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Computational Design and Experimental Characterization of Metallopeptides As Proteases for Bioengineering Applications

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Computational Design and Experimental Characterization of Metallopeptides As Proteases for Bioengineering Applications Henrique Daniel Figueiredo Carvalho Mestre em Bioquímica Computational design and experimental characterization of metallopeptides as proteases for bioengineering applications Dissertação para obtenção do Grau de Doutor em Bioengenharia (MIT Portugal) Orientador: Olga Iranzo, Doutora., Aix-Marseille Université Co-orientadores: Ana Cecília Roque, Prof. Doutora., Univ. NOVA de Lisboa Ricardo J. F. Branco, Doutor, Univ. NOVA de Lisboa Setembro 2017 ii Computational design and experimental characterization of metallopeptides as proteases for bioengineering applications Copyright © Henrique Daniel Figueiredo Carvalho, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa. A Faculdade de Ciências e Tecnologia e a Universidade Nova de Lisboa têm o direito, perpétuo e sem limites geográficos, de arquivar e publicar esta dissertação através de exemplares impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou que venha a ser inventado, e de a divulgar através de repositórios científicos e de admitir a sua cópia e distribuição com objectivos educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e editor. iii iv Agradecimentos Este trabalho marca o final de uma importante etapa no meu percurso académico e pessoal e portanto gostaria de agradecer a todos aqueles que de alguma forma estiveram envolvidos. À minha orientadora Doutora Olga Iranzo e co-orientadores Professora Doutora Ana Cecília A. Roque e Ricardo J. F. Branco por propocionarem as condições necessárias à realização deste trabalho nos grupo de Engenharia Biomolecular (UCIBIO/REQUIMTE, Faculdade de Ciências e Tecnologia da Universidade NOVA de Lisboa) e no grupo BiosCiences (Institut des Sciences Moléculaires de Marseille UMR CNRS 7313, Aix-Marseille Université), pela supervisão e apoio crítico na realização tarefas e discussão de resultados. Ao Doutor João Galamba Correia por me ter recebido no grupo de Ciências Radiofarmacológicas (Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico) para produção química de péptidos. Ao Doutor Manolis Matzapetakis por me ter recebibo do Laboratório de RMN Biomolecular (Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa) para realização de experiências de RMN. À Professora Doutora Rita Delgado e restantes membros do grupo de Química de Coordenação e Supramolecular (Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa) pela disponibilização de recursos e auxílio na realização de tarefas. Aos meus colegas e minhas colegas do grupo de Engenharia Biomolecular pelo companheirismo e convivência, assim como pela troca de ideias e auxílio na resolução de problemas. À minha família e amigos, pelo seu apoio incondicional e fundamental para que este trabalho pudesse ser realizado. Gostatia por ultimo de agradecer ao Programa Doutoral em Bioengenharia MIT/Portugal pela formação e à Fundação para a Ciência e Tecnologia pelo financiamento através da bolsa SFRH/BD/90644/2012 e ERA-IB-2. “The map is not the territory” Alfred Korzybski v vi Resumo Enzimas são versáteis catalisadoras presentes em sistemas biológicos e com elevado potencial tecnológico. Metaloproteases de zinco são uma classe de enzimas com aplicação corrente na indústria e.g. alimentar, biofarmacêutica e detergentes. A versatilidade química de diferentes metais combinada com modificações na sequência de metaloenzimas podem ser exploradas de forma a aumentar a sua robustez e leque de aplicações biológicas e tecnológicas, Neste trabalho, novos metaloproteases de zinco foram desenhados computacionalmente de forma a testar se atividade proteolítica pode ser reproduzida em proteínas pequenas adaptadas para bioengenharia. Análise de aspetos estruturais/dinâmicos de metaloproteases permitiram identificar interações cataliticamente relevantes. Modelos do centro ativo de zinco foram desenvolvidos para examinar com o software Rosetta um conjunto de 43 pequenas estruturas quanto ao seu potencial em recapitular a função nativa de enzimas. Duas estruturas foram selecionadas para design e caracterização experimental, nomeadamente o “zinc-finger” 2 da proteína Sp1 e o subdomínio cabeça da vilina. Embora coordenação com o metal tenha sido 5 -1 alcançada (constantes de afinidade KZnP,app na ordem 10 M ), as estruturas apresentam baixa estabilidade (temperatura de desnaturação inferior a 50 °C), refletindo perturbações provavelmente causadas por 4-10 modificações de sequência. Os metalopéptidos apresentam actividade catalítica para ésteres semelhante aos valores de literatura obtidos para outras -1 -1 -1 pequenas estruturas (constantes de segunda ordem k2 na