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Multitask NBDs of Bacterial ABC Type I importers: characterization of protein-protein interactions
Publication . Alves, Catarina de Jesus Martins; Nogueira, Isabel; Godinho, Lia
ATP-binding cassette (ABC) transporters are a family of membrane translocators ubiquitous to all domains of life. This supergroup is divided into importers and exporters, and both share a structure of two Nucleotide-Binding Domains (NBDs), responsible for the binding and hydrolysis of ATP and consequently powering the conformational changes in the two Transmembrane Domains (TMDs), that form the translocation pore. In the case of ABC type I importers, there is also a substrate-binding protein (SBP), responsible for the binding of substrate and its delivery to the TMDs. This subfamily of ABC transporters has been only identified in bacteria and plants, having an important part in pathogenesis and survival. Lately, several studies have shown the existence of a group of multitask ATPases, found in the Firmicutes phylum, capable of energizing several sugar transport systems. Intra- and inter-species exchangeability and exchangeability beyond the phylum were also found to be a characteristic of these multitask NBDs. To identify signature motifs associated with the multitasking ability of some NBDs, the AraNPQ-MsmX system of Bacillus subtilis was used as a model. A collection of mutant TMDs, AraP and AraQ was constructed by targeting several conserved residues in the EAA motifs and in the C-terminal tail of AraQ. Using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system, the effect of these mutations on interaction with the NBD MsmX was quantified. Our results show that all targeted residues have different effects on MsmX-TMDs interaction, demonstrating the importance of these conserved motifs as key contact points with this ATPase. Moreover, the BACTH system was also used to characterize the interaction between previously identified multitask ATPases of the Firmicutes phylum and beyond it and the AraPQ translocators. Here, a correlation between NBD-TMD interaction measured in E. coli and the respective functionality of the complete transporter in B. subtilis was found, establishing the BATCH system as a powerful tool to study protein-protein interactions in bacterial ABC type I importers.
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Fundação para a Ciência e a Tecnologia
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3599-PPCDT
Número da atribuição
PTDC/BIA-MIC/30696/2017