SPECIAL SECTION: MEMBRANE PROTEINS CURRENT SCIENCE, VOL. 87, NO. 2, 25 JULY 2004 190 *For correspondence. (e-mail: ben@cryst.bioc.cam.ac.uk) The structure and mechanism of the TolC outer membrane transport protein Luca Federici, Fabien Walas and Ben Luisi* Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK Gram-negative bacteria have evolved specialized multi- component systems that transport molecules from the cytoplasm to the extracellular environment in energy- dependent processes. Central to one of these systems is the TolC family of outer membrane proteins. TolC of Escherichia coli is a very versatile channel that can inter- act with a wide range of inner membrane, energy- driven pumps to export compounds ranging from small antibiotic molecules to large toxic proteins. Thus TolC and its associated partner proteins confer inva- sive virulence and drug resistance to Gram-negative bacterial pathogens. However TolC is also a source of vulnerability, as it is a conduit for the uptake of bac- tericidal proteins known as colicins. Recently the cry- stal structures have been reported of TolC and its inner-membrane partner protein AcrB, a proton anti- porter. The mechanisms of colicin uptake via TolC have also been extensively studied and the structures of some of the implicated protein components have been determined. In this review we focus on the cur- rent understanding of the structure and function rela- tionships in TolC-mediated transport systems. THE Gram-negative bacteria are characterized by a distin- ctive two-layered membrane system. The protective outer membrane, which is exposed to the external environment and contains special lipid components including lipopoly- saccharide, contains many different kinds of molecular pores that allow the free diffusion of water and ions. The inner membrane defines the cytoplasmic boundary, and the intervening space between the inner and outer mem- branes, known as the periplasm, is densely packed with peptidoglycan and other complex molecules. The physi- cal separation of inner and outer membranes may vary according to physiological conditions, and it is thought that these membranes may come closer together when- ever proteins are transported from the cell interior to the exterior 1,2 . Although the double-membrane system confers protec- tion and other benefits to the bacterium, the movement of molecules across the double-membrane enclosure pre- sents certain strategic problems to the cell. The periplasm contains no ATPase or other energy-providing processes, because this portion of the bacterium may be in equili- brium with the external environment. However, the energy required to do the work of moving molecules – either across a concentration gradient in the case of nutrients, or across a diffusion barrier in the case of secreted pro- teins – can be provided by inner membrane proteins that serve as engines fueled by ATP or proton electrochemical gradients. A number of distinctive transmembrane transport proc- esses have evolved in the Gram-negative bacteria to move proteins and other molecules across the membrane. In Esche- richia coli, five different systems have been characterized to date 3–9 . The system we shall focus on here is known as Type I transport, which employs a three-component pump that utilizes the free energy of ATP binding and hydroly- sis to move proteins vectorially across both inner and outer membranes in an apparently single-step process that requires no intermediates within the periplasm 10,11 . One component of this pump is represented by the TolC outer membrane protein. In the transport process, the TolC in- teracts with a periplasmic protein, known often as a mem- brane fusion protein, and an inner membrane ATPase. The best characterized example of this system is the transporter for the invasive toxin hemolysin 12 . The trans- port process is thought to proceed by the recruitment of the nascent hemolysin polypeptide by the inner membrane protein HylB, which may pre-exist in complex with the membrane fusion protein HlyD (Figure 1 a). On binding ATP, the TolC is engaged, and the protein passes through the assembly. The association of the components is tran- sient and reversible, and after the substrate molecule has been transported, the components dissociate. The inner membrane protein HlyB belongs to the wide family of ATP-binding cassette proteins, or ‘ABC’ proteins, which use the free energy of ATP binding and hydrolysis to translocate molecules. These proteins are widely dis- tributed in nature and are found in all three domains of life: archaebacteria, eubacteria and the eukaryotes 13 . In HlyB, the transmembrane and ATPase domains are contained within a single polypeptide chain, but in other systems, these domains are encoded in separate subunits 14 . An ex- ample of a multisubunit ABC ATPase is the vitamin B 12 receptor, BtuCD, which is a heterotetramer. The oli- gomeric construction of the ATPase suggests that it func- tions through allosteric transition, as might be expected for a mechanical pump. The crystal structure of the