cyclic di-GMP; diguanylate cyclase; phosphodiesterase; GGDEF; EAL; PilZ; bacteria; signaling; biofilm; Caulobacter crescentus; X-ray crystallography; single molecule FRET; bioinformatics; structural modeling; c-di-GMP; signal transduction; chronic infection
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 c-di-GMP, in Journal of biological Chemistry
, 289(10), 6978-6990.
Steiner Samuel, Lori Christian, Boehm Alex, Jenal Urs (2013), Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction, in EMBO Journal
, 32(3), 354-368.
Nesper Jutta Reinders Alberto Glatter Timo Schmidt Alexander Jenal Urs (2012), A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins, in Journal of Proteomics
, 75, 4874-4878.
Zähringer Franziska, Massa Claudia, Schrimer Tilman (2011), Efficient Enzymatic Production of the Bacterial Second Messenger c-di-GMP by the Diguanylate Cyclase YdeH from E. coli, in Applied Biochemistry and Biotechnology
, 163(1), 71-79.
Habazettl Judith, Allan Martin, Jenal Urs, Grzesiek Stephan (2011), Solution Structure of the PilZ Domain Protein PA4608 Complex with Cyclic di-GMP Identifies Charge Clustering as Molecular Readout, in Journal of Biological Chemistry
, 286(16), 14304-14314.
Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that emerges as a regulatory mastermind in orchestrating multicellular behavior, virulence, and biofilm-mediated persistence in a wide variety of bacteria. The cellular concentration of c-di-GMP is the result of the opposing activities of diguanylate cyclases (DGCs), which catalyze the condensation of two GTP molecules, and phosphodiesterases (PDEs) that degrade c-di-GMP to pGpG. C-di-GMP mediates downstream signaling by binding to different molecular switches, which in turn can influence cellular behavior on the transcriptional, translational, or allosteric level. The broad importance of this novel signaling molecule in pathogenic and non-pathogenic bacteria calls for a thorough analysis of the molecular mechanisms that control cellular levels of c-di-GMP and regulate its downstream targets. The c-di-GMP circuitry operating in most bacterial cells appears to be exceedingly complex. GGDEF (DGCs), EAL (PDEs), and PilZ (effectors) domain proteins belong to large protein families with multiple paralogs present in most bacteria, some of which have experienced functional diversification by adopting novel roles in cell signaling. To uncover structural, kinetic and dynamic properties of the c-di-GMP signaling network proteins, this project aims at a systematic analysis of its components on the atomic, molecular, and cellular level. A careful bioinformatics analysis will reveal conserved structural motifs of known members of the GGDEF, EAL, and PilZ protein families and will help identifying novel c-di-GMP signaling components. Crystal structure determination complemented by homology modeling will elucidate the detailed three-dimensional structures of representatives of the most important signaling components and its various regulatory conformations. These studies will be complemented by single molecule FRET experiments that address domain arrangements in solution and monitor the dynamics of conformational changes upon activation or ligand binding. Mechanistic models of catalysis and regulation that result from these studies will be validated and refined systematically and in a targeted manner by site directed mutagenesis and established in vitro and in vivo assays as readouts. In particular, we will make use of a well-developed model system for c-di-GMP signaling, Caulobacter crescentus, where cellular and molecular aspects are currently under intense investigation. Such an in depth analysis thrives on an interdisciplinary approach that combines different experimental skills and complementary methodology. Reaching a fundamental understanding of the basic mechanistic and quantitative aspects of c-di-GMP signaling components will lay the groundwork for a cell-wide systems level analysis of the c-di-GMP network, which eventually will lead to a full appreciation of the complex spatial and temporal control mechanisms involved in switching bacteria from motile and virulent single cells to persistent communities.