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Structural cell biology
We are using biochemical and biophysical methods, particularly X-ray crystallography, to ask mechanistic questions about protein function.
Structural foundations of intracellular transport and secretion.
Proteins are transported from one cellular location to another packaged within small vesicles. These vesicles bud from one compartment (for example, the endoplasmic reticulum) carrying a specific cargo which they deliver to a different compartment (for example, the Golgi apparatus) by docking and fusing with it. Exocytosis is similar: secretory vesicles dock and fuse with the plasma membrane. Many of the proteins that carry out cargo selection, budding, docking, and fusion have begun to be identified.
How intracellular traffic is controlled to bring about the biochemical architecture of eukaryotic cells is a central question in biology. To develop a molecular framework for investigating this question, we have been studying the protein machines that mediate vesicle docking and fusion. The active fusion apparatus is thought to be a complex between SNARE proteins, each anchored in one of the two membranes. Assembly of this SNARE complex is accompanied by large structural changes. We find that assembly is under stringent kinetic control, a feature that helps to explain the specificity of intracellular trafficking. Our recent results provide a structural foundation for this kinetic control mechanism and its functioning in vivo.
The initial recognition between a vesicle and its membrane target is mediated by large protein complexes called tethering factors. Tethering factors act upstream of SNARE complex assembly and likely play key roles in determining the specificity of trafficking. Furthermore, tethering factors are critical for organellar inheritance during cell division. Because little is known about the structures of tethering factors or the mechanism(s) by which they act, we have recently initiated biochemical and biophysical studies of these key protein complexes.
Bacterial Quorum Sensing
Cell-cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression and is important, for example, in virulence and biofilm formation. Our colleague Bonnie Bassler and her lab discovered an autoinducer (AI-2) that appears to serve as a 'universal' signal for inter-species communication. The chemical identity of AI-2 has, however, proved elusive. We have recently determined the crystal structure of an AI-2 sensor protein in a complex with autoinducer. The bound ligand is a furanosyl borate diester bearing no resemblance to previously characterized autoinducers. Our findings indicate a novel biological role for boron, an element required by a number of organisms but for unknown reasons. Together with Bonnie's lab, we are currently combining structural, biochemical, chemical, and genetic approaches to characterize AI-2 signaling in biology.
Selected Publications
Kelly RC, Bolitho ME, Higgins DA, Lu W, Ng WL, Jeffrey PD, Rabinowitz JD, Semmelhack MF, Hughson FM, Bassler BL. (2009) The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA. Nat Chem Biol. [Epub ahead of print]
Richardson BC, Smith RD, Ungar D, Nakamura A, Jeffrey PD, Lupashin VV, Hughson FM. (2009) Structural basis for a human glycosylation disorder caused by mutation of the COG4 gene. Proc Natl Acad Sci 106: 13329-13334. PubMed
Hughson FM. (2009) Structures of vesicle-tethering complexes. Annu Rev Cell Dev Biol. [Epub ahead of print]
Tripathi A, Ren Y, Jeffrey PD, Hughson FM. (2009) Structural characterization of Tip20p and Dsl1p, subunits of the Dsl1p vesicle tethering complex. Nat Struct Mol Biol. 16: 114-123. PubMed
Hughson FM. (2008) Both layers of the COPII coat come into view. Cell 134: 384-385. PubMed
Neiditch MB, Hughson FM. (2007) The regulation of histidine sensor kinase complexes by quorum sensing signal molecules. Methods Enzymol. 423: 250-263. PubMed
Cavanaugh LF, Chen X, Richardson BC, Ungar D, Pelczer I, Rizo J, Hughson FM. (2007) Structural analysis of conserved oligomeric golgi complex subunit 2. J Biol Chem 282: 23418-23426. PubMed
Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM. (2006) Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell 126: 1095-1108. PubMed
Ungar D, Oka T, Krieger M, Hughson FM. (2006) Retrograde transport on the COG railway. Trends Cell Biol 16: 113-120. PubMed
Ungar D, Oka T, Vasile E, Krieger M, Hughson FM. (2005) Subunit architecture of the conserved oligomeric Golgi complex. J Biol Chem 280: 32729-32735. PubMed
Oka T, Vasile E, Penman M, Novina CD, Dykxhoorn DM, Ungar D, Hughson FM, Krieger M. (2005) Genetic analysis of the subunit organization and function of the conserved oligomeric golgi (COG) complex: studies of COG5- and COG7-deficient mammalian cells. J Biol Chem 280: 32736-32745. PubMed
Neiditch MB, Federle MJ, Miller ST, Bassler BL, Hughson FM. (2005) Regulation of LuxPQ receptor activity by the quorum-sensing signal autoinducer-2. Mol Cell 18: 507-518. PubMed
Oka T, Ungar D, Hughson FM, Krieger M. (2004) The COG and COPI complexes interact to control the abundance of GEARs, a subset of Golgi integral membrane proteins. Mol Biol Cell 15: 2423-2435. PubMed
Miller ST, Xavier KB, Campagna SR, Taga ME, Semmelhack MF, Bassler BL, Hughson FM. (2004) Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2. Mol Cell 15: 677-687. PubMed
Zweifel ME, Leahy DJ, Hughson FM, Barrick D. (2003) Structure and stability of the ankyrin domain of the Drosophila Notch receptor. Protein Sci 12: 2622-2632. PubMed
Ungar D and Hughson FM. (2003) SNARE protein structure and function. Annu Rev Cell Dev Biol 19: 493-517. PubMed
Munson M, Hughson FM. (2002) Conformational regulation of SNARE assembly and disassembly in vivo. J Biol Chem 277: 9375-9381. PubMed
Chen X, Schauder S, Potier N, Van Dorsselaer A, Pelczer I, Bassler BL, Hughson FM. (2002) Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415: 545-549. PubMed
Barrick D, Hughson FM. (2002) Irreversible assembly of membrane fusion machines. Nat Struct Biol 9: 78-80. PubMed