Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure

The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published cross-linking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5), Ile340 (TMH6), Phe343 (TMH6), Phe728 (TMH7), and Val982 (TMH12), form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the site-directed mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.     

Molecular model of the outward facing state of the human multidrug resistance protein 4 (MRP4/ABCC4)

ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4, ABCC4) is involved in multidrug resistance (MDR), which is an increasing challenge to the treatment of cancer and infections. We have constructed a molecular model of ABCC4 based on the outward facing Sav1866 crystal structure using molecular modeling techniques. Amino acids reported by ICMPocketFinder to take part in substrate translocation were among others Glu103 (TMH1), Ser328 (TMH5), Gly359 (TMH6), Arg362 (TMH6), Val726 (TMH7), and Leu987 (TMH12), and their corresponding amino acids in ABCB1 (P-glycoprotein) have been reported to be involved in drug binding according to site-directed mutagenesis studies. The ABCC4 model may be used as a working tool for experimental studies on ABCC4 and design of more specific membrane transport modulating agents (MTMA).     

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.     

A structural model for the open conformation of the mdr1 P-glycoprotein based on the MsbA crystal structure

The validity of the structure of the Escherichia coli MsbA lipid transporter as a model from the mdr1 P-glycoprotein has been evaluated. Comparative sequence analyses, motif search and secondary structure prediction indicated that each of the two P-glycoprotein halves is structurally similar to the MsbA monomer and also suggested that the open dimer structure is valid for P-glycoprotein. Homology modeling was used to predict the structure of P-glycoprotein using MsbA as a template. The resulting modeled structure allowed a detailed study of the interactions between the intracellular domain and the nucleotide binding domain and suggested that these contacts are involved in mediating the coupling between nucleotide binding domain conformational changes and transmembrane helices reorientation during transport. In P-glycoprotein, the internal chamber open to the inner leaflet and the inner medium is significantly different in size and charge than in MsbA. These differences can be related to those of the transported substrates. Moreover an ensemble of 20 conserved aromatic residues appears to border the periphery of each side of the chamber in P-glycoprotein. These may be important for size selection and proper positioning of drugs for transport. The relevance of the modeled conformation to P-gp function is discussed.     

ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.     

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.ATP-binding cassette (ABC)² transporters transduce the energy of ATP hydrolysis to power the movement of a wide range of substrates across the cell membranes (1, 2). They constitute the largest family of prokaryotic transporters, import essential cell nutrients, flip lipids, and export toxic molecules (3). Forty eight human ABC transporters have been identified, including ABCB1, or P-glycoprotein, which is implicated in cross-resistance to drugs and cytotoxic molecules (4, 5). Inherited mutations in these proteins are linked to diseases such as cystic fibrosis, persistent hypoglycemia of infancy, and immune deficiency (6).The functional unit of an ABC transporter consists of four modules. Two highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport (7). ABCs drive the mechanical work of proteins with diverse functions ranging from membrane transport to DNA repair (3, 5). Substrate specificity is determined by two transmembrane domains (TMDs) that also provide the translocation pathway across the bilayer (7). Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one TMD, that dimerize to form the active transporter (3). The number of transmembrane helices and their organization differ significantly between ABC importers and exporters reflecting the divergent structural and chemical nature of their substrates (1, 8, 9). Furthermore, ABC exporters bind substrates directly from the cytoplasm or bilayer inner leaflet and release them to the periplasm or bilayer outer leaflet (10, 11). In contrast, bacterial importers have their substrates delivered to the TMD by a dedicated high affinity substrate-binding protein (12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on the inner membrane to its final destination in the outer membrane requires the ABC transporter MsbA (13). Although MsbA has not been directly shown to transport lipid A, suppression of MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits bacterial growth strongly suggesting a role in translocation (14-16). In addition to this role in lipid A transport, MsbA shares sequence similarity with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus lactis, and Sav1866 of Staphylococcus aureus (16-19). ABCB1, a prototype of the ABC family, is a plasma membrane protein whose overexpression provides resistance to chemotherapeutic agents in cancer cells (1). LmrA and MsbA have overlapping substrate specificity with ABCB1 suggesting that both proteins can function as drug exporters (18, 20). Indeed, cells expressing MsbA confer resistance to erythromycin and ethidium bromide (21). MsbA can be photolabeled with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342 ({"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342) across membrane vesicles in an energy-dependent manner (21).The structural mechanics of ABC exporters was revealed from comparison of the MsbA crystal structures in the apo- and nucleotide-bound states as well as from analysis by spin labeling EPR spectroscopy in liposomes (17, 19, 22, 23). The energy harnessed from ATP binding and hydrolysis drives a cycle of NBD association and dissociation that is transmitted to induce reorientation of the TMD from an inward- to outward-facing conformation (17, 19, 22). Large amplitude motion closes the cytoplasmic end of a chamber found at the interface between the two TMDs and opens it to the periplasm (23). These rearrangements lead to significant changes in chamber hydration, which may drive substrate translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile, wasting the energy of ATP hydrolysis without substrate extrusion (7). Consistent with this model, ATP binding reduces ABCB1 substrate affinity, potentially through binding site occlusion (24-26). Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP hydrolysis increasing transport efficiency (1, 27, 28). However, there is a paucity of information regarding the location of substrate binding, the transport pathway, and the structural basis of substrate recognition by ABC exporters. In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29, 30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model of substrate binding to ABC efflux exporters. This so-called “hydrophobic cleaner model” describes substrates binding from the inner leaflet of the bilayer and then translocating through the TMD (10, 31, 32). These studies also identified a large number of residues involved in substrate binding and selectivity (33). When these crucial residues are mapped onto the crystal structures of MsbA, a subset of homologous residues clusters to helices 3 and 6 lining the putative substrate pathway (34). Consistent with a role in substrate binding and specificity, simultaneous replacement of two serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport of ethidium and taxol, although {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 and erythromycin interactions remain unaffected (34).The tendency of lipophilic substrates to partition into membranes confounds direct analysis of substrate interactions with ABC exporters (35, 36). Such partitioning may promote dynamic collisions with exposed Trp residues and nonspecific cross-linking in photo-affinity labeling experiments. In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20, 29, 30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence. Residues that quench DNR cluster along the cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR. Furthermore, many of these residues are not lipid-exposed but face the putative substrate chamber formed between the two TMDs. These residues are proximal to two Trps, which likely explains the previously reported quenching (29). Our results suggest DNR partitions to the membrane and then binds MsbA in a manner consistent with the hydrophobic cleaner model. Interpretation in the context of the crystal structures of MsbA identifies a putative translocation pathway through the transmembrane segment.     

