Saroj Velamakanni

Structural and functional conservation of key domains in InsP3 and ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca2+ channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP3R gating is initiated by InsP3 binding to the InsP3-binding core (IBC, residues 224–604 of InsP3R1) and it requires the suppressor domain (SD, residues 1–223 of InsP3R1). Here we present structures of the amino-terminal region (NT, residues 1–604) of rat InsP3R1 with (3.6 Å) and without (3.0 Å) InsP3 bound. The arrangement of the three NT domains, 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 RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP3R and of the ABC domains docked into RyR 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 mutations. Binding of 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 (‘hotspot’ (HS) loop) that is essential for InsP3R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. 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 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 reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-β or B domain), to gate the pore.

An ABC transporter with a secondary-active multidrug translocator domain

Multidrug resistance, by which cells become resistant to multiple unrelated pharmaceuticals, is due to the extrusion of drugs from the cells 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.

Drug-Lipid A Interactions on the Escherichia coli ABC Transporter MsbA

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K(i) values of 16 and 11 microM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K(i) of 57 microM, in a similar range as the K(i) for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).

Multidrug transport by the ABC transporter Sav1866 from Staphylococcus aureus.

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.

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

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. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

The emerging pharmacotherapeutic significance of the breast cancer resistance protein (ABCG2).

The breast cancer resistance protein (also termed ABCG2) is an ATP‐binding cassette transporter, which mediates the extrusion of toxic compounds from the cell, and which was originally identified in relation to the development of multidrug resistance of cancer cells. ABCG2 interacts with a range of substrates including clinical drugs but also substances such as sterols, porphyrins and a variety of dietary compounds. Physiological functions of ABCG2 at both cellular and systemic levels are reviewed. For example, ABCG2 expression in erythrocytes may function in porphyrin homeostasis. In addition, ABCG2 expression at apical membranes of cells such as hepatocytes, enterocytes, endothelial and syncytiotrophoblast cells may correlate to protective barrier or secretory functions against environmental or clinically administered substances. ABCG2 also appears influential in the inter‐patient variation and generally poor oral bioavailability of certain chemotherapeutic drugs such as topotecan. As this often precludes an oral drug administration strategy, genotypic and environmental factors altering ABCG2 expression and activity are considered. Finally, clinical modulation of ABCG2 activity is discussed. Some of the more recent strategies include co‐administered modulating agents, hammerhead ribozymes or antisense oligonucleotides, and with specificity in cell targeting, these may be used to reduce drug resistance and increase drug bioavailability to improve the profile of chemotherapeutic efficacy versus toxicity. While many such strategies remain in relative infancy at present, increased knowledge of modulators of ABCG2 could hold the key to novel approaches in medical treatment.

IP3 receptors: some lessons from DT40 cells.

Summary:  Inositol‐1,4,5‐trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that are regulated by IP3 and Ca2+ and are modulated by many additional signals. These properties allow them to initiate and, via Ca2+‐induced Ca2+ release, regeneratively propagate Ca2+ signals evoked by receptors that stimulate formation of IP3. The ubiquitous expression of IP3R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP3R. DT40 cells are a pre‐B‐lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40‐knockout cells with both alleles of each of the three IP3R genes disrupted provide the only null‐background for analysis of homogenous recombinant IP3R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP3R behavior. Patch‐clamp recording from the nuclear envelope and Ca2+ release from intracellular stores loaded with a low‐affinity Ca2+ indicator address the mechanisms leading to activation of IP3R. We show that IP3 causes intracellular IP3R to cluster and re‐tune their responses to IP3 and Ca2+, better equipping them to mediate regenerative Ca2+ signals. Finally, we show that DT40 cells reliably count very few IP3R into the plasma membrane, where they mediate about half the Ca2+ entry evoked by the B‐cell antigen receptor.

A Multidrug ABC Transporter with a Taste for Salt

Background LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H+-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. Methodology/Principal Findings We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H+-Na+-Cl− symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. Conclusions/Significance The observations on H+-Na+-Cl− co-transport substantiate earlier suggestions of H+-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.

A functional steroid-binding element in an ATP-binding cassette multidrug transporter.

The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown. Here, we identify a steroid-binding element in the membrane domain of ABCG2 with a similarity to steroid hormone/nuclear receptors. The element facilitates steroid hormone binding and mediates modulation of ABCG2 activity. The identification of this element might provide an opportunity for the development of new therapeutic ligands for ABCG2.

A critical role of a carboxylate in proton conduction by the ATP-binding cassette multidrug transporter LmrA

The ATP binding cassette (ABC) transporter LmrA from the bacterium Lactococcus lactis is a homolog of the human multidrug resistance P‐glycoprotein (ABCB1), the activity of which impairs the efficacy of chemotherapy. In a previous study, LmrA was shown to mediate ethidium efflux by an ATP‐dependent proton‐ethidium symport reaction in which the carboxylate E314 is critical. The functional importance of this key residue for ABC proteins was suggested by its conservation in a wider family of related transporters; however, the structural basis of its role was not apparent. Here, we have used homology modeling to define the structural environment of E314. The residue is nested in a hydrophobic environment that probably elevates its pKa, accounting for the pH dependency of drug efflux that we report in this work. Functional analyses of wild‐type and mutant proteins in cells and proteoliposomes support our proposal for the mechanistic role of E314 in proton‐coupled ethidium transport. As the carboxylate is known to participate in proton translocation by secondary‐active transporters, our observations suggest that this substituent can play a similar role in the activity of ABC transporters.