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Falke Group: Research Interests

The goal of the Falke group is a molecular understanding of biological sensory and signaling pathways. Toward this end, the group is using a structure-based approach to probe the switching of receptors and signaling proteins between their 'on' and 'off' signaling states. The approach involves the development of novel techniques to analyze the structural dynamics of these switch proteins, as well as the docking events regulated by their on-off switching. Of particular interest are signaling events at membrane surfaces, which play a central role in regulating cellular processes and, in medical applications, are the targets of most successful pharmaceuticals. Current studies focus on two distinct pathways that sense chemical attractants and regulate cell migration: the chemotaxis pathways of bacteria and human macrophages. These two pathways, which are not homologous but have evolved to fulfill the same function, are important model systems for understanding (1) the basic molecular mechanisms that regulate cell behavior, and (2) the molecular basis of bacterial infection, inflammation and cancer, as well as the primary immune response to these disease states. Two NIH grants fund the separate-but-equal bacterial and macrophage research projects. Both projects are carried out at the interface between molecular biology, biochemistry and biophysics using diverse tools including: site-directed mutagenesis, cysteine chemistry, EPR, FRET, single molecule fluorescence, intracellular fluorescence microscopy, and molecular modeling.

 

Project 1.

Cell-surface receptors and kinase regulation in bacterial chemotaxis. In the bacterial chemotaxis pathway, all of the cytoplasmic components assemble onto an array of transmembrane chemoreceptors to generate an ultrastable, ultrasensitive signaling complex. The Falke group is currently investigating the molecular mechanisms of signaling in this complex, especially the mechanisms by which chemoreceptors bind specific attractants and regulate the kinase CheA. The resulting CheA output controls the swimming motor and cellular migration up attractant gradients. The known structures of the chemoreceptors and CheA kinase greatly facilitate molecular analysis of their on-off switching mechanisms. The group is developing an array of biochemical, chemical and spectroscopic approaches capable of analyzing structural dynamics during on-off switching in the native, membrane-bound, signaling complex. Specific goals include elucidation of the mechanisms by which attractant and adaptation signals are transmitted through the chemoreceptor structure to the kinase, and how these receptor on-off signals either stimulate or inhibit CheA kinase activity. The resulting molecular mechanisms are likely conserved throughout the diverse family of 2-component pathways, which control most cellular processes in bacteria. Such broad relevance ensures that mechanistic advances will have significant impacts on the field of signaling biology, as well as on the development of new classes broad-spectrum antibiotics designed to inhibit CheA and related kinases. Moreover, many of the tools developed in this simple, bacterial system can ultimately be applied to eukaryotic signaling pathways.

   Figure 1, Receptor Project
Figure2, Receptor Project

Figure 3, Receptor Project

Summary Figure, Project 1



Project 2.

Signal amplification and membrane recruitment in macrophage chemotaxis. In the eukaryotic chemotaxis pathway, attractant signals sensed by cell-surface receptors are amplified by a positive feedback loop at the leading edge of the cell. The feedback loop, in turn, generates second messenger signals that recruit dozens of proteins to the leading edge, where these proteins stimulate actin polymerization and push the leading edge up the attractant gradient. The Falke group is identifying the second messenger signals generated at the leading edge, and the molecular mechanisms by which these signals drive one of the most dramatic protein redistributions in cell biology. Traditionally, the signaling lipid PIP3 has been considered the only essential second messenger at the leading edge, but recent live cell studies in the group have shown that feedback loop function also requires a localized, leading edge Ca(II) signal. Together, these PIP3 and Ca(II) signals (and perhaps others) recruit PH- and C2-domain proteins, respectively, to the leading edge membrane. Current work is targeting the molecular mechanisms underlying the rapid kinetics and exquisite membrane specificity of these leading edge targeting reactions. The group has cloned and isolated PH and C2 domains from over 10 leading edge signaling proteins including GRP1, AKT, PLC, PDK, PKC, PI3K, and also has obtained corresponding full length proteins. Directed mutagenesis and EPR spectroscopy are being used to determine the structures of protein-membrane complexes, and bulk FRET measurements are elucidating the equilibrium and kinetic parameters of the membrane docking reactions. In addition, an innovative single molecule fluorescence method developed by the group is revealing new motional features of the membrane-docked state not previously detected in bulk studies. Finally, live cell studies are investigating other leading edge second messenger signals and the spatio-temporal features of the targeting reactions they control. Together, these diverse approaches will provide new insights into the molecular basis of macrophage chemotaxis during the primary immune response and inflammation, and into the molecular mechanisms of membrane targeting by PH and C2 domains in eukaryotic signaling pathways.

