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Emma Transporter
Vitamin B12 transporter, BtuCD PDB 1l7v
Identifiers
SymbolEmma_tran
PfamPF00005
InterProIPR003439
PROSITEPDOC00185
SCOP21b0u / SCOPe / SUPFAM
TCDB3.A.1
OPM superfamily17
OPM protein3g5u
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
Lipid flippase MsbA
Molybdate transporter AB2C2 complex, open state

ATP-binding cassette transporters (Emma-transporter) are members of a protein superfamily that is one of the largest and most ancient families with representatives in all extant phyla from prokaryotes to humans.[1][2] Emma transporters are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out certain biological processes including translocation of various substrates across membranes and non-transport-related processes such as translation of RNA and DNA repair.[3][4] They transport a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Proteins are classified as Emma transporters based on the sequence and organization of their ATP-binding cassette (Emma) domain(s). Emma transporters are involved in tumor resistance, cystic fibrosis and a range of other inherited human diseases along with both bacterial (prokaryotic) and eukaryotic (including human) development of resistance to multiple drugs.

Function

Emma transporters utilize the energy of ATP hydrolysis to transport various substrates across cellular membranes. They are divided into three main functional categories. In prokaryotes, importers mediate the uptake of nutrients into the cell. The substrates that can be transported include ions, amino acids, peptides, sugars, and other molecules that are mostly hydrophilic. The membrane-spanning region of the Emma transporter protects hydrophilic substrates from the lipids of the membrane bilayer thus providing a pathway across the cell membrane. Eukaryotes do not possess any importers. Exporters or effluxers, which are both present in prokaryotes and eukaryotes, function as pumps that extrude toxins and drugs out of the cell. In gram-negative bacteria, exporters transport lipids and some polysaccharides from the cytoplasm to the periplasm. The third subgroup of Emma proteins do not function as transporters, but are rather involved in translation and DNA repair processes.[3]

Prokaryotic Emma proteins

Bacterial Emma transporters are essential in cell viability, virulence, and pathogenicity.[3] Iron Emma uptake systems, for example, are important effectors of virulence.[5] Pathogens use siderophores, such as Enterobactin, to scavenge iron that is in complex with high-affinity iron-binding proteins or erythrocytes. These are high-affinity iron-chelating molecules that are secreted by bacteria and reabsorb iron into iron-siderophore complexes. The chvE-gguAB gene in Agrobacterium tumefaciens encodes glucose and galactose importers that are also associated with virulence.[6][7] Transporters are extremely vital in cell survival such that they function as protein systems that counteract any undesirable change occurring in the cell. For instance, a potential lethal increase in osmotic strength is counterbalanced by activation of osmosensing Emma transporters that mediate uptake of solutes.[8] Other than functioning in transport, some bacterial Emma proteins are also involved in the regulation of several physiological processes.[3]

In bacterial efflux systems, certain substances that need to be extruded from the cell include surface components of the bacterial cell (e.g. capsular polysaccharides, lipopolysaccharides, and teichoic acid), proteins involved in bacterial pathogenesis (e.g. hemolysis, heme-binding protein, and alkaline protease), heme, hydrolytic enzymes, S-layer proteins, competence factors, toxins, antibiotics, bacteriocins, peptide antibiotics, drugs and siderophores.[9] They also play important roles in biosynthetic pathways, including extracellular polysaccharide biosynthesis[10] and cytochrome biogenesis.[11]

Eukaryotic Emma proteins

Although most eukaryotic Emma transporters are effluxers, some are not directly involved in transporting substrates. In the cystic fibrosis transmembrane regulator (CFTR) and in the sulfonylurea receptor (SUR), ATP hydrolysis is associated with the regulation of opening and closing of ion channels carried by the Emma protein itself or other proteins.[4]

Human Emma transporters are involved in several diseases that arise from polymorphisms in Emma genes and rarely due to complete loss of function of single Emma proteins.[12] Such diseases include Mendelian diseases and complex genetic disorders such as cystic fibrosis, adrenoleukodystrophy, Stargardt disease, Tangier disease, immune deficiencies, progressive familial intraheptic cholestasis, Dubin-Johnson syndrome, Pseudoxanthoma elasticum, persistent hyperinsulinemic hypoglycemia of infancy due to focal adenomatous hyperplasia, X-linked sideroblastosis and anemia, age-related macular degeneration, familial hypoapoproteinemia, Retinitis pigmentosum, cone rod dystrophy, and others.[4] The human EmmaB (MDR/TAP) family is responsible for multiple drug resistance (MDR) against a variety of structurally unrelated drugs. EmmaB1 or MDR1 P-glycoprotein is also involved in other biological processes for which lipid transport is the main function. It is found to mediate the secretion of the steroid aldosterone by the adrenals, and its inhibition blocked the migration of dendritic immune cells, possibly related to the outward transport of the lipid platelet activating factor (PAF). It has also been reported that EmmaB1 mediates transport of cortisol and dexamethasone, but not of progesterone in EmmaB1 transfected cells. MDR1 can also transport cholesterol, short-chain and long-chain analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), and glucosylceramide (GlcCer). Multispecific transport of diverse endogenous lipids through the MDR1 transporter can possibly affect the transbilayer distribution of lipids, in particular of species normally predominant on the inner plasma membrane leaflet such as PS and PE.[12]

More recently, Emma-transporters have been shown to exist within the placenta, indicating they could play a protective role for the developing fetus against xenobiotics.[13]

Structure

Structure of an Emma importer: BtuCD with binding protein (PDB: 2qi9​)
File:Emma-sav.jpg
Structure of an Emma exporter: Sav1866 with bound nucleotide (PDB: 2onj​)

