title: Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals.
abstract: TolC is an outer membrane protein required for the export of virulence proteins and toxic compounds without a periplasmic intermediate. We show that TolC is an integral part of the translocator, interacting with inner membrane components, by demonstrating a need for TolC in protein export not only from intact cells but also from sphaeroplasts. To establish the structure of TolC, and thus gain information on how this might be achieved, the protein was purified from the Escherichia coli outer membrane, as a trimer, and crystallized in two-dimensional lattices by reconstitution in phospholipid bilayers. The projection structure at 12A resolution showed a threefold symmetric molecule of 58A outer diameter, and a single pool of stain filling its centre. Side views parallel to the membrane plane revealed an additional domain outside the membrane. Eighteen membrane-spanning beta-strands were predicted for the 51.5 kDa monomer, excluding a 7 kDa C-terminal segment, and this segment was shown to contain a proteinase K-sensitive site that was exposed in reconstituted membranes and sphaeroplasts, but which was protected in intact cells. The combined data suggest that TolC is a trimeric outer membrane protein with each monomer comprising a membrane domain, predicted to be beta-barrel, and a C-terminal periplasmic domain. The latter could form part of the bridge to the energized inner membrane component of the translocation complex.
authors.affiliation: Department of Pathology, Cambridge University, UK. vk103@mole.bio.cam.ac.uk
authors.forename: V
authors.initials: V
authors.lastname: Koronakis
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: E
authors.initials: E
authors.lastname: Koronakis
authors.forename: K
authors.initials: K
authors.lastname: Stauffer
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D003460:Crystallization; D004926:Escherichia coli / Q000737:chemistry / Q000502:physiology*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology; D026901:Membrane Transport Proteins; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli