Repository logo
 
No Thumbnail Available
Publication

Unconventional Cohesin-Dockerin binding mechanisms reveal the complexity of cellulosome assembly

Use this identifier to reference this record.

Abstract(s)

ABSTRACT - The plant cell wall is a vast reservoir of carbon and energy, yet its recalcitrant nature presents a significant challenge to efficient biomass conversion. In order to overcome this hurdle, certain anaerobic bacteria have evolved sophisticated multi-enzyme complexes for the efficient degradation of plant cell wall polysaccharides, termed cellulosomes. Cellulosomes are assembled via calcium-dependent, high-affinity interactions between the cohesin (Coh) modules present in non-catalytic scaffoldins and the enzyme-borne dockerin (Doc) modules. The majority of these interactions display a dual-binding mode (DBM), which enhances the structural flexibility of the cellulosome by allowing Docs to bind Cohs in two opposing orientations, thereby minimizing steric hindrance. This study focuses on the Coh-Doc systems of Bacteroides cellulosolvens and Ruminococcus flavefaciens, two bacteria with highly intricate cellulosomal architectures. While B. cellulosolvens makes extensive use of the DBM, facilitating the assembly of up to 110 enzymes into a single complex, R. flavefaciens was traditionally thought to rely solely on single-binding mode interactions, forming smaller, albeit complex, cellulosomes. Additionally, Ruminococcus albus, a ruminal bacterium that possesses several Doc-encoding genes with high homology to R. flavefaciens, lacks the necessary cohesins to form canonical cellulosomes, raising questions about its cellulose degrading mechanism. In this work, the molecular mechanisms underlying the Coh-Doc interactions in these three microorganisms were explored, revealing several key insights. First, it was discovered that type II Coh-Doc interactions in B. cellulosolvens, previously thought to function only in cell anchoring, also exhibit DBM, underscoring the organism's reliance on this versatile mechanism to assemble its highly complex cellulosome. Contrary to earlier reports, R. flavefaciens was also found to incorporate DBM through the action of the adaptor scaffoldin ScaH, introducing structural flexibility to avoid steric clashes between cellulosomal units. Furthermore, a novel binding mechanism was uncovered in which a single Coh domain could bind two Doc modules simultaneously. This novel 2:1 Coh-Doc interaction mode involves smaller, naturally truncated Doc modules. The study also demonstrated interspecies hybridization potential, with cross species Coh-Doc interactions occurring between R. flavefaciens and R. albus, suggesting a cooperative role in the rumen ecosystem. Additionally, through structure guided protein engineering, enhanced Coh-Doc binding affinities were achieved by modulating the electrostatic profiles of the binding surfaces. This research advances our understanding of cellulosome assembly, with implications for the efficient conversion of lignocellulosic biomass into biofuels, as well as potential biotechnological applications in protein engineering and molecular biology RESUMO - A biomassa vegetal representa a maior fonte de carbono orgânico do planeta. Contudo, a parede celular vegetal é altamente recalcitrante, o que dificulta o acesso e ação das enzimas envolvidas na sua degradação. Estas enzimas, genericamente designadas de Cazymes (carbohydrate active enzymes), são produzidas por diversos microrganismos e desempenham um papel crucial na conversão da biomassa vegetal. Elas encontram se organizadas em dois sistemas distintos. Os microrganismos aeróbios secretam enzimas livres no espaço extracelular, onde estas encontram e degradam o seu respetivo substrato. Em contraste, os microrganismos anaeróbios, que habitam ambientes altamente competitivos, desenvolveram uma estratégia distinta ao secretarem complexos multi-enzimáticos, denominados celulossomas, que mantêm as enzimas funcionalmente integradas, trabalhando em sinergia e acopladas à parede celular. Este mecanismo promove uma degradação mais eficiente e coordenada da parede celular vegetal, destacando-se como uma estratégia evolutiva para maximizar a conversão da biomassa. A montagem do celulossoma, que se baseia em interações proteína-proteína, tem vindo a ser alvo de estudos de natureza estrutural e funcional que conduziram à elucidação da forma como os celulossomas se organizam. A montagem destes complexos é mediada por interações de alta afinidade dependentes de cálcio entre duas proteínas modulares: as coesinas (Cohs) e as doquerinas (Docs). As coesinas são componentes modulares repetidas localizadas em proteínas estruturais não catalíticas, denominadas escafoldinas, enquanto as doquerinas estão predominantemente presentes na extremidade C-terminal das CAZymes. A interação Coh-Doc serve para fixar firmemente as CAZymes às escafoldinas, que por sua vez também podem conter um módulo doquerina que facilita a ancoragem à parede celular ou a articulação com outras escafoldinas portadoras de coesinas, formando estruturas ainda mais complexas. [...].

Description

Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Veterinárias na especialidade em Ciências Biológicas e Biomédicas

Keywords

Cohesin Dockerin Cellulosomes Protein Complexes Carbohydrates Celulossoma Coesina Doquerina Complexos Proteicos

Pedagogical Context

Citation

Santos MRCD. 2025. Unconventional Cohesin-Dockerin binding mechanisms reveal the complexity of cellulosome assembly [dissertation]. Lisboa: FMV-Universidade de Lisboa

Organizational Units

Journal Issue

Publisher

Universidade de Lisboa, Faculdade de Medicina Veterinária

CC License