ATP-binding cassette transporter genes

ATP-binding cassette
Gene cassette
A gene cassette is broadly a modular DNA sequence encoding one or more genes for a single biochemical function.In genetic engineering, a gene cassette refers to a manipulable fragment of DNA carrying, and capable of expressing, one or more genes of interest between one or more sets of restriction...

transporters (ABC-transporter) are members of a protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

superfamily that is one of the largest and most ancient families with representatives in all extant phyla

Phylum

In biology, a phylum The term was coined by Georges Cuvier from Greek φῦλον phylon, "race, stock," related to φυλή phyle, "tribe, clan." is a taxonomic rank below kingdom and above class. "Phylum" is equivalent to the botanical term division....

from prokaryote

Prokaryote

The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...

s to humans. ABC transporters are transmembrane protein

Transmembrane protein

A transmembrane protein is a protein that goes from one side of a membrane through to the other side of the membrane. Many TPs function as gateways or "loading docks" to deny or permit the transport of specific substances across the biological membrane, to get into the cell, or out of the cell as...

s that utilize the energy of adenosine triphosphate

Adenosine triphosphate

Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

(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. They transport a wide variety of substrates across extra- and intracellular membranes

Cell membrane

The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...

, including metabolic products, lipids and sterols, and drugs

Medication

A pharmaceutical drug, also referred to as medicine, medication or medicament, can be loosely defined as any chemical substance intended for use in the medical diagnosis, cure, treatment, or prevention of disease.- Classification :...

. Proteins are classified as ABC transporters based on the sequence and organization of their ATP-binding cassette (ABC) domain(s). ABC transporters are involved in tumour resistance, cystic fibrosis

Cystic fibrosis

Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...

, bacterial multidrug resistance, and a range of other inherited human diseases.

Function

ABC transporters utilize the energy of ATP hydrolysis to transport various substrates across cellular membranes

Cell membrane

. 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 ABC transporter protects hydrophilic substrates from the lipids of the membrane bilayer

Lipid bilayer

The lipid bilayer is a thin membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around cells. The cell membrane of almost all living organisms and many viruses are made of a lipid bilayer, as are the membranes surrounding the cell nucleus...

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

Cytoplasm

The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...

to the periplasm. The third subgroup of ABC proteins do not function as transporters, but are rather involved in translation and DNA repair processes.

Prokaryotic ABC proteins

Bacterial ABC transporters are essential in cell viability, virulence

Virulence

Virulence is by MeSH definition the degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenicity of an organism - its ability to cause disease - is determined by its...

, and pathogenicity. Iron ABC uptake systems, for example, are important effectors of virulence. Pathogens use siderophores to scavenge iron that is in complex with high-affinity iron-binding proteins or erythrocytes. These are high-affinity iron-complexing molecules that are secreted by bacteria and reabsorb iron into iron-siderophore complexes. The chvE-gguAB gene

Gene

A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

in Agrobacterium tumefaciens encodes glucose

Glucose

Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...

and galactose

Galactose

Galactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....

importers that are also associated with virulence. 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

Osmosis

Osmosis is the movement of solvent molecules through a selectively permeable membrane into a region of higher solute concentration, aiming to equalize the solute concentrations on the two sides...

strength is counterbalanced by activation of osmosensing ABC transporters that mediate uptake of solutes. Other than functioning in transport, some bacterial ABC proteins are also involved in the regulation of several physiological processes.

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

Teichoic acid

Teichoic acids are bacterial polysaccharides of glycerol phosphate or ribitol phosphate linked via phosphodiester bonds.-Location and structure:...

), proteins involved in bacterial pathogenesis (e.g. hemolysis

Hemolysis

Hemolysis —from the Greek meaning "blood" and meaning a "loosing", "setting free" or "releasing"—is the rupturing of erythrocytes and the release of their contents into surrounding fluid...

, heme

Heme

A heme or haem is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. Not all porphyrins contain iron, but a substantial fraction of porphyrin-containing metalloproteins have heme as their prosthetic group; these are...

-binding protein, and alkaline protease

Protease

A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....

), heme, hydrolytic enzymes, S-layer proteins, competence factors, toxins, lantibiotics

Lantibiotics

Lantibiotics are a class of peptide antibiotics that contain the characteristic polycyclic thioether amino acids lanthionine or methyllanthionine, as well as the unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid....

