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Molecular mechanisms of c-di-GMP signaling and of target AMPylation

English title Molecular mechanisms of c-di-GMP signaling and of target AMPylation
Applicant Schirmer Tilman
Number 138414
Funding scheme Project funding
Research institution Abteilung Strukturbiologie und Biophysik Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Biophysics
Start/End 01.02.2012 - 31.01.2016
Approved amount 560'000.00
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Keywords (6)

crystal structure analysis; pathogenic bacteria; signaling; second messenger; molecular mechanism; novel targets for antibiotics

Lay Summary (English)

Lead
Lay summary
This research project investigates the mechanisms of two bacterial proteins on the molecular level. For this we are combining X-ray structure analysis, functional and biophysical characterization, and site-directed mutagenesis. Since in both projects fundamental bacterial activities are investigated, the resulting knowledge may help in the development of novel strategies to combat pathogenic bacteria. (A) Bacteria use small molecules as "second messengers" to relay information from sensory or regulatory protein domains to specific receptors. Thereby, the messenger molecules can interact with several receptors to control their activity (catalysis, motion, etc.) simultaneously. In his way, bacteria can react to various input signals like light or the presence of certain nutrients. Via the second messenger cyclic di-GMP, a small nucleotide, a large variety of cell surface associated traits are regulated in response to internal and external stimuli. We aim to unravel the mechanisms through which cyclic-di-GMP specific phosphodiesterases, enzymes that degrade cyclic-di-GMP, are regulated by their associated regulatory domains, such as Rec, PAS, and DNA binding domains. Furthermore, we want to determine the atomic structures of novel c-di-GMP receptor proteins. This will provide insight into mechanisms of down-stream signaling. Elucidation of the cyclic-di-GMP signal network may lead to the identification of targets for the development of novel antibiotics. (B) AMPylation, i.e. transfer of a AMP moiety onto a protein target molecule, is a recently discovered strategy of bacteria to subvert the proper functioning of mammalian host cells. This process is catalyzed by the Fic domain and we propose to continue our studies on the molecular mechanism of this activity. Most recently it has been discovered that certain effector proteins of the type 4 secretion system (T4SS) with a Fic domain are inhibited by so-called antitoxins. We have started to elucidate the molecular basis of this interaction and want to unravel the inhibitory mechanism. Furthermore, we want to extend our studies to stand-alone Fic proteins from E. coli and N. meningitis that probably AMPylate targets in their own cell. Identification of Fic targets and structure determination of Fic/target structures will follow to reveal structural determinants of target affinity and specificity. Discovering the molecular basis for the toxicity of Fic proteins may also help to combat bacteria with their own weapons.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli
Reinders Alberto, Hee Chee-Seng, Ozaki Shogo, Mazur Adam, Boehm Alex, Schirmer Tilman, Jenal Urs (2016), Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli, in Journal of Bacteriology, 198(3), 448-462.
Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification.
Stanger Frédéric V, Burmann Björn M, Harms Alexander, Aragão Hugo, Mazur Adam, Sharpe Timothy, Dehio Christoph, Hiller Sebastian, Schirmer Tilman (2016), Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification., in Proceedings of the National Academy of Sciences of the United States of America, 113(5), 529-37.
Adenylylation of Gyrase and Topo IV by FicT Toxins Disrupts Bacterial DNA Topology.
Harms Alexander, Stanger Frédéric Valentin, Scheu Patrick Daniel, de Jong Imke Greet, Goepfert Arnaud, Glatter Timo, Gerdes Kenn, Schirmer Tilman, Dehio Christoph (2015), Adenylylation of Gyrase and Topo IV by FicT Toxins Disrupts Bacterial DNA Topology., in Cell reports, 12(9), 1497-507.
Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control.
Ozaki Shogo, Schalch-Moser Annina, Zumthor Ludwig, Manfredi Pablo, Ebbensgaard Anna, Schirmer Tilman, Jenal Urs (2014), Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control., in Molecular microbiology, 94(3), 580-94.
Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP.
Sundriyal Amit, Massa Claudia, Samoray Dietrich, Zehender Fabian, Sharpe Timothy, Jenal Urs, Schirmer Tilman (2014), Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP., in The Journal of biological chemistry, 289(10), 6978-90.
Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold.
Goepfert Arnaud, Stanger Frédéric V, Dehio Christoph, Schirmer Tilman (2013), Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold., in PloS one, 8(5), 64901-64901.
Structure and signaling mechanism of a zinc-sensory diguanylate cyclase.
Zähringer Franziska, Lacanna Egidio, Jenal Urs, Schirmer Tilman, Boehm Alex (2013), Structure and signaling mechanism of a zinc-sensory diguanylate cyclase., in Structure (London, England : 1993), 21(7), 1149-57.
Type II toxin - antitoxin loci: the fic family.
Goepfert Arnaud, Harms Alexander, Schirmer Tilman, Dehio Christoph (2013), Type II toxin - antitoxin loci: the fic family., in Gerdes Ken (ed.), Springer, Berlin, Heidelberg, 177-187.

Collaboration

Group / person Country
Types of collaboration
Prof. U. Jenal, Biozentrum, Universität Basel, Abt. Molekulare Mikrobiologie, 4056 Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Ch. Dehio, Biozentrum, Universität Basel, Abt. Molekulare Mikrobiologie, 4056 Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Associated projects

Number Title Start Funding scheme
105587 Structure and function of prokaryotic membrane permeases and pores and of a two-component response regulator 01.10.2004 Project funding
150814 Purchase of a 900 MHz high-resolution NMR instrument 01.12.2013 R'EQUIP
145023 Advanced Imaging System for Biomolecular Crystallization Screening 01.12.2012 R'EQUIP
127433 Mechanisms of cyclic di-GMP signaling 01.02.2010 Sinergia
166652 Molecular mechanisms of c-di-GMP signaling 01.04.2016 Project funding
166652 Molecular mechanisms of c-di-GMP signaling 01.04.2016 Project funding

Abstract

Our general aim is to contribute to the understanding, on the molecular level, of the mechanisms by which proteins perform their action. For this we are combining X-ray structure analysis, functional and biophysical characterization, and site-directed mutagenesis. Here, we propose research on components of the bacterial c-di-GMP signaling pathway, which we are studying for several years, and on adenylyl transferases with a Fic domain.(A) Via the second messenger cyclic di-GMP, a large variety of cell surface associated traits are regulated in response to internal and external stimuli. We aim to unravel the mechanisms through which c-di-GMP specific phosphodiesterases are regulated by their associated regulatory domains, such as Rec, PAS, and DNA binding domains. A working hypothesis has previously been derived from our study on the YkuI protein that predicts coupling of quarternary state and active site geometry of the phoshodiesterase. Furthermore, we want to determine the structures of novel c-di-GMP binding proteins (effector proteins) that are putatively involved in the control of transcription and in the synthesis and export of capsular polysaccharide. This will provide insight into mechanisms of down-stream signaling. (B) AMPylation, i.e. transfer of a AMP moiety, is a recently discovered strategy of bacteria to subvert host cell function. This process is catalyzed by the Fic domain and we propose to continue our studies on the molecular mechanism of this activity. Most recently it has been discovered that effector proteins of the type 4 secretion system (T4SS) with a Fic domain are inhibited by cognate antitoxins, probably to avoid AMPylation of endogenous proteins. We have started to elucidate the molecular basis of this interaction and want to unravel the inhibitory mechanism. Furthermore, we want to extend our studies to stand-alone Fic proteins from E. coli and N. meningitis that probably AMPylate endogenous targets. Interestingly, some of these proteins appear to utilize a related, but autoinhibitory, mechanism. Identification of Fic targets and structure determination Fic/target structures will follow to reveal structural determinants of target affinity and specificity.
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