Inositol 1,4,5-trisphosphate receptors (InsP3R) and ryanodine receptors (RyR) are tetrameric intracellular Ca2+ channels1. For each, the pore is formed by C-terminal transmembrane domains and regulated by signals detected by the large cytosolic structures. InsP3R gating is initiated by InsP3 binding to the InsP3-binding core (IBC, residues 224-604 of InsP3R1)2 and it requires the suppressor domain (SD, residues 1-223)2-8. We present structures of the N-terminal region (NT) of InsP3R1 with (3.6 Å) and without (3.0 Å) InsP3 bound. The arrangement of the three NT domains, the SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR19. The orientations of the three domains docked into a tetrameric structure of InsP3R10 and of the ABC domains in RyR9 are remarkably similar. The importance of the α-interface for activation of InsP3R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations9,11,12. InsP3 causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop (HS-loop) that is essential for InsP3R activation7. The loop is conserved in RyR and includes mutations associated with malignant hyperthermia and central core disease9,11,12. The HS-loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A-domain of RyR functionally replaced the SD in a full-length InsP3R, and an InsP3R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP3 and blocked by ryanodine. Activation mechanisms are conserved between InsP3R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit re-orients the first domain (SD or A-domain), allowing it, via interactions of the second domain of an adjacent subunit (IBC-β or B-domain), to gate the pore.
Multidrug resistance, by which cells become resistant to multiple unrelated pharmaceuticals, is due to the extrusion of drugs from the cell's interior by active transporters such as the human multidrug resistance P-glycoprotein. Two major classes of transporters mediate this extrusion. Primary-active transporters are dependent on ATP hydrolysis, whereas secondary-active transporters are driven by electrochemical ion gradients that exist across the plasma membrane. The ATP-binding cassette (ABC) transporter LmrA is a primary drug transporter in Lactococcus lactis that can functionally substitute for P-glycoprotein in lung fibroblast cells. Here we have engineered a truncated LmrA protein that lacks the ATP-binding domain. Surprisingly, this truncated protein mediates a proton-ethidium symport reaction without the requirement for ATP. In other words, it functions as a secondary-active multidrug uptake system. These findings suggest that the evolutionary precursor of LmrA was a secondary-active substrate translocator that acquired an ATP-binding domain to enable primary-active multidrug efflux in L. lactis.
Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP.
Sav1866 is an ATP-binding cassette (ABC) protein from the pathogen Staphylococcus aureus and is a homologue of bacterial and human multidrug ABC transporters. Recently, the three-dimensional crystal structure of Sav1866 was determined at 3.0 A resolution [Dawson, R. J., and Locher, K. P. (2006) Nature 443, 180-185]. Although this structure is frequently used to homology model human and microbial ABC multidrug transporters by computational methods, the ability of Sav1866 to transport multiple drugs has not been described. We obtained functional expression of Sav1866 in the drug-sensitive, Gram-positive bacterium Lactococcus lactis Delta lmrA Delta lmrCD lacking major endogenous multidrug transporters. Sav1866 displayed a Hoechst 33342, verapamil, tetraphenylphosphonium, and vinblastine-stimulated ATPase activity. In growing cells, Sav1866 expression conferred resistance to Hoechst 33342. In transport assays in intact cells, Sav1866 catalyzed the translocation of amphiphilic cationic ethidium. Additionally, Sav1866 mediated the active transport of Hoechst 33342 in membrane vesicles and proteoliposomes containing purified and functionally reconstituted protein. Sav1866-mediated resistance and transport were inhibited by the human ABCB1 and ABCC1 modulator verapamil. This work represents the first demonstration of multidrug transport by Sav1866 and suggests that Sav1866 can serve as a well-defined model for studies on the molecular bases of drug-protein interactions in ABC transporters. Our methods for the overexpression, purification, and functional reconstitution of Sav1866 are described in detail.
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