Characterization of protein-protein interactions in the Bacillus subtilis AraNPQ-MsmX transporter

• Autor(a) principal: Almeida, Joana Raquel Encarnação
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/111323
• topic: ABC carbohydrate importers; Multitask ATPases; MsmX; AraNPQ importer; NBD-TMD interactions; BACTH system; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The ATP-binding cassette (ABC) transporters are one of the largest and most diverse transporter superfamilies, widespread among all domains of life. They share the same modular architecture, comprising two nucleotide-binding domains (NBDs) responsible for energizing the system via ATP hydrolysis, two transmembrane domains (TMDs), which form a substrate translocation pathway, and in the case of importers, a substrate-binding protein (SBP). In ABC importers, upon ATP binding to the intracellular NBDs, conformational modifications are coupled to the TMDs, which lead to ATP hydrolysis and subsequent substrate translocation. Lately, it has been suggested that bacteria evolved towards the sharing of ATPases between ABC importers involved in carbohydrate utilization. To identify putative signature motifs associated with the multitasking role of this family of NBDs, the MsmX ATPase from Bacillus subtilis, which is the NBD partner of several sugar importers, such as AraNPQ, was used as a model to characterize interactions between multitask ATPases and their cognate transmembrane domains of ABC importers. Preliminary data gave an insight about putative residues important for the contact between MsmX and AraPQ. In this work we further characterize the NBD-TMD interaction region, by targeting several amino acids in distinct combinations. The ability of these mutant ATPases to act as an energy generating component of the AraNPQ importer in a B. subtilis msmX-null mutant was assessed. The results show that all targeted amino acids may have a putative role in MsmX-TMDs interaction, although each mutant MsmX exhibits a different degree of capacity to energize the AraNPQ importer. Furthermore, the bacterial adenylate cyclase two hybrid system (BACTH), used to screen NBD-TMD interaction in vivo, was established as suitable for detecting changes in the interacting ability of mutant MsmX proteins with TMDs AraP and AraQ.