: STRUCTURE AND FUNCTION STUDIES IN COHESIN-DOCKERIN COMPLEXES OF RUMINOCOCCUS FLAVEFACIENS PEDRO MIGUEL BULE GOMES Orientadores: Doutor Carlos Mendes Godinho de Andrade Fontes Doutor Shabir Najmudin Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Veterinárias na Especialidade de Produção Animal 2017 UNIVERSIDADE DE LISBOA FACULDADE DE MEDICINA VETERINÁRIA DIVERGENT CELLULOSOME ARCHITECTURE IN RUMEN BACTERIA: STRUCTURE AND FUNCTION STUDIES IN COHESIN-DOCKERIN COMPLEXES OF RUMINOCOCCUS FLAVEFACIENS PEDRO MIGUEL BULE GOMES Orientadores: Professor Doutor Carlos Mendes Godinho de Andrade Fontes Doutor Shabir Najmudin Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Veterinárias na Especialidade de Produção Animal Júri: Presidente: Doutor Rui Manuel de Vasconcelos e Horta Caldeira Vogais: Doutor Harry J. Gilbert Doutor Gideon Davies Doutor Luís Manuel dos Anjos Ferreira Doutor José António Mestre Prates Doutor Carlos Mendes Godinho de Andrade Fontes 2017 Para a coautora das minhas aventuras Joana Dias I II Agradecimentos Ao entrar na reta final de uma das fases mais desafiantes da minha vida, não poderia deixar de aproveitar esta oportunidade para agradecer a todos os que estiveram envolvidos no meu percurso como estudante de doutoramento. Embora as grandes conquistas científicas estejam invariavelmente associadas a um nome, o sucesso científico está longe de ser um esforço individual. Uma equipa que nos apoie, motive e estimule a persistir mesmo nos momentos de maior frustração, é essencial para alcançar os nossos objetivos. Nesse aspeto considero-me bastante afortunado pois o grupo de trabalho a que pertenci ao longo destes anos foi fundamental para o meu sucesso como investigador. Assim, gostaria de agradecer: Ao meu orientador, Professor Doutor Carlos Fontes, que é o exemplo perfeito de um grande cientista e que irei para sempre ter como referência e modelo. Uma fonte inesgotável de genialidade, pragmatismo e perseverança tornam-no no líder perfeito do grupo de investigação. Muito obrigado pelo seu constante entusiamo, boa disposição, transmissão de conhecimento, amizade e também pela confiança que depositou no meu trabalho, o que tanto me motivou. Ao longo destes anos o Carlos foi um “chefe” que nunca me levou a olhar para o trabalho como uma obrigação mas sim como uma emocionante tarefa; To my other supervisor, Dr. Shabir Najmudin, for all his teachings and for always pushing me to go further. He has given me the habit of always questioning everything, something that is at the very heart of science. His free and surprising adventurous spirit is inspiring and make him great company. Thank you, Shabir, for becoming a dear friend!; Ao Professor Doutor Luís Ferreira pela sua incrível simpatia e sentido crítico. O seu entusiasmo e capacidade de transmitir conhecimento em forma de elogios são extraordinariamente motivadores; Ao Professor Doutor José Prates pelos seus ensinamentos e pela natureza metódica do seu trabalho, a qual me inspirou a ser mais exigente e organizado; Ao Professor Victor Alves pelos seus ensinamentos e pela excelente colaboração que mantivemos. A excelente forma com que os nossos resultados estão ilustrados devem-se maioritariamente à sua proficiência; À Helena Santos, a alma do laboratório, pela sua incondicional disponibilidade para ajudar, pela qualidade do seu trabalho, organização e perseverança. Sem a presença da Lena jamais teríamos as condições necessárias para produzir boa ciência. Obrigado também pela sua simpatia e amizade; À Virgínia Pires, a minha derradeira colega de laboratório, pela sua ajuda, pelos seus ensinamentos, pela sua capacidade de aturar a minha rabugice, por adicionar mais um ou dois protocolos à sua já enorme dose diária e me salvar do desespero da falta de tempo, pelos géis, placas e pipetas que lhe surripiei mas principalmente pela sua amizade; À Joana Brás e à Teresa Ribeiro por me terem guiado na minha introdução ao mundo da ciência, pela vossa ajuda e simpatia. As vossas dicas preveniram uma série de eventos potencialmente catastróficos e com cheiro a queimado. III To Kate Cameron, my Coh-Doc buddy, for sharing with me the thrills of ITC runs, crystal fishing sessions and trips to the plate robot! Her sense of humor has kept me sane through the most tedious protocols. Thank you for your friendship, Kate! To Immacolata Venditto for her help and friendship. Her efficiency and organization skills never stopped to amaze me. À Dona Paula pela sua disponibilidade, bom humor e amizade. É um elemento fundamental do DPASA! Ao grupo de Biologia Estrutural da Faculdade de Ciências e Tecnologia, com especial destaque para a Professora Ana Luísa Carvalho e a Cecília Bonifácio, pela sua ajuda, disponibilidade, paciência