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Molecular mechanisms of c-di-GMP signal transduction and AMP transferases

We are employing crystallographic and biochemical/biophysical techniques to reveal the structural basis for the catalysis and regulation of c-di-GMP related proteins. Our second focus is on bacterial type IV secretion system (T4SS) effector proteins with AMP transferase activity.

Make, break and recognition of c-di-GMP
Recent discoveries show that a novel second messenger, c-di-GMP, is extensively used by bacteria to control multicellular behavior, such as biofilm formation. Condensation of two GTP to the dinucleotide is catalyzed by GGDEF domains that usually occur in combination with sensory and/or regulatory modules. The opposing phosphodiesterase activity is provided by EAL domains that are similarly regulated.

In collaboration with the Jenal group (Biozentrum) and based on crystallographic and functional studies, we have studied the catalytic and regulatory mechanisms of diguanylate cyclases, the enzymes that synthesize the second messenger. It appears that the general mechanism of activation relies on signal induced dimerization of its regulatory domains that ensures productive encounter of the two GTP loaded catalytic GGDEF domains. Currently, we are studying the molecular basis of phosphodiesterase regulation that are the antagonistic enzymes that degrade c-di-GMP. Although the active site is completely contained within their EAL domain, the domain is active only as a homodimer. This generic property of the catalytic domain is probably utilized in many of the full-length proteins to control their activity, very much the same as in diguanylate cyclases. Thus, our results provide clues about how this class of enzymes can be regulated in a modular and universal fashion by their sensory domains.

Recently, we have started to elucidate the structures and binding modes of newly discovered c-di-GMP receptors. Amongst these, there are c-di-GMP regulated histidine kinases (see below) and a re-purposed TIM barrel structure that is capable of binding c-di-GMP as well as ppGpp with inverse down-stream effects. The investigations will contribute to our knowledge of the complete c-di-GMP signal cascade.

c-di-GMP regulated histidine kinases
Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We are studying the regulation and the structure of the bifunctional histidine kinase CckA from C. crescentus and were able to reveal the mechanism that allows c-di-GMP to switch the enzyme from its default kinase to the phosphatase state. Non-covalent domain cross-linking by c-di-GMP freezes the protein in a domain constellation that allows docking and dephosphorylation of the target Rec domain, but prevents movement of the CA domain relative to the dimeric DHp stem required for auto-phosphorylation. Sequence comparisons suggest that c-di-GMP regulation is wide-spread amongst bacterial histidine kinases. 

Regulation of AMP transferases with Fic fold
Type IV secretion systems (T4SS) are utilized by many bacterial pathogens for the delivery of virulence proteins or protein-DNA complexes into their eukaryotic target cells. Together with the Dehio group (Biozentrum) we are working on a class of effector proteins that are composed of a FIC and a BID domain responsible for pathogenic action in the host cell (AMPylation of specific target proteins) and translocation, respectively.

Based on crystallographic analyses, we have found that Fic proteins are expressed in an inhibited form and are, thus, catalytically silent under normal circumstances. Inhibition is caused by partial obstruction of the ATP binding site by a helix that, depending on the Fic class, is provided by a cognate anti-toxin or is part of the enzyme itself. For the latter class, we have shown that auto-inhibition can get relieved by auto-modification of a tyrosine residue of this helix. We are interested in the structural basis of target recognition and, particular, target specificity. This knowledge may be utilized for drug development to target Fic proteins of bacterial pathogens.

 

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