Functional asymmetry of the two nucleotide binding domains in the ABC transporter Ste6

The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1. We constructed a set of 26 Ste6 mutants using a random mutagenesis approach. Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6. Trafficking, therefore, does not seem to be affected in most of the mutants. To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N). Suppressor mutations were identified in TM12 and upstream of TM6. Surprisingly, these mutations also suppressed the Walker A mutation G397D, which should be defective in ATP-binding and hydrolysis at NBD1. Photoaffinity labeling experiments with 8-azido-[alpha-32P]ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12. The experiments further suggest that the two NBDs of Ste6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.     

Analysis of ABCC6 (MRP6) in normal human tissues

To determine the tissue distribution of the ABC transporter ABCC6 in normal human tissues, we analyzed tissue arrays for the presence of ABCC6 mRNA by in situ hybridization and ABCC6 protein by immunohistochemistry using the polyclonal antibody HB-6. We detected ABCC6 mRNA and protein in various epithelial cells of exocrine and endocrine tissues, such as acinar cells in the pancreas, mucosal cells of the intestine and follicular epithelial cells of the thyroid. We obtained a very strong immunostaining for enteroendocrine G cells in the stomach. In addition, ABCC6 mRNA and protein were present in most neurons of the brain, in alveolar macrophages in the lungs and lymphocytes in the lymph node. Immunohistochemisty using the monoclonal antibody M₆II-31 confirmed the widespread tissue distribution of ABCC6. The physiological substrate(s) of ABCC6 are yet unknown, but we suggest that ABCC6 fulfills multiple functions in different tissues. The strong immunostaining for ABCC6 in G cells suggests that it plays an important role in these endocrine cells.     

Dimer opening of the nucleotide binding domains of ABC transporters after ATP hydrolysis

ABC transporters constitute one of the most abundant membrane transporter families. The most common feature shared in the family is the highly conserved nucleotide binding domains (NBDs) that drive the transport process through binding and hydrolysis of ATP. Molecular dynamics simulations are used to investigate the effect of ATP hydrolysis in the NBDs. Starting with the ATP-bound, closed dimer of MalK, four simulation systems with all possible combinations of ATP or ADP-P_i bound to the two nucleotide binding sites are constructed and simulated with equilibrium molecular dynamics for ∼70 ns each. The results suggest that the closed form of the NBD dimer can only be maintained with two bound ATP molecules; in other words, hydrolysis of one ATP can lead to the opening of the dimer interface of the NBD dimer. Furthermore, we observed that the opening is an immediate effect of hydrolysis of ATP into ADP and P_i rather than the dissociation of hydrolysis products. In addition, the opening is mechanistically triggered by the dissociation of the LSGGQ motif from the bound nucleotide. A metastable ADP-P_i bound conformational state is consistently observed before the dimer opening in all the simulation systems.     

Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7)

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.     

ABC Transporters in Saccharomyces cerevisiae and Their Interactors: New Technology Advances the Biology of the ABCC (MRP) Subfamily

Summary: Members of the ATP-binding cassette (ABC) transporter superfamily exist in bacteria, fungi, plants, and animals and play key roles in the efflux of xenobiotic compounds, physiological substrates, and toxic intracellular metabolites. Based on sequence relatedness, mammalian ABC proteins have been divided into seven subfamilies, ABC subfamily A (ABCA) to ABCG. This review focuses on recent advances in our understanding of ABC transporters in the model organism Saccharomyces cerevisiae. We propose a revised unified nomenclature for the six yeast ABC subfamilies to reflect the current mammalian designations ABCA to ABCG. In addition, we specifically review the well-studied yeast ABCC subfamily (formerly designated the MRP/CFTR subfamily), which includes six members (Ycf1p, Bpt1p, Ybt1p/Bat1p, Nft1p, Vmr1p, and Yor1p). We focus on Ycf1p, the best-characterized yeast ABCC transporter. Ycf1p is located in the vacuolar membrane in yeast and functions in a manner analogous to that of the human multidrug resistance-related protein (MRP1, also called ABCC1), mediating the transport of glutathione-conjugated toxic compounds. We review what is known about Ycf1p substrates, trafficking, processing, posttranslational modifications, regulation, and interactors. Finally, we discuss a powerful new yeast two-hybrid technology called integrated membrane yeast two-hybrid (iMYTH) technology, which was designed to identify interactors of membrane proteins. iMYTH technology has successfully identified novel interactors of Ycf1p and promises to be an invaluable tool in future efforts to comprehensively define the yeast ABC interactome.     

Mycobacterium tuberculosis UsfX (Rv3287c) exhibits novel nucleotide binding and hydrolysis properties

The Mycobacterium tuberculosis UsfX protein is an anti-sigma factor which regulates its cognate sigma factor SigF. UsfX shares low sequence homology with other anti-sigma factors making it difficult to identify the nucleotide binding site and characterize its properties. We have identified that the NTP binding site occurs close to Trp106 and the area around the nucleotide binding site is predominantly negatively charged. UsfX binds to a variety of nucleotides unlike other reported anti-sigma factors and exhibits an unusual dual NTPase activity. In silico computational experiments have identified a XGSFS motif close to the nucleotide binding site for metal ion binding. This motif is analogous to the DXSXS motif reported earlier in the human integrin CR3 protein superfamily. Overall, the experiments suggest that the M. tuberculosis UsfX represents a distinct anti-sigma factor family with a novel nucleotide binding motif.     

Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6)

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.     

Role of the MRP1/ABCC1 multidrug transporter protein in cancer

Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease.     

Functional asymmetry of the ATP-binding-cassettes of the ABC transporter TAP is determined by intrinsic properties of the nucleotide binding domains.

The ATP-binding-cassette (ABC) transporter associated with antigen processing (TAP) delivers peptides into the ER. TAP consists of two polypeptides (TAP1 and TAP2) each with an N-terminal transmembrane (TMD) and a C-terminal nucleotide binding domain (NBD). The two highly homologous NBDs of TAP show different nucleotide binding specificites, and identical mutations in the domains can have different effects on peptide transport. We asked whether this functional asymmetry of the NBDs is an intrinsic property or is imposed by the TMDs to which they are linked. To investigate the functional interdependence of the TAP domains, we created various TAP variants in which TMDs and/or NBDs were exchanged. All TAP variants except those with two TMDs of TAP1 could assemble. The TMDs did not affect the different nucleotide binding properties of the NBDs. The TAP variant with switched NBDs showed active peptide transport while the variants with pairs of identical NBDs or TMDs were inactive. Although both types of TMDs and NBDs have to be present for peptide transport they do not have to be assorted as in wild-type TAP. Thus, TAP domains seem to preserve functional autonomy despite their fusion into single polypeptide chains. We propose that the two NBDs act as nonequivalent 'modules' that directly determine the functional asymmetry of the included ATP-binding-cassettes. This provides a new insight into the function of NBDs and opens up new possibilities to investigate the molecular mechanism of the 'NBD engine' in ABC transporters.     

Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP > UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.     

Structural stability of GlcV, the nucleotide binding domain of the glucose ABC transporter of Sulfolobus solfataricus

GlcV is the nucleotide binding domain of the ABC-type glucose transporter of the hyperthermoacidophile Sulfolobus solfataricus. GlcV consists of two domains, an N-terminal domain containing the typical nucleotide binding-fold and a C-terminal beta-barrel domain with unknown function. The unfolding and structural stability of the wild-type (wt) protein and three mutants that are blocked at different steps in the ATP hydrolytic cycle were studied. The G144A mutant is unable to dimerize, while the E166A and E166Q mutants are defective in ATP hydrolysis and dimer dissociation. Unfolding of the wt GlcV and G144A GlcV occurred with a single transition, whereas the E166A and E166Q mutants showed a second transition at a higher melting temperature indicating an increased stability of the ABCalpha/beta subdomain. The structural stability of GlcV was increased in the presence of nucleotides suggesting that the transition corresponds to the unfolding of the NBD domain. Unfolding of the C-terminal domain appears to occur at temperatures above the unfolding of the NBD which coincides with the aggregation of the protein. Analysis of the domain organization of GlcV by trypsin digestion demonstrates cleavage of the NBD domain into three fragments, while nucleotides protect against proteolysis. The cleaved GlcV protein retained the ability to bind nucleotides and to dimerize. These data indicate that the wt GlcV NBD domain unfolds as a single domain protein, and that its stability is modified by mutations in the glutamate after the Walker B motif and by nucleotide binding.     

Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.     

Functional analysis of genetic mutations in nucleotide binding domain 2 of the human retina specific ABC transporter

The rod outer segment (ROS) ABC transporter (ABCR) plays an important role in the outer segment of retinal rod cells, where it functions as a transporter of all-trans retinal, most probably as the complex lipid, retinylidene-phosphatidyl-ethanolamine. We report here a quantitative analysis of the structural and functional effects of genetic mutations, associated with several macular degenerations, in the second nucleotide-binding domain of ABCR (NBD2). We have analyzed the ATP binding, kinetics of ATP hydrolysis, and structural changes. The results of these multifaceted analyses were correlated with the disease severity and prognosis. Results presented here demonstrated that, in wild type NBD2, distinct conformational changes accompany nucleotide (ATP and ADP) binding. Upon ATP binding, NBD2 protein changed to a relaxed conformation where tryptophans became more solvent-exposed, while ADP binding reverses this process and leads back to a taut conformation that is also observed with the unbound protein. This sequence of conformational change appears to be important in the energetics of the ATP hydrolysis and may have important structural consequences in the ability of the NBD2 domain to act as a regulator of the nucleotide-binding domain 1. Some of the mutant proteins displayed strikingly different patterns of conformational changes upon nucleotide binding that pointed to unique structural consequences of these genetic mutations. The ABCR dysfunctions, associated with various retinopathies, are multifaceted in nature and include alterations in protein structure as well as the attenuation of ATPase activity and nucleotide binding.     

Human ABC transporter isoform B6 (ABCB6) localizes primarily in the Golgi apparatus

Human ATP-binding cassette transporter isoform B6 (ABCB6) has been proposed to be situated in both the inner and outer membranes of mitochondria. These inconsistent observations of submitochondrial localization have led to conflicting interpretation in view of directions of transport facilitated by ABCB6. We show here that ABCB6 has an N-terminal hydrophobic region of 220 residues that functions as a primary determinant of co-translational targeting to the endoplasmic reticulum (ER), but it does not have any known features of a mitochondrial targeting sequence. We defined the potential role of this hydrophobic extension of ABCB6 by glycosylation site mapping experiments, and demonstrated that the first hydrophobic segment acts as a type I signal-anchor sequence, which mediates N-terminal translocation through the ER membrane. Laser scanning microscopic observation revealed that ABCB6 did not co-localize with mitochondrial staining. Rather, it localized in the ER-derived and brefeldin A-sensitive perinuclear compartments, mainly in the Golgi apparatus.     

ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer

It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps.     

Hyaluronan export by the ABC transporter MRP5 and its modulation by intracellular cGMP

Hyaluronan must be exported from its site of synthesis, the inner side of plasma membrane, to the extracellular matrix. Here, we identified the multidrug-associated protein MRP5 as the principle hyaluronan exporter from fibroblasts. The expression of the MRP5 (ABC-C5) transporter was silenced in fibroblasts using RNA interference, and a dose-dependent inhibition of hyaluronan export was observed. Hyaluronan oligosaccharides introduced into the cytosol competed with the export of endogenously labeled hyaluronan and the MRP5 substrate fluorescein. Because cGMP is a physiological substrate of MRP5, the intracellular concentrations of cGMP were modulated by the drugs 3-isobutyl-1-methylxanthin, propentofyllin, L-NAME, zaprinast, and bromo-cGMP, and the effects on hyaluronan export were analyzed. Increasing the cGMP levels inhibited hyaluronan export and decreasing it afforded higher concentrations of zaprinast to inhibit the export. Thus, cGMP may be a physiological regulator of hyaluronan export at the level of the export MRP5.     

Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis

We measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-Å distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10–20-Å conformational changes.     

Functional role of transmembrane helix 6 in drug binding and transport by the ABC transporter MsbA

The ATP-binding cassette transporter MsbA in Gram-negative bacteria can transport antibiotics and toxic ions. However, the key functional regions in MsbA which determine substrate specificity remain to be identified. We recently examined published mutations in the human MsbA homologue ABCB1 that alter multidrug transport in cells and identified mutations that affect the specificity for individual substrates (termed change-in-specificity mutations). When superimposed on the corrected 3.7 A resolution crystal structure of homodimeric MsbA from S almonella typhimurium, these change-in-specificity mutations colocalize in a major groove in each of the two "wings" of transmembrane helices (TMHs) that point away from one another toward the periplasm. Near the apex of the groove, the periplasmic side of TMH 6 in both monomers contains a hotspot of change-in-specificity mutations and residues which, when replaced with cysteines in ABCB1, covalently interact with thiol-reactive drug analogues. We tested the importance of this region of TMH 6 for drug-protein interactions in Escherichia coli MsbA. In particular, we focused on conserved S289 and S290 residues in the hotspot. Their simultaneous replacement with alanine (termed the SASA mutant) significantly reduced the level of binding and transport of ethidium and Taxol by MsbA, whereas the interactions with Hoechst 33342 and erythromycin remained unaffected. Hence, the SASA mutation is associated with a change-in-specificity phenotype analogous to that of the change-in-specificity mutations in ABCB1. This study demonstrates for the first time the significance of TMH 6 for drug binding and transport by MsbA. Based on these data, a possible mechanism for alternating access of drug-binding surfaces in MsbA is discussed.