ordem 10 M s ). A metodologia desenvolvida neste trabalho foi bem sucedida em desenhar metalopéptidos catalíticos, embora a atividade alvo de metaloprotease não tenha sido alcançada. Simulações de dinâmica molecular na escala de microsegundo foram usadas posteriormente para detectar falhas nos designs relacionadas com elevada flexibilidade estrutural. Este trabalho contribui para o melhoramento de métodos de design computacional de enzimas, ao demonstrar a necessidade de considerar aspectos dinâmicos dos designs em escalas de tempo maiores, e no desenvolvimento de métodos rápidos para classificar e avaliar um vasto leque de potenciais biocatalisadores. Termos-chave: design de enzimas; biocatalisadores: bioengenharia; péptidos; metaloproteases vii viii Abstract Enzymes are highly versatile catalysts present in biological systems and with high technolog- ical potential. Zinc metalloproteases are a major class of enzymes currently being employed in e.g. food, detergent, biopharmaceutical industries. In order to increase their robustness and range of biological and technological applications, metalloenzymes can be redesigned by exploring the chemical versatility of different metals along with protein sequence modifications. In this work, the computational design of new zinc metalloproteases was approached to test if proteolytic activity can be recapitulated in small scaffolds tailored for bioengineering applications. Structural and dynamical aspects of metalloproteases were first addressed to identify catalyti- cally-relevant interactions. Models of the active site were developed to screen a set of 43 small scaffolds with the Rosetta software for their ability to recapitulate the native enzyme functionality. Two candidate scaffolds were selected for enzyme design and experimental characterization, namely the Sp1 zinc finger 2 and the villin headpiece subdomain. While metal coordination was 5 -1 achieved (binding constants KZnP,app in the 10 M range), the scaffolds presented low stabilities (thermal unfolding bellow 50 °C) most likely due to perturbations introduced by the 4 to 10 se- quence modifications. The metallopeptides presented catalytic activity towards ester substrates within the range of values found for other small scaffolds in the literature (second-order rate con- -1 -1 -1 stants k2 in the 10 M s range). The design approach developed in this work was successful in achieving catalytically-active metallopeptides, although target metalloprotease activity could not be achieved. Molecular dy- namics simulations in microsecond regimes were subsequently used to detect design flaws re- lated with high scaffold flexibility. This work contributes to the improvement of the computational enzyme design approaches by pointing out the need for a dynamical treatment of the designs in longer time-scales, and through the development of fast methods to rank and evaluate a large number of potential biocatalysts. Keywords: enzyme design; biocatalysts; bioengineering; peptides; metalloproteases ix x Table of Contents AGRADECIMENTOS .........................................................................................................................V RESUMO .......................................................................................................................................... VII ABSTRACT ........................................................................................................................................ IX INDEX OF FIGURES ..................................................................................................................... XIII INDEX OF TABLES ......................................................................................................................... XV LIST OF ABBREVIATIONS ........................................................................................................ XVII 1. GENERAL INTRODUCTION .................................................................................................. 1 1.1 COMPUTATIONAL ENZYME DESIGN ............................................................................................. 3 1.2 METALLOPROTEASES ..................................................................................................................... 4 1.3 CATALYTIC MECHANISM OF METALLOPROTEASES..................................................................... 6 1.4 TECHNOLOGICAL APPLICATIONS OF METALLOPROTEASES ...................................................... 8 1.5 SMALL SCAFFOLDS FOR BIOENGINEERING APPLICATIONS ........................................................ 9 1.6 OBJECTIVES AND PROJECT LAYOUT ............................................................................................ 10 2. COMPUTATIONAL ENZYME DESIGN OF SMALL SCAFFOLDS ................................. 13 2.1 INTRODUCTION
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