, bacteriocins, peptide antibiotics, drugs and siderophores. They also play important roles in biosynthetic pathways, including extracellular polysaccharide biosynthesis and cytochrome

Cytochrome

Cytochromes are, in general, membrane-bound hemoproteins that contain heme groups and carry out electron transport.They are found either as monomeric proteins or as subunits of bigger enzymatic complexes that catalyze redox reactions....

biogenesis.

Eukaryotic ABC proteins

Although most eukaryotic ABC transporters are effluxers, some are not directly involved in transporting substrates. In the cystic fibrosis

Cystic fibrosis

Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...

transmembrane regulator (CFTR

Cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator is a protein that in humans is encoded by the CFTR gene.CFTR is a ABC transporter-class ion channel that transports chloride and thiocyanate ions across epithelial cell membranes...

) and in the sulfonylurea

Sulfonylurea

Sulfonylurea derivatives are a class of antidiabetic drugs that are used in the management of diabetes mellitus type 2. They act by increasing insulin release from the beta cells in the pancreas.-First generation:* Carbutamide...

receptor (SUR), ATP hydrolysis is associated with the regulation of opening and closing of ion channels carried by the ABC protein itself or other proteins.

Human ABC transporters are involved in several diseases that arise from polymorphisms

Polymorphism (biology)

Polymorphism in biology occurs when two or more clearly different phenotypes exist in the same population of a species — in other words, the occurrence of more than one form or morph...

in ABC genes and rarely due to complete loss of function of single ABC proteins. Such diseases include Mendelian diseases and complex genetic disorders such as cystic fibrosis, adrenoleukodystrophy

Adrenoleukodystrophy

Adrenoleukodystrophy is a rare, inherited disorder that leads to progressive brain damage, failure of the adrenal glands and eventually death. ALD is a disease in a group of genetic disorders called leukodystrophies, whose chief feature is damage to myelin...

, Stargardt disease, Tangier disease

Tangier disease

Tangier disease is a rare inherited disorder characterized by a severe reduction in the amount of high density lipoprotein , often referred to as "good cholesterol," in the bloodstream.-Diagnosis:...

, immune deficiencies, progressive familial intraheptic cholestasis

Cholestasis

In medicine, cholestasis is a condition where bile cannot flow from the liver to the duodenum. The two basic distinctions are an obstructive type of cholestasis where there is a mechanical blockage in the duct system such as can occur from a gallstone or malignancy and metabolic types of...

, Dubin-Johnson syndrome

Dubin-Johnson syndrome

Dubin–Johnson syndrome is an autosomal recessive disorder that causes an increase of conjugated bilirubin in the serum without elevation of liver enzymes . This condition is associated with a defect in the ability of hepatocytes to secrete conjugated bilirubin into the bile, and is similar to...

, Pseudoxanthoma elasticum

Pseudoxanthoma elasticum

Pseudoxanthoma elasticum , also known as Grönblad–Strandberg syndrome, is a genetic disease that causes fragmentation and mineralization of elastic fibers in some tissues. The most common problems arise in the skin and eyes, and later in blood vessels in the form of premature atherosclerosis...

, persistent hyperinsulinemic hypoglycemia

Hyperinsulinemic hypoglycemia

Hyperinsulinemic hypoglycemia describes the condition and effects of low blood glucose caused by excessive insulin. Hypoglycemia due to excess insulin is the most common type of serious hypoglycemia. It can be due to endogenous or injected insulin.-Causes:...

of infancy due to focal adenomatous hyperplasia

Hyperplasia

Hyperplasia means increase in number of cells/proliferation of cells. It may result in the gross enlargement of an organ and the term is sometimes mixed with benign neoplasia/ benign tumor....

, X-linked sideroblastosis and anemia

Sideroblastic anemia

Sideroblastic anemia or sideroachrestic anemia is a disease in which the bone marrow produces ringed sideroblasts rather than healthy red blood cells . It may be caused either by a genetic disorder or indirectly as part of myelodysplastic syndrome, which can evolve into hematological malignancies...

, age-related macular degeneration

Macular degeneration

Age-related macular degeneration is a medical condition which usually affects older adults and results in a loss of vision in the center of the visual field because of damage to the retina. It occurs in “dry” and “wet” forms. It is a major cause of blindness and visual impairment in older adults...