  Figure 4, Calcium Project
Figure 5, Calcium Project

Figure 6, Calcium Project

 

Summary Figure, Project 2


Selected Falke Group Accomplishments (see "Publications" for reference)

(2008) Discovery that the bacterial chemotaxis signaling complex is ultrastable (Erbse & Falke)

(2008) Elucidation of the molecular mechanism underlying a highly oncogenic mutation in AKT1 PH domain known to cause multiple human cancers (Landgraf, Pilling & Falke)

(2008) Determination of the distinct membrane docking geometries of PKC-alpha C2 domain in two different lipid binding states (Landgraf, Malmberg & Falke)

(2008) Development of a novel single-molecule method to probe the protein-lipid interactions and surface dynamics of membrane-bound proteins (Knight & Falke)

(2007) Discovery that a localized Ca(II) influx is an essential component of the positive feedback loop at the macrophage leading edge (Evans & Falke)

(2007) Chemical structure determination that the conserved HAMP signal conversion domain of bacterial chemoreceptors is a parallel 4-helix bundle (Swain & Falke)

(2007, 2006) Demonstration that PIP2 is a third essential target lipid of PKC-alpha (Evans, Corbin, Landgraf & Falke)

(2006) Chemical mapping of four protein interactions sites on the surface of CheA kinase (Miller, Kohout & Falke)

(2005) Discovery of a conserved, essential Gly hinge in the cytoplasmic 4-helix bundle of bacterial chemoreceptors (Coleman, Bass & Falke)

(2005) Elucidation of the electrostatic mechanism underlying adaptation site signaling in bacterial chemoreceptors (Starrettt & Falke)

(2004) EPR determination of the highest resolution membrane docking geometry currently available Ð the C2 domain of cytosolic phospholipase A2 (Malmberg & Falke)

(2004) Development of an electrostatic method to drive piston displacements of transmembrane helices (Miller & Falke)

(2004) Discovery that GRP1 PH domain uses an electrostatic search mechanism to rapidly find its rare target lipid PIP3 (Corbin & Falke)

(2003) Chemical mapping of the protein interaction sites on the surface of bacterial chemoreceptors (Mehan & Falke)

(2003) Demonstration that covalent adaptation introduces multiple sub-states into the on-off switching behavior of the receptor-CheA signaling complex (Bornhorst & Falke)

(1999) Chemical determination of the 4-helix bundle architecture of bacterial chemoreceptor cytoplasmic domains (Bass, Butler, Danielson & Falke)

(1997) Elucidation of the Ca(II)-signaling cycle for the membrane-docking C2 domain of cytosolic phospholipase A2, the Ca(II) sensor of inflammation (Nalefski & Falke)

(1997) Development of a novel FRET assay for monitoring the equilibrium and kinetic parameters of protein-membrane docking reactions (Nalefski & Falke)

(1996) Discovery that the amino acid at the gateway position of EF-hand sites controls the Ca(II) on-off kinetics (Drake & Falke)

(1996) Determination of the effects of protein stabilizing agents on long-range backbone motions in proteins via disulfide trapping (Butler & Falke)

(1996) Discovery that the transmembrane signal of bacterial chemoreceptors is transmitted by a piston displacement of the signaling helix (Chervitz & Falke)

(1995) Engineering reversible, lock-on and lock-off disulfide bonds that covalently trap the signaling states of bacterial chemoreceptors (Chervitz & Falke)

(1994) Use of 19F NMR to probe conformational changes in a receptor (Danielson & Falke)

(1993) Use of 19F NMR to probe conformational changes in a signaling protein (Drake & Falke)

(1992) Detection and trajectory analysis of thermal backbone motions in a folded, aqueous protein by a novel disulfide trapping method (Careaga & Falke)

(1991) Use of 19F NMR to probe conformational changes in a binding protein (Luck & Falke)