Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 14, Pages -Publisher
NATL ACAD SCIENCES
Keywords
KhtT; KhtTU; B. subtilis; RCK domain; K+/H+ antiporter
Categories
Funding
- Biochemical and Biophysical Technologies, Cell Culture and Genotyping, and X-ray Crystallography scientific platforms of i3S (Porto, Portugal)
- Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance [ROTEIRO/0028/2013, LISBOA-01-0145-FEDER-022125]
- Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020
- Portuguese funds through Fundacao para a Ciencia e a Tecnologia (FCT)/Ministerio da Ciencia, Tecnologia e Ensino Superior [POCI-01-0145-FEDER-029863(PTDC/BIA-BQM/29863/2017)]
- Fundacao Luso-Americana para o Desenvolvimento through the FLAD Life Science 2020 award Bacterial K+ transporters are potential antimicrobial targets: mechanisms of transport and regulation
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bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that plays various roles in bacteria, including regulation of virulence, cell wall formation, biofilm formation, and DNA integrity surveillance. It has been shown to have a regulatory effect on the activity of K+ homeostasis machinery, inhibiting K+ import and activating K+ export. Experimental data suggest that c-di-AMP activates KhtTU through a specific mechanism.
bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.
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