e ensinamentos. Foram fundamentais para a concretização do meu trabalho e também para a minha progressão enquanto investigador; To Professor Harry Gilbert and all the support he has given me throughout my PhD, especially during my stay at Newcastle University’s Institute for Cell and Molecular Biosciences. His input has been invaluable for my work. To Professor Ed Bayer for being always ready to help and for being such an inspiring scientist with a wonderful personality. Our collaborations have been particularly fruitful! To Professor Arun Goyal for his teachings, kind nature, hospitality and friendship. Also to all his students in IIT Guwahati who have made me feel at home during my stay at the institute. To Professor Steven Smith for his fundamental help and contribution that guaranteed the quality of our work. Ao grupo da NZYtech pela sua enorme ajuda. Com o vosso apoio consegui estabelecer a base sobre o qual todo o trabalho aqui apresentado foi construído. À Susana Martins, Cristina Monteiro e Inês Viegas pela boa disposição e sentido de humor e por me terem apresentado ao fantástico mundo da parentalidade e todos os cremes a ele associados. Num percurso repleto de altos e baixos, momentos de êxtase mas também de frustração, foi essencial partilhar essas experiências com aquelas pessoas que para mim são as mais importantes do mundo. Família e amigos são capazes de tornar um problema numa memória distante e uma pequena conquista num grande avanço científico. Mais uma vez, também neste aspeto, me considero uma pessoa sortuda. Quando estamos rodeados das pessoas que adoramos, amamos e admiramos, tudo se torna muito mais simples e é por isso que quero agradecer: A todos os meus amigos pelos bons momentos que passamos, pela sua disponibilidade para ir beber um copo e ouvir as minhas queixas e por saber que irão estar sempre presentes. Nunca poderia de deixar de vos agradecer. Até porque vocês possuem demasiada informação sensível para que eu arrisque deixar-vos ofendidos... Em particular agradeço ao João Ferreira, Joana IV Lobo, Rui Mascarenhas e Mafalda Coelho por me terem ajudado a manter a saúde mental ao longo destes anos. À minha família, por todo o seu apoio e carinho, com destaque especial para os meus pais e irmã. Mãe e Pai, vocês fizeram de mim aquilo que eu sou, sempre me apoiaram nas minhas decisões e sempre me deram o vosso amor incondicional. Ensinaram-me que a vida não é uma série de problemas mas sim de soluções e que apenas podemos ser respeitados se respeitarmos os outros. São as duas melhores pessoas que eu conheço e não poderia ter tido melhores pais. Mana, obrigado por todas as parvoíces que partilhámos. Até fomos umas crianças felizes! Estou muito orgulhoso que também tu estejas a terminar uma etapa importante na tua vida. À Joana Dias, a quem dedico esta tese, pela sua ajuda, apoio e amor incondicionais. Obrigado pelos teus conselhos, por me manteres focado e me inspirares, por me aturares nos dias mais rabugentos, por não te importares com jantares fora de hora, por aguentares as ausências mais prolongadas e por manteres a calma numa casa sem roupa lavada e comida no frigorífico. Tenho muita sorte em poder partilhar contigo, não só uma vida, mas também a paixão e o entusiasmo pela ciência: “That’s our thing” e é aquilo que vamos passar a quem aí vier. Obrigado meu Amor! V This work was supported by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-PRO/103980/2008, EXPL/BIA-MIC/1176/2012 and PTDC/BIA- MIC/5947/2014, by the European Union Seventh Framework Programme (FP7 2007-2013) under the WallTraC project (grant agreement No. 263916) and BioStruct-X (grant agreement No. 283570) VI Resumo Arquitetura celulossomal divergente em bactérias ruminais: estudos de estrutura e função em complexos coesina-doquerina do Ruminococcus flavefaciens As interacções proteína-proteína desempenham um papel essencial em vários processos celulares, sendo exemplo disso a estruturação do celulosoma, um complexo bacteriano multienzimático altamente eficiente na degradação da celulose e hemicelulose. A montagem do celulosoma envolve interações de alta afinidade entre doquerinas do tipo I, presentes em enzimas, e os módulos coesina presentes em proteínas estruturais não catalíticas denominadas de escafoldinas. Adicionalmente, todo o complexo é ancorado à superfície bacteriana através da ligação de uma dockerina do tipo II, presente numa escafoldina, a coesinas ligadas à célula. Inicialmente, pensava-se que a arquitectura e organização dos celulosomas assentava exclusivamente em interacções coesina-doquerina do tipo I e II. Recentemente, foi sugerido que a microbiota ruminal contém
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