, familial hypoapoproteinemia, Retinitis pigmentosum, cone rod dystrophy

Corneal dystrophy

Corneal dystrophy is a group of disorders, characterised by a noninflammatory, inherited, bilateral opacity of the transparent front part of the eye called the cornea.The distrophies could be subdivided based on specie affected:...

, and others. The human ABCB (MDR/TAP) family is responsible for multiple drug resistance (MDR) against a variety of structurally unrelated drugs. ABCB1 or MDR1 P-glycoprotein

P-glycoprotein

P-glycoprotein 1 also known as multidrug resistance protein 1 or ATP-binding cassette sub-family B member 1 or cluster of differentiation 243 is a glycoprotein that in humans is encoded by the ABCB1 gene...

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

Aldosterone

Aldosterone is a hormone that increases the reabsorption of sodium ions and water and the release of potassium in the collecting ducts and distal convoluted tubule of the kidneys' functional unit, the nephron. This increases blood volume and, therefore, increases blood pressure. Drugs that...

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 ABCB1 mediates transport of cortisol

Cortisol

Cortisol is a steroid hormone, more specifically a glucocorticoid, produced by the adrenal gland. It is released in response to stress and a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis; suppress the immune system; and aid in fat,...

and dexamethasone

Dexamethasone

Dexamethasone is a potent synthetic member of the glucocorticoid class of steroid drugs. It acts as an anti-inflammatory and immunosuppressant...

, but not of progesterone

Progesterone

Progesterone also known as P4 is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy and embryogenesis of humans and other species...

in ABCB1 transfected cells. MDR1 can also transport cholesterol

Cholesterol

Cholesterol is a complex isoprenoid. Specifically, it is a waxy steroid of fat that is produced in the liver or intestines. It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals. It is an essential structural component of mammalian cell membranes...

, short-chain and long-chain analogs of phosphatidylcholine

Phosphatidylcholine

Phosphatidylcholines are a class of phospholipids that incorporate choline as a headgroup.They are a major component of biological membranes and can be easily obtained from a variety of readily available sources such as egg yolk or soy beans from which they are mechanically extracted or chemically...

(PC), phosphatidylethanolamine

Phosphatidylethanolamine

Phosphatidylethanolamine is a lipid found in biological membranes. It is synthesized by the addition of CDP-ethanolamine to diglyceride, releasing CMP. S-adenosyl methionine can subsequently methylate the amine of phosphatidyl ethanolamine to yield phosphatidyl choline.Cephalin is a phospholipid,...

(PE), phosphatidylserine

Phosphatidylserine

Phosphatidylserine is a phospholipid component, usually kept on the inner-leaflet of cell membranes by an enzyme called flippase...

(PS), sphingomyelin

Sphingomyelin

Sphingomyelin is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphorylcholine and ceramide...

(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.

More recently, ABC-transporters have been shown to exist within the placenta

Placenta

The placenta is an organ that connects the developing fetus to the uterine wall to allow nutrient uptake, waste elimination, and gas exchange via the mother's blood supply. "True" placentas are a defining characteristic of eutherian or "placental" mammals, but are also found in some snakes and...

, indicating they could play a protective role for the developing fetus against xenobiotic

Xenobiotic

A xenobiotic is a chemical which is found in an organism but which is not normally produced or expected to be present in it. It can also cover substances which are present in much higher concentrations than are usual...

Structure

The common feature of all ABC transporters is that they consist of two distinct domains, the transmembrane domain (TMD) and the nucleotide-binding domain (NBD)
ATP-binding domain of ABC transporters
In molecular biology, ATP-binding domain of ABC transporters is a water-soluble domain of transmembrane ABC transporters.ABC transporters belong to the ATP-Binding Cassette superfamily, which uses the hydrolysis of ATP to translocate a variety of compounds across biological membranes...

. 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 (ABC) domain, on the other hand, is located in the cytoplasm and has a highly conserved sequence. The NBD is the site for ATP binding. In most exporters, the N-terminal transmembrane domain and the C-terminal ABC 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 ABC domain is N-terminal whereas the TMD is C-terminal, such as in the E. coli MacB protein responsible for macrolide

Macrolide

The macrolides are a group of drugs whose activity stems from the presence of a macrolide ring, a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered...

resistance.

The structural architecture of ABC transporters consists minimally of two TMDs and two ABCs. 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 importer involved in the uptake of vitamin B₁₂

Vitamin B12

Vitamin B12, vitamin B12 or vitamin B-12, also called cobalamin, is a water-soluble vitamin with a key role in the normal functioning of the brain and nervous system, and for the formation of blood. It is one of the eight B vitamins...

. Most exporters, such as in the multidrug exporter Sav1866 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 ABC 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 ABC transporter. Exporters do not have the binding protein but have an intracellular domain (ICD) that joins the membrane-spanning helices and the ABC domain. The ICD is believed to be responsible for communication between the TMD and NBD.

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 ABC importer, type II ABC importer and ABC exporter folds. The classification of importer folds is based on detailed characterization of the sequences. The type I ABC importer fold was originally observed in the ModB TM subunit of the molybdate

Molybdate

In chemistry a molybdate is a compound containing an oxoanion with molybdenum in its highest oxidation state of 6. Molybdenum can form a very large range of such oxoanions which can be discrete structures or polymeric extended structures, although the latter are only found in the solid state.The...

transporter. This diagnostic fold can also be found in the MalF and MalG TM subunits of MalFGK₂ and the Met transporter MetI. 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 ABC importer fold is observed in the twenty TM helix-domain of BtuCD and in Hi1471, 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 ABC 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.

Nucleotide-binding domain (NBD)

The ABC domain consists of two domains, the catalytic core domain similar to RecA

RecA

RecA is a 38 kilodalton Escherichia coli protein essential for the repair and maintenance of DNA. RecA has a structural and functional homolog in every species in which it has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins...

-like motor ATPases and a smaller, structurally diverse α-helical subdomain that is unique to ABC transporters. The larger domain typically consists of two β-sheets and six α helices, where the catalytic Walker A motif
Walker motifs
The Walker A and Walker B motifs are protein sequence motifs. These were first reported in ATP-binding proteins by Walker and co-workers in 1982.-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 ABC signature motif, also known as LSGGQ motif, linker peptide or C motif. The ABC domain also has a glutamine residue residing in a flexible loop called Q loop, lid or γ-phosphate switch, that connects the TMD and ABC. The Q loop is presumed to be involved in the interaction of the NBD and TMD, particularly in the coupling of nucleotide hydrolysis

Hydrolysis

Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

to the conformational changes of the TMD during substrate translocation. The H motif or switch region contains a highly conserved histidine

Histidine

Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...

residue that is also important in the interaction of the ABC 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.

ATP binding and hydrolysis

Dimer formation of the two ABC domains of transporters requires ATP binding. It is generally observed that the ATP bound state is associated with the most extensive interface between ABC domains, whereas the structures of nucleotide-free transporters exhibit conformations with greater separations between the ABC domains. Structures of the ATP-bound state of isolated NBDs have been reported for importers including HisP, GlcV, MJ1267, E. coli MalK (E.c.MalK), T. litoralis MalK (TlMalK), and exporters such as TAP, HlyB, MJ0796, Sav1866, and MsbA. In these transporters, ATP is bound to the ABC 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. This was first observed in Rad50 and reported in structures of MJ0796, the NBD subunit of the LolD transporter from Methanococcus jannaschii and E.c.MalK of a maltose transporter. 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

Catalysis

Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....

.

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. 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,(2) hydrogen-bonds between a conserved lysine

Lysine

Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....

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, and (3) γ-phosphate coordination with side chain of serine

Serine

Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...

and backbone amide

Amide

In chemistry, an amide is an organic compound that contains the functional group consisting of a carbonyl group linked to a nitrogen atom . The term refers both to a class of compounds and a functional group within those compounds. The term amide also refers to deprotonated form of ammonia or an...

groups of glycine

Glycine

Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...

residues in the LSGGQ motif. 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.

The enzymatic hydrolysis of ATP requires proper binding of the phosphates and positioning of the γ-phosphate to the attacking water. 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 and coordinate with Mg²⁺. This Mg²⁺ ion also coordinates with the terminal aspartate residue in the Walker B motif through the attacking H₂O. A general base, which may be the glutamate residue adjacent to the Walker B motif, glutamine

Glutamine

Glutamine is one of the 20 amino acids encoded by the standard genetic code. It is not recognized as an essential amino acid but may become conditionally essential in certain situations, including intensive athletic training or certain gastrointestinal disorders...

in the Q-loop, 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. The precise molecular mechanism of ATP hydrolysis is still controversial.

Mechanism of transport

ABC transporters are active transport

Active transport

Active transport is the movement of a substance against its concentration gradient . In all cells, this is usually concerned with accumulating high concentrations of molecules that the cell needs, such as ions, glucose, and amino acids. If the process uses chemical energy, such as from adenosine...

ers, 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. Both ABC 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. 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

Phosphate

A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...

(P_i) and adenosine diphosphate

Adenosine diphosphate

Adenosine diphosphate, abbreviated ADP, is a nucleoside diphosphate. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine....

(ADP). Switching between the open and closed dimer conformations induces conformational changes in the TMD resulting in substrate translocation.

The general mechanism for the transport cycle of ABC 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 ABC transporters has the NBDs in an open dimer configuration, with low affinity for ATP but high affinity for substrate binding site. 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 P_i 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.

Several groups studying ABC 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 possibly involved in different steps in the transport cycle. 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 suggesting a concurrent function of the two NBDs in the transport cycle.

Some evidences to show that ATP binding is indeed the power stroke of the transport cycle were reported. It has been shown that ATP binding induces changes in the substrate-binding properties of the TMDs. The affinity of ABC 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

Vinblastine

Vinblastine is an antimicrotubule drug used to treat certain kinds of cancer, including Hodgkin's lymphoma, non-small cell lung cancer, breast cancer, head and neck cancer, and testicular cancer. It is also used to treat Langerhan cell histiocytosis....

binding to permease

Permease

The permeases are membrane transport proteins, a class of multipass transmembrane proteins that facilitate the diffusion of a specific molecule in or out of the cell by passive transport...

-glycoprotein (P-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. Also, ATP binding induces substantial conformational changes in the TMDs. Spectroscopic

Spectroscopy

Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...

, protease

Protease

accessibility and crosslinking studies have shown that ATP binding to the NBDs induces conformational changes in multidrug resistance-associated protein-1 (MRP1), HisPMQ, LmrA, and Pgp. 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. 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 ABC 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.

ABC importers

Most ABC 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

Gram-positive

Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain and appearing red or pink...

microorganisms lack a periplasm such that their binding protein is often a lipoprotein

Lipoprotein

A lipoprotein is a biochemical assembly that contains both proteins and lipids water-bound to the proteins. Many enzymes, transporters, structural proteins, antigens, adhesins, and toxins are lipoproteins...

bound to the external face of the cell membrane

Cell membrane

. Some gram-positive bacteria have BPs fused to the transmembrane domain of the transporter itself. The first successful x-ray crystal

X-ray crystallography

X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...

structure of an intact ABC importer is the molybdenum

Molybdenum

Molybdenum , is a Group 6 chemical element with the symbol Mo and atomic number 42. The name is from Neo-Latin Molybdaenum, from Ancient Greek , meaning lead, itself proposed as a loanword from Anatolian Luvian and Lydian languages, since its ores were confused with lead ores...

transporter (ModBC-A) from Archaeoglobus fulgidus. Atomic-resolution structures of three other bacterial importers, E. coli BtuCD, E. coli maltose

Maltose

Maltose , or malt sugar, is a disaccharide formed from two units of glucose joined with an αbond, formed from a condensation reaction. The isomer "isomaltose" has two glucose molecules linked through an α bond. Maltose is the second member of an important biochemical series of glucose chains....

transporter (MalFGK₂-E), and the putative metal-chelate transporter of Haemophilus influenza, HI1470/1, have also been determined. The structures provided detailed pictures of the interaction of the transmembrane and ABC 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 ABC subdomains and lies approximately parallel to the membrane bilayer.

Large ABC importers

The BtuCD and HI1470/1 are classified as large ABC 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 ABC. In the absence of nucleotide, the two ABC 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 ABC 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.

Small ABC importers

Structures of the ModBC-A and MalFGK₂-E, which are in complex with their binding protein, correspond to small ABC 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 ABC 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

Transition state

The transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...

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 which is closed toward the cytoplasm but with an opening that faces outward. The structure suggests a possibility that MBP may stimulate the ATPase

ATPase

ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...

activity of the transporter upon binding.

Mechanism of transport for importers

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 P_i 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.

ABC exporters

Prokaryotic ABC 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

Hydrolase

In biochemistry, a hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond. For example, an enzyme that catalyzed the following reaction is a hydrolase:-Nomenclature:...

, S-layer proteins, lantibiotics

Lantibiotics

, bacteriocins, and competence factors) export and the other in drug efflux. ABC 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.

