Regulation of the magnesium channel MgtE by cyclic-di-AMP

• Autor(a) principal: Costa, Marta Bonifácio Pinho e
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/36522
• topic: Magnesium; c-di-AMP; MgtE; Ion channel; CBS domain

Cells are complex, adaptable systems that maintain a tight control over the concentration gradients of inorganic ions like K⁺, Na⁺, Cl⁻, Ca²⁺ and Mg²⁺, vital for a host of cellular functions like pH regulation, osmolarity control, electrical activity, and cellular communication. For this purpose, cells possess a variety of transporters and channels that facilitate their passage through the cell membrane. Magnesium (Mg²⁺) is the second most abundant intracellular cation, an important enzyme cofactor, structural component, and stabilizer of DNA. In Bacteria, several channels and transporters regulate Mg²⁺ influx and efflux, including the MgtE channel. MgtE possesses a cystathionine-β-synthase domain, the CBS domain, that can bind adenosine-containing ligands, with regulatory functions in other transporters. Among such ligands, we find cyclic-di-AMP, a cyclic nucleotide second messenger that modulates K⁺ transport at several levels and has been proposed to bind to MgtE. Recently, changes in intracellular Mg²⁺, K⁺ and cyclic-di-AMP concentrations were observed during osmotic stress response. The main goal of this work is to prove that cyclic-di-AMP binds to MgtE from B. subtilis, a model Gram-position bacteria, and to explore its effects on Mg²⁺ transport through MgtE. In this work, functional assays were performed, confirming MgtE’s role as a Mg²⁺ channel, but no conclusive results about cyclic-di-AMP’s effects were obtained. Large-scale expression and purification of MgtE’s N-terminal domain (containing the CBS sub-domain) were performed, followed by structural assays. Differential scanning fluorimetry assays identified cyclic-di-AMP as a ligand, and isothermal titration calorimetry determined that binding occurs with a dissociation constant KD of 1.75 μM and a stoichiometry of 1:4 cyclic-di-AMP to MgtE molecules. Size-exclusion chromatography revealed that MgtE’s cytosolic domain undergoes a conformational change, supporting the oligomerization of the domain in the presence of cyclic-di-AMP. Finally, crystallography assays yielded several crystals for analysis, but the structure of the protein-ligand complex could not be resolved due to poor crystal quality.