STRUCTURE OF TOLC, THE OUTER-MEMBRANE COMPONENT OF THE BACTERIAL TYPE-I EFFLUX SYSTEM, DERIVED FROM 2-DIMENSIONAL CRYSTALS

TolC is an outer membrane protein required for the export of virulence proteins and toxic compounds without a periplasmic intermediate. We s how that TolC is an integral part of the translocator, interacting wit h inner membrane components, by demonstrating a need for TolC in prote in export not only from intact cells but also from sphaeroplasts. To e stablish 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 latti ces by reconstitution in phospholipid bilayers. The projection structu re at 12 Angstrom resolution showed a threefold symmetric molecule of 58 Angstrom outer diameter, and a single pool of stain filling its cen tre. Side views parallel to the membrane plane revealed an additional domain outside the membrane. Eighteen membrane-spanning beta-strands w ere predicted for the 51.5 kDa monomer, excluding a 7 kDa C-terminal s egment, and this segment was shown to contain a proteinase K-sensitive site that was exposed in reconstituted membranes and sphaeroplasts, b ut which was protected in intact cells. The combined data suggest that TolC is a trimeric outer membrane protein with each monomer comprisin g a membrane domain, predicted to be beta-barrel, and a C-terminal per iplasmic domain. The latter could form part of the bridge to the energ ized inner membrane component of the translocation complex.