Characterization of the LSGGQ and H motifs from the Escherichia coli lipid A transporter MsbA

ATP-binding cassette (ABC) transporters make up one of the largest superfamilies of proteins known and have been shown to transport substrates ranging from lipids and antibiotics to sugars and amino acids. The dysfunction of ABC transporters has been linked to human pathologies such as cystic fibrosis, hyperinsulinemia, and macular dystrophy. Several bacterial ABC transporters are also necessary for bacterial survival and transport of virulence factors in an infected host. MsbA is a 65 kDa protein that forms a functional homodimer consisting of two six-helix transmembrane domains and two approximately 250 amino acid nucleotide-binding domains (NBD). The NBDs contain several conserved regions such as the Walker A, LSGGQ, and H motif that bind directly to ATP and align it for hydrolysis. MsbA transports lipid A, its native substrate, across the inner membrane of Gram-negative bacteria. The loss or dysfunction of MsbA results in a toxic accumulation of lipid A inside the cell, leading to cell-membrane instability and cell death. Using site-directed spin labeling electron paramagnetic resonance spectroscopy, conserved motifs within the MsbA NBD have been evaluated for structure and dynamics upon substrate binding. It has been determined that the LSGGQ NBD consensus sequence is consistent with an alpha-helical conformation and that these residues maintain extensive tertiary contacts throughout hydrolysis. The dynamics of the LSGGQ and the H-motif region have been studied in the presence of ATP, ADP, and ATP plus vanadate to identify the residues that are directly affected by interactions with the substrate before, after, and during hydrolysis, respectively.     

Molecular dynamics of buspirone analogues interacting with the 5-HT1A and 5-HT2A serotonin receptors

Three-dimensional (3-D) models of the human serotonin 5-HT1A and 5-HT2A receptors were constructed, energy refined, and used to study the interactions with a series of buspirone analogues. For both receptors, the calculations showed that the main interactions of the ligand imide moieties were with amino acids in transmembrane helix (TMH) 2 and 7, while the main interactions of the ligand aromatic moieties were with amino acids in TMH5, 6 and 7. Differences in binding site architecture in the region of highly conserved serine and tyrosine residues in TMH7 gave slightly different binding modes of the buspirone analogues at the 5-HT1A and 5-HT2A receptors. Molecular dynamics simulations of receptor-ligand interactions indicated that the buspirone analogues did not alter the interhelical hydrogen bonding patterns upon binding to the 5-HT2A receptor, while interhelical hydrogen bonds were broken and others were formed upon ligand binding to the 5-HT1A receptor. The ligand-induced changes in interhelical hydrogen bonding patterns of the 5-HT1A receptor were followed by rigid body movements of TMH2, 4 and 6 relative to each other and to the other TMHs, which may reflect the structural conversion into an active receptor structure.     

Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA

Recent crystal structures of the multidrug ATP‐binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208′) in the intracellular domains of its two half‐transporters, close to the nucleotide‐binding domains (NBDs), which are in close proximity of each other in the outward‐facing state but not in the inward‐facing state. Using intermolecular cysteine crosslinking between E208C and E208C′ in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide‐free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half‐transporters like a nucleotide‐free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Dynamic Elements at Both Cytoplasmically and Extracellularly Facing Sides of the UapA Transporter Selectively Control the Accessibility of Substrates to Their Translocation Pathway

In the UapA uric acid-xanthine permease of Aspergillusnidulans, subtle interactions between key residues of the putative substrate binding pocket, located in the TMS8-TMS9 loop (where TMS is transmembrane segment), and a specificity filter, implicating residues in TMS12 and the TMS1-TMS2 loop, are critical for function and specificity. By using a strain lacking all transporters involved in adenine uptake (ΔazgA ΔfcyB ΔuapC) and carrying a mutation that partially inactivates the UapA specificity filter (F528S), we obtained 28 mutants capable of UapA-mediated growth on adenine. Seventy-two percent of mutants concern replacements of a single residue, R481, in the putative cytoplasmic loop TMS10-TMS11. Five missense mutations are located in TMS9, in TMS10 or in loops TMS1-TMS2 and TMS8-TMS9. Mutations in the latter loops concern residues previously shown to enlarge UapA specificity (Q113L) or to be part of a motif involved in substrate binding (F406Y). In all mutants, the ability of UapA to transport its physiological substrates remains intact, whereas the increased capacity for transport of adenine and other purines seems to be due to the elimination of elements that hinder the translocation of non-physiological substrates through UapA, rather than to an increase in relevant binding affinities. The additive effects of most novel mutations with F528S and allele-specific interactions of mutation R481G (TMS10-TMS11 loop) with Q113L (TMS1-TMS2 loop) or T526M (TMS12) establish specific interdomain synergy as a critical determinant for substrate selection. Our results strongly suggest that distinct domains at both sides of UapA act as selective dynamic gates controlling substrate access to their translocation pathway.     

The multidrug resistance pump ABCB1 is a substrate for the ubiquitin ligase NEDD4-1

Abstract

The ATP Binding Cassette transporter ABCB1 can export the neurotoxic peptide β-amyloid from endothelial cells that line the blood-brain barrier (BBB). This has the potential to lower cerebral levels of β-amyloid, but ABCB1 expression in the BBB appears to be progressively reduced in patients with Alzheimer’s disease. The surface density of many membrane proteins is regulated by ubiquitination catalyzed by ubiquitin E3 ligases. In brain capillaries of mice challenged with β-amyloid ex vivo, we show that the level of the ubiquitin ligase Nedd4 increases concomitant with reduction in Abcb1. In vitro we show that human ABCB1 is a substrate for human NEDD4-1 ligase. Recombinant ABCB1 was purified from Sf21 insect cells and incubated with recombinant NEDD4-1 purified from Escherichia coli. The treated ABCB1 had reduced mobility on SDS-PAGE, and mass spectrometry identified eight lysine residues, K271, K272, K575, K685, K877, K885, K887 and K1062 that were ubiquitinated by NEDD4-1. Molecular modelling showed that all of the residues are exposed on the surface of the intracellular domains of ABCB1. K877, K885 and K887 in particular, are located in the intracellular loop of transmembrane helix 10 (TMH10) in close proximity, in the tertiary fold, to a putative NEDD4-1 binding site in the intracellular helix extending from TMH12 (PxY motif, residues 996–998). Transient expression of NEDD4-1 in HEK293 Flp-In cells stably expressing ABCB1 was shown to reduce the surface density of the transporter. Together, the data identify this ubiquitin ligase as a potential target for intervention in the pathophysiology of Alzheimer’s disease.     

Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot

We explore the thermodynamic basis for high affinity binding and specificity in conserved protein complexes using colicin endonuclease-immunity protein complexes as our model system. We investigated the ability of each colicin-specific immunity protein (Im2, Im7, Im8 and Im9) to bind the endonuclease (DNase) domains of colicins E2, E7 and E8 in vitro and compared these to the previously studied colicin E9. We find that high affinity binding (Kd < or = 10(-14) M) is a common feature of cognate colicin DNase-Im protein complexes as are non-cognate protein-protein associations, which are generally 10(6)-10(8)-fold weaker. Comparative alanine scanning of Im2 and Im9 residues involved in binding the E2 DNase revealed similar behaviour to that of the two proteins binding the E9 DNase; helix III forms a conserved binding energy hotspot with specificity residues from helix II only contributing favourably in a cognate interaction, a combination we have termed as "dual recognition". Significant differences are seen, however, in the number and side-chain chemistries of specificity sites that contribute to cognate binding. In Im2, Asp33 from helix II dominates colicin E2 specificity, whereas in Im9 several hydrophobic residues, including position 33 (leucine), help define its colicin specificity. A similar distribution of specificity sites was seen using phage display where, with Im2 as the template, a library of randomised sequences was generated in helix II and the library panned against either the E2 or E9 DNase. Position 33 was the dominant specificity site recovered in all E2 DNase-selected clones, whereas a number of Im9 specificity sites were recovered in E9 DNase-selected clones, including position 33. In order to probe the relationship between biological specificity and in vitro binding affinity we compared the degree of protection afforded to bacteria against colicin E9 toxicity by a set of immunity proteins whose affinities for the E9 DNase differed by up to ten orders of magnitude. This analysis indicated that the Kd required for complete biological protection is <10(-10)M and that the "affinity window" over which the selection of novel immunity protein specificities likely evolves is 10(-6)-10(-10)M. This comprehensive survey of colicin DNase-immunity protein complexes illustrates how high affinity protein-protein interactions can be very discriminating even though binding is dominated by a conserved hotspot, with single or multiple specificity sites modulating the overall binding free energy. We discuss these results in the context of other conserved protein complexes and suggest that they point to a generic specificity mechanism in divergently evolved protein-protein interactions.     

Tryptophan-mediated Dimerization of the TssL Transmembrane Anchor Is Required for Type VI Secretion System Activity

The type VI secretion system (T6SS) is a multiprotein complex used by bacteria to deliver effectors into target cells. The T6SS comprises a bacteriophage-like contractile tail structure anchored to the cell envelope by a membrane complex constituted of the TssJ outer-membrane lipoprotein and the TssL and TssM inner-membrane proteins. TssJ establishes contact with the periplasmic domain of TssM whereas the transmembrane segments of TssM and its cytoplasmic domain interact with TssL. TssL protrudes in the cytoplasm but is anchored by a C-terminal transmembrane helix (TMH). Here, we show that TssL TMH dimerization is required for the stability of the protein and for T6SS function. Using the TOXCAT assay and point mutations of the 23 residues of the TssL TMH, we identified Thr194 and Trp199 as necessary for TssL TMH dimerization. NMR hydrogen–deuterium exchange experiments demonstrated the existence of a dimer with the presence of Trp185 and Trp199 at the interface. A structural model based on molecular dynamic simulations shows that TssL TMH dimer formation involves π–π interactions resulting from the packing of the two Trp199 rings at the C-terminus and of the six aromatic rings of Tyr184, Trp185 and Trp188 at the N-terminus of the TMH.     

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (K_d = 0.18 μm) and an ATPase activity with a K_m of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.     

Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. The molecular basis for SNAP25 recognition and cleavage by BoNT serotype E is currently unclear. Here we define the multiple pocket recognition of SNAP25 by LC/E. The initial recognition of SNAP25 is mediated by the binding of the B region of SNAP25 to the substrate-binding (B) region of LC/E comprising Leu166, Arg167, Asp127, Ala128, Ser129, and Ala130. The mutations at these residues affected substrate binding and catalysis. Three additional residues participate in scissile bond cleavage of SNAP25 by LC/E. The P3 site residues, Ile178, of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile47, Ile164, and Ile182 and appeared to align the P1' and P2 residues of SNAP25 with the S1' and S2 pockets of LC/E. The S1' pocket of LC/E included three residues, Phe191, Thr159, and Thr208, which contribute hydrophobic and steric interactions with the SNAP25 P1' residue Ile181. The S2 pocket residue of LC/E, Lys224, binds the P2 residue of SNAP25, Asp179, through ionic interactions. Deletion mapping indicates that main chain interaction(s) of residues 182-186 of SNAP25 contribute to substrate recognition by LC/E. Understanding the mechanism for substrate specificity provides insight for the development of inhibitors against the botulinum neurotoxins.     

Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates

The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of approximately 2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 microm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25-50 microm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates.     

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).     

TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli

The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.     

Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1

Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.     

Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.