Falke Lab, Research Interests

Falke Group: Research Interests

The broad goal of the Falke group is a molecular understanding of biological signaling pathways, using chemosensory pathways as a model. Motile cells possess chemosensory pathways that control cell movement, enabling migration up or down gradients of chemical attractants and repellents, respectively. The chemosensory pathway is a biological integrated circuit in which receptors detect specific attractants or repellents to activate the circuit. Each protein component of the circuit serves as an on-off switch that controls the activity of the next component in the pathway, and the output signal of the curcuit ultimately regulates the motility machinery that propels the cell. Current studies in the Falke group, funded by two separate but equal NIH grants, focus on the chemosensory circuits that (i) bacteria use to find their way to a food source or wound, and (ii) human macrophage (a type of white blood cell) use to track down a bacterial infection or tissue damage.

More specifically, the Falke group is using a structure-based approach to probe the switching of receptors and signaling proteins between their 'on' and 'off' signaling states at the molecular level in the working biological circuit. The approach involves the development of novel techniques to analyze the structural dynamics of switch proteins, as well as the molecular docking events regulated by their on-off switching. Of particular interest are on-off switching events at membrane surfaces, which play a central role in regulating chemsensory and most other biological signaling circuits and, in medical applications, are the targets of most pharmaceuticals. The group employs biophysical, biochemical, and molecular biological methods including: FRET, bulk and single molecule fluorescence, EPR, X-ray crystallography, mass spec, site-directed mutagenesis, cysteine chemistry, molecular modeling, and intracellular fluorescence microscopy.

Project 1.

Cell-surface receptors and kinase regulation in bacterial chemosensing. In the bacterial chemosensory pathway, all of the cytoplasmic components assemble onto an array of transmembrane chemoreceptors to generate an ultrastable, ultrasensitive signaling complex. This complex is one of the simplest, yet highly efficient, biological integrated circuits.

The Falke group is currently investigating the molecular mechanisms of on-off switching of components within the bacterial chemosensory circuit, especially the mechanisms by which transmembrane receptors bind specific attractants and regulate the output kinase activity of the circuit. This kinase activity controls the swimming motor and cellular migration up attractant gradients. The known structures of the pathway components 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 functional, membrane-bound, signaling circuit. Specific goals include elucidation of the mechanisms by which attractant and adaptation signals are transmitted through receptor structure to the kinase, and how these receptor on-off signals switch the kinase on and off. 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 bacterial receptors and kinases. Moreover, many of the tools developed in this simple, bacterial system can ultimately be applied to eukaryotic signaling circuits.

Project 2.

Signal amplification and membrane recruitment in macrophage chemosensing. In the eukaryotic chemosensory signaling circuit, 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 relevant second messenger at the leading edge, but recent live cell studies in the group have shown that normal circuit 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. X-ray crystallography 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, for the first time, the surface diffusion behaviors and interactions of membrane-docked signaling proteins. 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 workings of the macrophage chemosensory circuit during the primary immune response and inflammation, and, more broadly, into the molecular mechanisms of membrane targeting by PH and C2 domains in eukaryotic signaling pathways.

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

(2009) Discovery that the conserved cytoplasmic domain of bacterial chemoreceptors transmits signals through its long four-helix bundle via a novel yin-yang mechanism (Swain & Falke)

(2009) Discovery that the bacterial chemosensory signaling complex is ultrastable (Erbse & Falke)

(2009) Development of a novel single-molecule method to probe the protein-lipid interactions and surface dynamics of membrane-bound proteins (Knight & 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)

(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 the bacterial chemosensory kinase CheA (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)