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Projekt Druckansicht

Structural, biochemical and biophysical analysis of DisA and DAC-domain proteins

Fachliche Zuordnung Biochemie
Förderung Förderung von 2012 bis 2016
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 218802008
 
Erstellungsjahr 2016

Zusammenfassung der Projektergebnisse

In the project we successfully analysed the mechanism of c-di-AMP synthesis by the DisA protein. We crystallized different Thermotoga maritma DisA proteins (wildtype, inactive mutants) in presence of ATP or non-reactive nucleotides in order to trap the enzyme in reaction intermediate steps. Using the structural data from X-ray crystallography we could identify the catalytically essential metal-ion binding site, describe the substrate coordination and activation that facilitates the reaction of 2 ATP to c-di-AMP between the two facing DAC- domains of DisA. In biochemical assays we tested the effects of active-site mutants and also provided a model for the main kinetical step, i.e. diffusion of substrate and product in and out of the rigid enzyme, by a mutant that created an additional tunnel from the bulk solvent to the active site. The respective mutant shows an almost doubled turnover rate of the enzyme. In addition, we show that a commercially available compound is a potent inhibitor of c-di-AMP synthesis. With respect to the fact that c-di-AMP signalling is essential for the bacteria producing it and the occurrence of c-di-AMP in various pathogenic diseases, the altering of cellular c-di-AMP levels might be a promising target for future antimicrobial therapies. In a second approach we biophysically and structurally characterized the c-di-AMP binding PII-like protein S.aureus PstA. We were able to solve the crystal structures of PstA in absence and presence of c-di-AMP. The putative signaling of the c-di-AMNP bound state is probably achieved via loop rearrangements that occur upon c-di-AMP is bound. We furthermore characterized c-di-AMP binding in biophysical assays and could show the high affinity and specificity of PstA for c-di-AMP.

Projektbezogene Publikationen (Auswahl)

 
 

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