One main focus of the research in our group is to understand the protein machinery that generates the interior architecture of cells by guiding the movement and fusion of intracellular transport vesicles. All eukaryotic cells contain a profusion of membrane-bounded compartments. Traffic among these compartments is brisk. Cargo is transported in membrane vesicles that bud from one compartment, travel through the cell, and deliver their contents by fusing with another compartment. Underlying this intricate choreography is a set of proteins and protein complexes responsible for the creation, movement, docking, and fusion of vesicles. Our lab seeks to understand the design principles that endow these protein nanomachines with the ability to manipulate membrane vesicles, thereby powering a bustling intracellular transportation network.
Several of our current projects involve structural and mechanistic studies of large multi-subunit protein complexes that orchestrate the docking and fusion of transport vesicles. These complexes guide cargo-laden vesicles to their destinations and coordinate the activities of other components of the trafficking machinery, including the ‘SNARE’ proteins that catalyze membrane fusion itself. We are investigating the structure and function of these complexes using an array of technologies including x-ray crystallography, electron microscopy, site-directed mutagenesis, in vitro reconstitution, and a suite of spectroscopic techniques.
A second major focus within our group is bacteria, and the remarkable finding that these single-celled organisms communicate with one another by emitting and receiving small-molecule signals. We are interested in cataloging the signal molecules and in understanding their biosynthesis and detection. Moreover, since bacteria often respond to these signals in undesirable ways – like forming antibiotic-resistant biofilms or mounting an attack on a human host – we are interested in discovering and characterizing molecules that interfere with bacterial communication. We are investigating these issues using a range of biochemical and biophysical approaches. For example, we are attempting to determine the crystal structures of the enzymes responsible for synthesizing the signal molecules, and of the receptors responsible for detecting them. We have also begun to identify antagonists – molecules that inhibit signaling – and we are interested in using biochemical and structural methods to figure out how they work and how they might be improved by rational design. Finally, in collaboration with other Princeton labs in the molecular biology, chemistry, and physics departments, we are trying to understand how bacteria integrate the information they receive via multiple different signal molecules to craft an appropriate response.
Selected Recent Publications
- Neiditch, M.B., Federle, M.J., Pompeani, A.J., Kelly, R.C., Swem, D.L., Jeffrey, P.D., Bassler, B.L., and Hughson, F.M. (2006) Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell 126, 1095-1108.
- Tripathi, A., Ren, Y., Jeffrey, P.D., and Hughson, F.M. (2009) Structural characterization of Tip20p and Dsl1p, subunits of the Dsl1p vesicle tethering complex. Nat. Struct. Mol. Biol. 16, 114-123.
- Richardson, B.C., Smith, R.D., Ungar, D., Nakamura, A., Jeffrey, P.D., Lupashin, V.V., and Hughson, F.M. (2009) Structural basis for a human glycosylation disorder caused by mutation of the COG4 gene. Proc. Natl. Acad. Sci. USA 106, 13329-13334.
- Kelly, R.C., Bolitho, M.E., Higgins, D.A., Lu, W., Ng, W.-L., Jeffrey, P.D., Rabinowitz, J.D., Semmelhack, M.F., Hughson, F.M., and Bassler, B.L. (2009) The Vibrio cholerae quorum-sensing autoinducer CAI-1: Analysis of the biosynthetic enzyme CqsA. Nature Chem. Biol. 5, 891-895.
- Ren, Y., Yip, C.K., Tripathi, A., Huie, D., Jeffrey, P.D., Walz, T., and Hughson, F.M. (2009) A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. Cell 139, 1119-1129.