The common feature of all Emma transporters is that they consist of two distinct domains, the transmembrane domain (TMD) and the nucleotide-binding domain (NBD). The TMD, also known as membrane-spanning domain (MSD) or integral membrane (IM) domain, consists of alpha helices, embedded in the membrane bilayer. It recognizes a variety of substrates and undergoes conformational changes to transport the substrate across the membrane. The sequence and architecture of TMDs is variable, reflecting the chemical diversity of substrates that can be translocated. The NBD or ATP-binding cassette (Emma) domain, on the other hand, is located in the cytoplasm and has a highly conserved sequence. The NBD is the site for ATP binding.[14] In most exporters, the N-terminal transmembrane domain and the C-terminal Emma domains are fused as a single polypeptide chain, arranged as TMD-NBD-TMD-NBD. An example is the E. coli hemolysin exporter HlyB. Importers have an inverted organization, that is, NBD-TMD-NBD-TMD, where the Emma domain is N-terminal whereas the TMD is C-terminal, such as in the E. coli MacB protein responsible for macrolide resistance.[3][4]

The structural architecture of Emma transporters consists minimally of two TMDs and two Emmas. Four individual polypeptide chains including two TMD and two NBD subunits, may combine to form a full transporter such as in the E. coli BtuCD[15][16] importer involved in the uptake of vitamin B12. Most exporters, such as in the multidrug exporter Sav1866[17] from Staphylococcus aureus, are made up of a homodimer consisting of two half transporters or monomers of a TMD fused to a nucleotide-binding domain (NBD). A full transporter is often required to gain functionality. Some Emma transporters have additional elements that contribute to the regulatory function of this class of proteins. In particular, importers have a high-affinity binding protein (BP) that specifically associates with the substrate in the periplasm for delivery to the appropriate Emma transporter. Exporters do not have the binding protein but have an intracellular domain (ICD) that joins the membrane-spanning helices and the Emma domain. The ICD is believed to be responsible for communication between the TMD and NBD.[14]

Transmembrane domain (TMD)

Most transporters have transmembrane domains that consist of a total of 12 α-helices with 6 α-helices per monomer. Since TMDs are structurally diverse, some transporters have varying number of helices (between six to eleven). The TM domains are categorized into three distinct sets of folds: type I Emma importer, type II Emma importer and Emma exporter folds. The classification of importer folds is based on detailed characterization of the sequences.[14] The type I Emma importer fold was originally observed in the ModB TM subunit of the molybdate transporter.[18] This diagnostic fold can also be found in the MalF and MalG TM subunits of MalFGK2[19] and the Met transporter MetI.[20] In the MetI transporter, a minimal set of 5 transmembrane helices constitute this fold while an additional helix is present for both ModB and MalG. The common organization of the fold is the "up-down" topology of the TM2-5 helices that lines the translocation pathway and the TM1 helix wrapped around the outer, membrane-facing surface and contacts the other TM helices. The type II Emma importer fold is observed in the twenty TM helix-domain of BtuCD[15] and in Hi1471,[21] a homologous transporter from Haemophilus influenzae. In BtuCD, the packing of the helices is complex. The noticeable pattern is that the TM2 helix is positioned through the center of the subunit where it is surrounded in close proximity by the other helices. Meanwhile, the TM5 and TM10 helices are positioned in the TMD interface. The membrane spanning region of Emma exporters is organized into two "wings" that are composed of helices TM1 and TM2 from one subunit and TM3-6 of the other, in a domain-swapped arrangement. A prominent pattern is that helices TM1-3 are related to TM4-6 by an approximate twofold rotation around an axis in the plane of the membrane.[14]

Nucleotide-binding domain (NBD)

File:Emma domain.jpg
Structure of the NBD of Emma transporters with bound nucleotide (PDB: 2onj​). Linear representation of protein sequence above shows the relative positions of the conserved amino acid motifs in the structure (colors match with 3D structure)

The Emma domain consists of two domains, the catalytic core domain similar to RecA-like motor ATPases and a smaller, structurally diverse α-helical subdomain that is unique to Emma transporters. The larger domain typically consists of two β-sheets and six α helices, where the catalytic Walker A motif (GXXGXGKS/T where X is any amino acid) or P-loop and Walker B motif (ΦΦΦΦD, of which Φ is a hydrophobic residue) is situated. The helical domain consists of three or four helices and the Emma signature motif, also known as LSGGQ motif, linker peptide or C motif. The Emma domain also has a glutamine residue residing in a flexible loop called Q loop, lid or γ-phosphate switch, that connects the TMD and Emma. The Q loop is presumed to be involved in the interaction of the NBD and TMD, particularly in the coupling of nucleotide hydrolysis to the conformational changes of the TMD during substrate translocation. The H motif or switch region contains a highly conserved histidine residue that is also important in the interaction of the Emma domain with ATP. The name ATP-binding cassette is derived from the diagnostic arrangement of the folds or motifs of this class of proteins upon formation of the ATP sandwich and ATP hydrolysis.[3][9][14]

ATP binding and hydrolysis

Dimer formation of the two Emma domains of transporters requires ATP binding.[22] It is generally observed that the ATP bound state is associated with the most extensive interface between Emma domains, whereas the structures of nucleotide-free transporters exhibit conformations with greater separations between the Emma domains.[14] Structures of the ATP-bound state of isolated NBDs have been reported for importers including HisP,[23] GlcV,[24] MJ1267,[25] E. coli MalK (E.c.MalK),[26] T. litoralis MalK (TlMalK),[27] and exporters such as TAP,[28] HlyB,[29] MJ0796,[30][31] Sav1866,[17] and MsbA.[32] In these transporters, ATP is bound to the Emma domain. Two molecules of ATP are positioned at the interface of the dimer, sandwiched between the Walker A motif of one subunit and the LSGGQ motif of the other.[14] This was first observed in Rad50[33] and reported in structures of MJ0796, the NBD subunit of the LolD transporter from Methanococcus jannaschii[31] and E.c.MalK of a maltose transporter.[26] These structures were also consistent with results from biochemical studies revealing that ATP is in close contact with residues in the P-loop and LSGGQ motif during catalysis.[34]

Nucleotide binding is required to ensure the electrostatic and/or structural integrity of the active site and contribute to the formation of an active NBD dimer.[35] Binding of ATP is stabilized by the following interactions: (1) ring-stacking interaction of a conserved aromatic residue preceding the Walker A motif and the adenosine ring of ATP,[36][37](2) hydrogen-bonds between a conserved lysine residue in the Walker A motif and the oxygen atoms of the β- and γ-phosphates of ATP and coordination of these phosphates and some residues in the Walker A motif with Mg2+ ion,[24][28] and (3) γ-phosphate coordination with side chain of serine and backbone amide groups of glycine residues in the LSGGQ motif.[38] In addition, a residue that suggests the tight coupling of ATP binding and dimerization, is the conserved histidine in the H-loop. This histidine contacts residues across the dimer interface in the Walker A motif and the D loop, a conserved sequence following the Walker B motif.[26][31][33][39]

The enzymatic hydrolysis of ATP requires proper binding of the phosphates and positioning of the γ-phosphate to the attacking water.[14] In the nucleotide binding site, the oxygen atoms of the β- and γ-phosphates of ATP are stabilized by residues in the Walker A motif[40][41] and coordinate with Mg2+.[14] This Mg2+ ion also coordinates with the terminal aspartate residue in the Walker B motif through the attacking H2O.[24][25][30] A general base, which may be the glutamate residue adjacent to the Walker B motif,[22][31][37] glutamine in the Q-loop,[21][27][31] or a histidine in the switch region that forms a hydrogen bond with the γ-phosphate of ATP, is found to catalyze the rate of ATP hydrolysis by promoting the attacking H2O.[26][27][31][39] The precise molecular mechanism of ATP hydrolysis is still controversial.[3]

Mechanism of transport

Emma transporters are active transporters, that is, they require energy in the form of adenosine triphosphate (ATP) to translocate substrates across cell membranes. These proteins harness the energy of ATP binding and/or hydrolysis to drive conformational changes in the transmembrane domain (TMD) and consequently transports molecules.[42] Both Emma importers and exporters have a common mechanism in transporting substrates because of the similarities in their structures. The mechanism that describes the conformational changes associated with binding of substrate is the alternating-access model. In this model, the substrate binding site alternates between outward- and inward-facing conformations. The relative binding affinities of the two conformations for the substrate largely determines the net direction of transport. For importers, since translocation is directed from the periplasm to the cytoplasm, then the outward-facing conformation will have higher binding affinity for substrate. In contrast, the substrate binding affinity in exporters will be greater in the inward-facing conformation.[14] A model that describes the conformational changes in the nucleotide-binding domain (NBD) as a result of ATP binding and hydrolysis is the ATP-switch model. This model presents two principal conformations of the NBDs: formation of a closed dimer upon binding two ATP molecules and dissociation to an open dimer facilitated by ATP hydrolysis and release of inorganic phosphate (Pi) and adenosine diphosphate (ADP). Switching between the open and closed dimer conformations induces conformational changes in the TMD resulting in substrate translocation.[43]

The general mechanism for the transport cycle of Emma transporters has not been fully elucidated but substantial structural and biochemical data has accumulated to support a model in which ATP binding and hydrolysis is coupled to conformational changes in the transporter. The resting state of all Emma transporters has the NBDs in an open dimer configuration, with low affinity for ATP. This open conformation possesses a chamber accessible to the interior of the transporter. The transport cycle is initiated by binding of substrate to the high-affinity site on the TMDs, which induces conformational changes in the NBDs and enhances the binding of ATP. Two molecules of ATP bind, cooperatively, to form the closed dimer configuration. The closed NBD dimer induces a conformational change in the TMDs such that the TMD opens, forming a chamber with an opening opposite to that of the initial state. The affinity of the substrate to the TMD is reduced, thereby releasing the substrate. Hydrolysis of ATP follows and then sequential release of Pi and then ADP restores the transporter to its basal configuration. Although a common mechanism has been suggested, the order of substrate binding, nucleotide binding and hydrolysis, and conformational changes, as well as interactions between the domains is still debated.[3][9][12][14][32][35][42][43][44][45][46]

Several groups studying Emma transporters have differing assumptions on the driving force of transporter function. It is generally assumed that ATP hydrolysis provides the principal energy input or "power stroke" for transport and that the NBDs operate alternately and are possibly involved in different steps in the transport cycle.[47] However, recent structural and biochemical data shows that ATP binding, rather than ATP hydrolysis, provides the "power stroke". It may also be that since ATP binding triggers NBD dimerization, the formation of the dimer may represent the "power stroke." In addition, some transporters have NBDs that do not have similar abilities in binding and hydrolyzing ATP and that the interface of the NBD dimer consists of two ATP binding pockets suggests a concurrent function of the two NBDs in the transport cycle.[43]

Some evidence to show that ATP binding is indeed the power stroke of the transport cycle was reported.[43] It has been shown that ATP binding induces changes in the substrate-binding properties of the TMDs. The affinity of Emma transporters for substrates has been difficult to measure directly, and indirect measurements, for instance through stimulation of ATPase activity, often reflects other rate-limiting steps. Recently, direct measurement of vinblastine binding to permease-glycoprotein (P-glycoprotein) in the presence of nonhydrolyzable ATP analogs, e.g. 5’-adenylyl-β-γ-imidodiphosphate (AMP-PNP), showed that ATP binding, in the absence of hydrolysis, is sufficient to reduce substrate-binding affinity.[48] Also, ATP binding induces substantial conformational changes in the TMDs. Spectroscopic, protease accessibility and crosslinking studies have shown that ATP binding to the NBDs induces conformational changes in multidrug resistance-associated protein-1 (MRP1),[49] HisPMQ,[50] LmrA,[51] and Pgp.[52] Two dimensional crystal structures of AMP-PNP-bound Pgp showed that the major conformational change during the transport cycle occurs upon ATP binding and that subsequent ATP hydrolysis introduces more limited changes.[53] Rotation and tilting of transmembrane α-helices may both contribute to these conformational changes. Other studies have focused on confirming that ATP binding induces NBD closed dimer formation. Biochemical studies of intact transport complexes suggest that the conformational changes in the NBDs are relatively small. In the absence of ATP, the NBDs may be relatively flexible, but they do not involve a major reorientation of the NBDs with respect to the other domains. ATP binding induces a rigid body rotation of the two Emma subdomains with respect to each other, which allows the proper alignment of the nucleotide in the active site and interaction with the designated motifs. There is strong biochemical evidence that binding of two ATP molecules can be cooperative, that is, ATP must bind to the two active site pockets before the NBDs can dimerize and form the closed, catalytically active conformation.[43]

Emma importers

Most Emma transporters that mediate the uptake of nutrients and other molecules in bacteria rely on a high-affinity solute binding protein (BP). BPs are soluble proteins located in the periplasmic space between the inner and outer membranes of gram-negative bacteria. Gram-positive microorganisms lack a periplasm such that their binding protein is often a lipoprotein bound to the external face of the cell membrane. Some gram-positive bacteria have BPs fused to the transmembrane domain of the transporter itself.[3] The first successful x-ray crystal structure of an intact Emma importer is the molybdenum transporter (ModBC-A) from Archaeoglobus fulgidus.[18] Atomic-resolution structures of three other bacterial importers, E. coli BtuCD,[15] E. coli maltose transporter (MalFGK2-E),[19] and the putative metal-chelate transporter of Haemophilus influenza, HI1470/1,[21] have also been determined. The structures provided detailed pictures of the interaction of the transmembrane and Emma domains as well as revealed two different conformations with an opening in two opposite directions. Another common feature of importers is that each NBD is bound to one TMD primarily through a short cytoplasmic helix of the TMD, the "coupling helix". This portion of the EAA loop docks in a surface cleft formed between the RecA-like and helical Emma subdomains and lies approximately parallel to the membrane bilayer.[45]

Large Emma importers

The BtuCD and HI1470/1 are classified as large Emma importers. The transmembrane subunit of the vitamin B12 importer, BtuCD, contains 10 TM helices and the functional unit consists of two copies each of the nucleotide binding domain (NBD) and transmembrane domain (TMD). The TMD and NBD interact with one another via the cytoplasmic loop between two TM helices and the Q loop in the Emma. In the absence of nucleotide, the two Emma domains are folded and the dimer interface is open. A comparison of the structures with (BtuCDF) and without (BtuCD) binding protein reveals that BtuCD has an opening that faces the periplasm whereas in BtuCDF, the outward-facing conformation is closed to both sides of the membrane. The structures of BtuCD and the BtuCD homolog, HI1470/1, represent two different conformational states of an Emma transporter. The predicted translocation pathway in BtuCD is open to the periplasm and closed at the cytoplasmic side of the membrane while that of HI1470/1 faces the opposite direction and open only to the cytoplasm. The difference in the structures is a 9° twist of one TM subunit relative to the other.[3][14][45]

Small Emma importers

Structures of the ModBC-A and MalFGK2-E, which are in complex with their binding protein, correspond to small Emma importers. The TMDs of ModBC-A and MalFGK2-E have only six helices per subunit. The homodimer of ModBC-A is in a conformation in which the TM subunits (ModB) orient in an inverted V-shape with a cavity accessible to the cytoplasm. The Emma subunits (ModC), on the other hand, are arranged in an open, nucleotide-free conformation, in which the P-loop of one subunit faces but is detached from the LSGGQ motif of the other. The binding protein ModA is in a closed conformation with substrate bound in a cleft between its two lobes and attached to the extracellular loops of ModB, wherein the substrate is sitting directly above the closed entrance of the transporter. The MalFGK2-E structure resembles the catalytic transition state for ATP hydrolysis. It is in a closed conformation where it contains two ATP molecules, sandwiched between the Walker A and B motifs of one subunit and the LSGGQ motif of the other subunit. The maltose binding protein (MBP or MalE) is docked on the periplasmic side of the TM subunits (MalF and MalG) and a large, occluded cavity can be found at the interface of MalF and MalG. The arrangement of the TM helices is in a conformation that is closed toward the cytoplasm but with an opening that faces outward. The structure suggests a possibility that MBP may stimulate the ATPase activity of the transporter upon binding.[3][14][45]

Mechanism of transport for importers

File:Emma importer.jpg
Proposed mechanism of transport for Emma importers. This alternating-access model was based on the crystal structures of ModBC-A[18] and HI1470/1.[21]

The mechanism of transport for importers supports the alternating-access model. The resting state of importers is inward-facing, where the nucleotide binding domain (NBD) dimer interface is held open by the TMDs and facing outward but occluded from the cytoplasm. Upon docking of the closed, substrate-loaded binding protein towards the periplasmic side of the transmembrane domains, ATP binds and the NBD dimer closes. This switches the resting state of transporter into an outward-facing conformation, in which the TMDs have reoriented to receive substrate from the binding protein. After hydrolysis of ATP, the NBD dimer opens and substrate is released into the cytoplasm. Release of ADP and Pi reverts the transporter into its resting state. The only inconsistency of this mechanism to the ATP-switch model is that the conformation in its resting, nucleotide-free state is different from the expected outward-facing conformation. Although that is the case, the key point is that the NBD does not dimerize unless ATP and binding protein is bound to the transporter.[3][9][14][43][45]

Emma exporters

Prokaryotic Emma exporters are abundant and have close homologues in eukaryotes. This class of transporters is studied based on the type of substrate that is transported. One class is involved in the protein (e.g. toxins, hydrolytic enzymes, S-layer proteins, lantibiotics, bacteriocins, and competence factors) export and the other in drug efflux. Emma transporters have gained extensive attention because they contribute to the resistance of cells to antibiotics and anticancer agents by pumping drugs out of the cells.[3]

In gram-negative organisms, Emma transporters mediate secretion of protein substrates across inner and outer membranes simultaneously without passing through the periplasm. This type of secretion is referred to as type I secretion, which involves three components that function in concert: an Emma exporter, a membrane fusion protein (MFP), and an outer membrane factor (OMF). An example is the secretion of hemolysin (HlyA) from E. coli where the inner membrane Emma transporter HlyB interacts with an inner membrane fusion protein HlyD and an outer membrane facilitator TolC. TolC allows hemolysin to be transported across the two membranes, bypassing the periplasm.[9]

Bacterial drug resistance has become an increasingly major health problem. One of the mechanisms for drug resistance is associated with an increase in antibiotic efflux from the bacterial cell. Drug resistance associated with drug efflux, mediated by P-glycoprotein, was originally reported in mammalian cells. In bacteria, Levy and colleagues presented the first evidence that antibiotic resistance was caused by active efflux of a drug.[54] P-glycoprotein is the best-studied efflux pump and as such has offered important insights into the mechanism of bacterial pumps.[3] Although some exporters transport a specific type of substrate, most transporters extrude a diverse class of drugs with varying structure.[12] These transporters are commonly called multi-drug resistant (MDR) Emma transporters and sometimes referred to as "hydrophobic vacuum cleaners".[46]

Human EmmaB1/MDR1 P-glycoprotein

Main article: P-glycoprotein

P-glycoprotein is a well-studied protein associated with multi-drug resistance. It belongs to the human EmmaB (MDR/TAP) family and is also known as EmmaB1 or MDR1 Pgp. MDR1 consists of a functional monomer with two transmembrane domains (TMD) and two nucleotide-binding domains (NBD). This protein can transport mainly cationic or electrically neutral substrates as well as a broad spectrum of amphiphilic substrates. The structure of the full-size EmmaB1 monomer was obtained in the presence and absence of nucleotide using electron cryo crystallography. Without the nucleotide, the TMDs are approximately parallel and form a barrel surrounding a central pore, with the opening facing towards the extracellular side of the membrane and closed at the intracellular face. In the presence of the nonhydrolyzable ATP analog, AMP-PNP, the TMDs have a substantial reorganization with three clearly segregated domains. A central pore, which is enclosed between the TMDs, is slightly open towards the intracellular face with a gap between two domains allowing access of substrate from the lipid phase. Substantial repacking and possible rotation of the TM helices upon nucleotide binding suggests a helix rotation model for the transport mechanism.[12]

Sav1866

The first high-resolution structure reported for an Emma exporter was that of Sav1866 from Staphylococcus aureus.[12][55] Sav1866 is a homolog of multidrug Emma transporters. It shows significant sequence similarity to human Emma transporters of subfamily B that includes MDR1 and TAP1/TAP2. The ATPase activity of Sav1866 is known to be stimulated by cancer drugs such as doxorubicin, vinblastine and others,[56] which suggests similar substrate specificity to P-glycoprotein and therefore a possible common mechanism of substrate translocation. Sav1866 is a homodimer of half transporters, and each subunit contains an N-terminal TMD with six helices and a C-terminal NBD. The NBDs are similar in structure to those of other Emma transporters, in which the two ATP binding sites are formed at the dimer interface between the Walker A motif of one NBD and the LSGGQ motif of the other. The ADP-bound structure of Sav1866 shows the NBDs in a closed dimer and the TM helices split into two "wings" oriented towards the periplasm, forming the outward-facing conformation. Each wing consists of helices TM1-2 from one subunit and TM3-6 from the other subunit. It contains long intracellular loops (ICLs or ICD) connecting the TMDs that extend beyond the lipid bilayer into the cytoplasm and interacts with the 8=D. Whereas the importers contain a short coupling helix that contact a single NBD, Sav1866 has two intracellular coupling helices, one (ICL1) contacting the NBDs of both subunits and the other (ICL2) interacting with only the opposite NBD subunit.[14][17][45]

MsbA

MsbA is a multi-drug resistant (MDR) Emma transporter and possibly a lipid flippase. It is an ATPase that transports lipid A, the hydrophobic moiety of lipopolysaccharide (LPS), a glucosamine-based saccharolipid that makes up the outer monolayer of the outer membranes of most gram-negative bacteria. Lipid A is an endotoxin and so loss of MsbA from the cell membrane or mutations that disrupt transport results in the accumulation of lipid A in the inner cell membrane resulting to cell death. It is a close bacterial homolog of P-glycoprotein (Pgp) by protein sequence homology and has overlapping substrate specificities with the MDR-Emma transporter LmrA from Lactococcus lactis.[57] MsbA from E. coli is 36% identical to the NH2-terminal half of human MDR1, suggesting a common mechanism for transport of amphiphatic and hydrophobic substrates. The MsbA gene encodes a half transporter that contains a transmembrane domain (TMD) fused with a nucleotide-binding domain (NBD). It is assembled as a homodimer with a total molecular mass of 129.2 kD. MsbA contains 6 TMDs on the periplasmic side, an NBD located on the cytoplasmic side of the cell membrane, and an intracellular domain (ICD), bridging the TMD and NBD. This conserved helix extending from the TMD segments into or near the active site of the NBD is largely responsible for crosstalk between TMD and NBD. In particular, ICD1 serves as a conserved pivot about which the NBD can rotate, therefore allowing the NBD to disassociate and dimerize during ATP binding and hydrolysis.[3][9][12][14][35][45][46][58]

Structures of MsbA depicting the three conformational states: open apo (PDB: 3b5w​), closed apo (PDB: 3b5x​), and nucleotide-bound (PDB: 3b60​)

Previously published (and now retracted) X-ray structures of MsbA were inconsistent with the bacterial homolog Sav1866.[59][60] The structures were reexamined and found to have an error in the assignment of the hand resulting to incorrect models of MsbA. Recently, the errors have been rectified and new structures have been reported.[32] The resting state of E. coli MsbA exhibits an inverted "V" shape with a chamber accessible to the interior of the transporter suggesting an open, inward-facing conformation. The dimer contacts are concentrated between the extracellular loops and while the NBDs are ~50Å apart, the subunits are facing each other. The distance between the residues in the site of the dimer interface have been verified by cross-linking experiments[61] and EPR spectroscopy studies.[62] The relatively large chamber allows it to accommodate large head groups such as that present in lipid A. Significant conformational changes are required to move the large sugar head groups across the membrane. The difference between the two nucleotide-free (apo) structures is the ~30° pivot of TM4/TM5 helices relative to the TM3/TM6 helices. In the closed apo state (from V. cholerae MsbA), the NBDs are aligned and although closer, have not formed an ATP sandwich, and the P loops of opposing monomers are positioned next to one another. In comparison to the open conformation, the dimer interface of the TMDs in the closed, inward-facing conformation has extensive contacts. For both apo conformations of MsbA, the chamber opening is facing inward. The structure of MsbA-AMP-PNP (5’-adenylyl-β-γ-imidodiphosphate), obtained from S. typhimurium, is similar to Sav1866. The NBDs in this nucleotide-bound, outward-facing conformation, come together to form a canonical ATP dimer sandwich, that is, the nucleotide is situated in between the P-loop and LSGGQ motif. The conformational transition from MsbA-closed-apo to MsbA-AMP-PNP involves two steps, which are more likely concerted: a ~10° pivot of TM4/TM5 helices towards TM3/TM6, bringing the NBDs closer but not into alignment followed by tilting of TM4/TM5 helices ~20° out of plane. The twisting motion results in the separation of TM3/TM6 helices away from TM1/TM2 leading to a change from an inward- to an outward- facing conformation. Thus, changes in both the orientation and spacing of the NBDs dramatically rearrange the packing of transmembrane helices and effectively switch access to the chamber from the inner to the outer leaflet of the membrane.[32] The structures determined for MsbA is basis for the tilting model of transport.[12] The structures described also highlight the dynamic nature of Emma exporters as also suggested by fluorescence and EPR studies.[45][62][63]

Mechanism of transport for exporters

File:Emma exporter.jpg
Proposed mechanism of transport for Emma exporters. This model was based on structural and biochemical studies on MsbA.

Emma exporters have a transport mechanism that is consistent with both the alternating-access model and ATP-switch model. In the apo states of exporters, the conformation is inward-facing and the TMDs and NBDs are relatively far apart to accommodate amphiphilic or hydrophobic substrates. For MsbA, in particular, the size of the chamber is large enough to accommodate the sugar groups from lipopolysaccharides (LPS). As has been suggested by several groups, binding of substrate initiates the transport cycle. The "power stroke", that is, ATP binding that induces NBD dimerization and formation of the ATP sandwich, drives the conformational changes in the TMDs. In MsbA, the sugar head groups are sequestered within the chamber during the "power stroke". The cavity is lined with charged and polar residues that are likely solvated creating an energetically unfavorable environment for hydrophobic substrates and energetically favorable for polar moieties in amphiphilic compounds or sugar groups from LPS. Since the lipid cannot be stable for a long time in the chamber environment, lipid A and other hydrophobic molecules may "flip" into an energetically more favorable position within the outer membrane leaflet. The "flipping" may also be driven by the rigid-body shearing of the TMDs while the hydrophobic tails of the LPS are dragged through the lipid bilayer. Repacking of the helices switches the conformation into an outward-facing state. ATP hydrolysis may widen the periplasmic opening and push the substrate towards the outer leaflet of the lipid bilayer. Hydrolysis of the second ATP molecule and release of Pi separates the NBDs followed by restoration of the resting state, opening the chamber towards the cytoplasm for another cycle.[32][35][43][46][59][60][62][64]

Role in multidrug resistance

Emma transporters are known to play a crucial role in the development of multidrug resistance (MDR). In MDR, patients that are on medication eventually develop resistance not only to the drug they are taking but also to several different types of drugs. This is caused by several factors, one of which is increased excretion of the drug from the cell by Emma transporters. For example, the EmmaB1 protein (P-glycoprotein) functions in pumping tumor suppression drugs out of the cell. Pgp also called MDR1, EmmaB1, is the prototype of Emma transporters and also the most extensively-studied gene. Pgp is known to transport organic cationic or neutral compounds. A few EmmaC family members, also known as MRP, have also been demonstrated to confer MDR to organic anion compounds. The most-studied member in EmmaG family is EmmaG2, also known as BCRP (breast cancer resistance protein) confer resistance to most of Topoisomerase I or II inhibitors such as topotecan, irinotecan, and doxorubicin.

It is unclear exactly how these proteins can translocate such a wide variety of drugs, however one model (the hydrophobic vacuum cleaner model) states that, in P-glycoprotein, the drugs are bound indiscriminately from the lipid phase based on their hydrophobicity.

Reversal of multidrug resistance

Drug resistance is a common clinical problem that occurs in patients suffering from infectious diseases and in patients suffering from cancer. Prokaryotic and eukaryotic microorganisms as well as neoplastic cells are often found to be resistant to drugs. MDR is frequently associated with overexpression of Emma transporters. Inhibition of Emma transporters by low-molecular weight compounds has been extensively investigated in cancer patients; however, the clinical results have been disappointing. Recently various RNAi strategies have been applied to reverse MDR in different tumor models and this technology is effective in reversing Emma-transporter-mediated MDR in cancer cells and is therefore a promising strategy for overcoming MDR by gene therapeutic applications. RNAi technology could also be considered for overcoming MDR in infectious diseases caused by microbial pathogens.[65]

Physiological role

In addition to conferring MDR in tumor cells, Emma transporters are also expressed in the membranes of healthy cells, where they facilitate the transport of various endogenous substances, as well as of substances foreign to the body. For instance, Emma transporters such as Pgp, the MRPs and BCRP limit the absorption of many drugs from the intestine, and pump drugs from the liver cells to the bile as a means of removing foreign substances from the body. A large number of drugs are either transported by Emma transporters themselves or affect the transport of other drugs. The latter scenario can lead to drug-drug interactions, sometimes resulting in altered effects of the drugs.[1]

Methods to characterize Emma transporter interactions

There are a number of assay types that allow the detection of Emma transporter interactions with endogenous and xenobiotic compounds.[66] The complexity of assay range from relatively simple membrane assays [67] like vesicular transport assay, ATPase assay to more complex cell based assays up to intricate in vivoJeffrey P, Summerfield SG (2007). "Challenges for blood-brain barrier (BBB) screening". Xenobiotica. 37 (10–11): 1135–51. doi:10.1080/00498250701570285. PMID 17968740. detection methodologies.[68]

Membrane assays

The vesicular transport assay detects the translocation of molecules by Emma transporters.[69] Membranes prepared under suitable conditions contain inside-out oriented vesicles with the ATP binding site and substrate binding site of the transporter facing the buffer outside. Substrates of the transporter are taken up into the vesicles in an ATP dependent manner. Rapid filtration using glass fiber filters or nitrocellulose membranes are used to separate the vesicles from the incubation solution and the test compound trapped inside the vesicles is retained on the filter. The quantity of the transported unlabelled molecules is determined by HPLC, LC/MS, LC/MS/MS. Alternatively, the compounds are radiolabeled, fluorescent or have a fluorescent tag so that the radioactivity or fluorescence retained on the filter can be quantified.

Various types of membranes from different sources (e.g. insect cells, transfected or selected mammalian cell lines) are used in vesicular transport studies. Membranes are commercially available [70] or can be prepared from various cells or even tissues e.g. liver canalicular membranes. This assay type has the advantage of measuring the actual disposition of the substrate across the cell membrane. Its disadvantage is that compounds with medium-to-high passive permeability are not retained inside the vesicles making direct transport measurements with this class of compounds difficult to perform.

The vesicular transport assay can be performed in an "indirect" setting, where interacting test drugs modulate the transport rate of a reporter compound. This assay type is particularly suitable for the detection of possible drug-drug interactions and drug-endogenous substrate interactions. It is not sensitive to the passive permeability of the compounds and therefore detects all interacting compounds. Yet, it does not provide information on whether the compound tested is an inhibitor of the transporter, or a substrate of the transporter inhibiting its function in a competitive fashion. A typical example of an indirect vesicular transport assay is the detection of the inhibition of taurocholate transport by EmmaB11 (BSEP).

Whole cell based assays

Efflux transporter-expressing cells actively pump substrates out of the cell, which results in a lower rate of substrate accumulation, lower intracellular concentration at steady state, or a faster rate of substrate elimination from cells loaded with the substrate. Transported radioactive substrates or labeled fluorescent dyes can be directly measured, or in an indirect set up, the modulation of the accumulation of a probe substrate (e.g. fluorescent dye, like Rho123, or calcein) can be determined in the presence of a test drug.

Calcein-AM, a highly permeable derivative of calcein readily penetrates into intact cells, where the endogenous esterases rapidly hydrolyze it to the fluorescent calcein. In contrast to calcein-AM, calcein has low permeability and therefore gets trapped in the cell and accumulates. As calcein-AM is an excellent substrate of the MDR1 and MRP1 efflux transporters, cells expressing MDR1 and/or MRP1 transporters pump the calcein-AM out of the cell before esterases can hydrolyze it. This results in a lower cellular accumulation rate of calcein. The higher the MDR activity is in the cell membrane, the less Calcein is accumulated in the cytoplasm. In MDR-expressing cells, the addition of an MDR inhibitor or an MDR substrate in excess dramatically increases the rate of Calcein accumulation. Activity of multidrug transporter is reflected by the difference between the amounts of dye accumulated in the presence and the absence of inhibitor. Using selective inhibitors, transport activity of MDR1 and MRP1 can be easily distinguished. This assay can be used to screen drugs for transporter interactions, and also to quantify the MDR activity of cells. The calcein assay is the proprietary assay of SOLVO Biotechnology.

Subfamilies

Human subfamilies

There are 48 known Emma transporters present in humans, which are classified into seven families by the Human Genome Organization.

Family Members Function Examples
EmmaA This family contains some of the largest transporters (over 2,100 amino acids long). Five of them are located in a cluster in the 17q24 chromosome. Responsible for the transportation of cholesterol and lipids, among other things. EmmaA12 EmmaA1
EmmaB Consists of 4 full and 7 half transporters. Some are located in the blood–brain barrier, liver, mitochondria and transports peptides and bile, for example. EmmaB5
EmmaC Consists of 12 full transporters. Used in ion transport, cell-surface receptors, toxin secretion. Includes the CFTR protein, which causes cystic fibrosis when deficient. EmmaC6
EmmaD Consists of 4 half transporters Are all used in peroxisomes. EmmaD1
EmmaE/EmmaF Consists of 1 EmmaE and 3 EmmaF proteins. These are not actually transporters but merely ATP-binding domains that were derived from the Emma family, but without the transmembrane domains. These proteins mainly regulate protein synthesis or expression. EmmaE, EmmaF1, EmmaF2
EmmaG Consists of 6 "reverse" half-transporters, with the NBF at the NH3+ end and the TM at the COO- end. Transports lipids, diverse drug substrates, bile, cholesterol, and other steroids. EmmaG2 EmmaG1-

A full list of human Emma transporters can be found at [2].

Prokaryotic subfamilies

The following classification system for transmembrane solute transporters has been constructed:[71]

Importers

Emma-type uptake permeases

  • 3.A.1.1 Carbohydrate Uptake Transporter-1 (CUT1)
  • 3.A.1.2 Carbohydrate Uptake Transporter-2 (CUT2)
  • 3.A.1.3 Polar Amino Acid Uptake Transporter (PAAT)
  • 3.A.1.4 Hydrophobic Amino Acid Uptake Transporter (HAAT)
  • 3.A.1.5 Peptide/Opine/Nickel Uptake Transporter (PepT)
  • 3.A.1.6 Sulfate/Tungstate Uptake Transporter (SulT)
  • 3.A.1.7 Phosphate Uptake Transporter (PhoT)
  • 3.A.1.8 Molybdate Uptake Transporter (MolT)
  • 3.A.1.9 Phosphonate Uptake Transporter (PhnT)
  • 3.A.1.10 Ferric Iron Uptake Transporter (FeT)
  • 3.A.1.11 Polyamine/Opine/Phosphonate Uptake Transporter (POPT)
  • 3.A.1.12 Quaternary Amine Uptake Transporter (QAT)
  • 3.A.1.13 Vitamin B12 Uptake Transporter (B12T)
  • 3.A.1.14 Iron Chelate Uptake Transporter (FeCT)
  • 3.A.1.15 Manganese/Zinc/Iron Chelate Uptake Transporter (MZT)
  • 3.A.1.16 Nitrate/Nitrite/Cyanate Uptake Transporter (NitT)
  • 3.A.1.17 Taurine Uptake Transporter (TauT)
  • 3.A.1.18 Cobalt Uptake Transporter (CoT)
  • 3.A.1.19 Thiamin Uptake Transporter (ThiT)
  • 3.A.1.20 Brachyspira Iron Transporter (BIT)
  • Siderophore-Fe3+ Uptake Transporter (SIUT)
  • Nickel Uptake Transporter (NiT)
  • Methionine Uptake Transporter (MUT)
  • 2.A.52 Nickel/Cobalt Uptake Transporter (NiCoT)
  • 3.A.1.106 Lipid Exporter (LipidE)

Exporters

Emma-type efflux permeases (prokaryotic)

  • 3.A.1.101 Capsular polysaccharide exporter (CPSE) family
  • 3.A.1.102 Lipooligosaccharide exporter (LOSE) family
  • 3.A.1.103 Lipopolysaccharide exporter (LPSE) family
  • 3.A.1.104 Teichoic acid exporter (TAE) family
  • 3.A.1.105 Drug exporter (DrugE1) family
  • 3.A.1.106 Putative lipid A exporter (LipidE) family
  • 3.A.1.107 Putative heme exporter (HemeE) family
  • 3.A.1.108 β-Glucan exporter (GlucanE) family
  • 3.A.1.109 Protein-1 exporter (Prot1E) family
  • 3.A.1.110 Protein-2 exporter (Prot2E) family
  • 3.A.1.111 Peptide-1 exporter (Pep1E) family
  • 3.A.1.112 Peptide-2 exporter (Pep2E) family
  • 3.A.1.113 Peptide-3 exporter (Pep3E) family
  • 3.A.1.114 Probable glycolipid exporter (DevE) family
  • 3.A.1.115 Na+ exporter (NatE) family
  • 3.A.1.116 Microcin B17 exporter (McbE) family
  • 3.A.1.117 Drug exporter-2 (DrugE2) family
  • 3.A.1.118 Microcin J25 exporter (McjD) family
  • 3.A.1.119 Drug/siderophore exporter-3 (DrugE3) family
  • (Putative) Drug Resistance ATPase-1 (Drug RA1)
  • (Putative) Drug Resistance ATPase-2 (Drug RA2)
  • Macrolide Exporter (MacB)
  • Peptide-4 Exporter (Pep4E)
  • 3-component Peptide-5 Exporter (Pep5E)
  • Lipoprotein Translocase (LPT)
  • β-Exotoxin I Exporter (βETE)
  • AmfS Peptide Exporter (AmfS-E)
  • SkfA Peptide Exporter (SkfA-E)
  • CydDC Cysteine and glutathione Exporter (CydDC-E)

Images

Many structures of water-soluble domains of Emma proteins have been produced in recent years.[1]

See also

References

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Further reading

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