In gram-negative organisms, ABC 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 ABC exporter, a membrane fusion protein
Membrane fusion protein
Membrane fusion proteins are membrane proteins which cause more than one membrane to combine.Examples include:* SNARE , such as VAMP* Gp41...

(MFP), and an outer membrane factor (OMF). An example is the secretion of hemolysin

Hemolysin

Hemolysins are exotoxins produced by bacteria that cause lysis of red blood cells in vitro. Visualization of hemolysis of red blood cells in agar plates facilitates the categorization of some pathogenic bacteria such as Streptococcus and Staphylococcus...

(HlyA) from E. coli where the inner membrane ABC 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.

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

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. P-glycoprotein is the best-studied efflux pump and as such has offered important insights into the mechanism of bacterial pumps. Although some exporters transport a specific type of substrate, most transporters extrude a diverse class of drugs with varying structure. These transporters are commonly called multi-drug resistant (MDR) ABC transporters and sometimes referred to as “hydrophobic vacuum cleaners”.

Human ABCB1/MDR1 P-glycoprotein

P-glycoprotein is a well-studied protein associated with multi-drug resistance. It belongs to the human ABCB (MDR/TAP) family and is also known as ABCB1 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 ABCB1 monomer was obtained in the presence and absence of nucleotide using electron cryo crystallography

Electron crystallography

Electron crystallography is a method to determine the arrangement of atoms in solids using a transmission electron microscope .- Comparison with X-ray 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.

Sav1866

The first high-resolution structure reported for an ABC exporter was that of Sav1866 from Staphylococcus aureus. Sav1866 is a homolog of multidrug ABC transporters. It shows significant sequence similarity to human ABC 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

Doxorubicin

Doxorubicin INN is a drug used in cancer chemotherapy. It is an anthracycline antibiotic, closely related to the natural product daunomycin, and like all anthracyclines, it works by intercalating DNA....

, vinblastine

Vinblastine

and others, 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 ABC 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.

MsbA

MsbA is a multi-drug resistant (MDR) ABC transporter and possibly a lipid flippase

Flippase

Flippases are a family of transmembrane lipid transporter enzymes located in the membrane responsible for aiding the movement of phospholipid molecules between the two leaflets that compose a cell's membrane...

. It is an ATPase

ATPase

that transports lipid A

Lipid A

Lipid A is a lipid component of an endotoxin held responsible for toxicity of Gram-negative bacteria. It is the innermost of the three regions of the lipopolysaccharide molecule, and its hydrophobic nature allows it to anchor the LPS to the outer membrane...

, the hydrophobic moiety of lipopolysaccharide

Lipopolysaccharide

Lipopolysaccharides , also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals.-Functions:LPS is the major...

(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

Endotoxin

Endotoxins are toxins associated with some Gram-negative bacteria. An "endotoxin" is a toxin that is a structural molecule of the bacteria that is recognized by the immune system.-Gram negative:...

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-ABC transporter LmrA from Lactococcus lactis. 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.

Previously published X-ray structures of MsbA were inconsistent with the bacterial homolog Sav1866. 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. 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 and EPR spectroscopy studies. 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. The structures determined for MsbA is basis for the tilting model of transport. The structures described also highlight the dynamic nature of ABC exporters as also suggested by fluorescence

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

and EPR studies.

Mechanism of transport for exporters

ABC 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 P_i separates the NBDs followed by restoration of the resting state, opening the chamber towards the cytoplasm for another cycle.

Role in multidrug resistance

ABC transporters are known to play a crucial role in the development of multidrug resistance

Multidrug resistance

Multiple drug resistance or Multidrug resistance is a condition enabling a disease-causing organism to resist distinct drugs or chemicals of a wide variety of structure and function targeted at eradicating the organism...

(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 ABC transporters. For example, the ABCB1 protein (P-glycoprotein

P-glycoprotein

) functions in pumping tumor suppression drugs out of the cell. Pgp also called MDR1, ABCB1, is the prototype of ABC transporters and also the most extensively-studied gene. Pgp is known to transport organic cationic or neutral compounds. A few ABCC family members, also known as MRP, have also been demonstrated to confer MDR to organic anion compounds. The most-studied member in ABCG family is ABCG2, 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 ABC transporters. Inhibition of ABC transporters by low-molecular weight compounds has been extensively investigated in cancer patients; however, the clinical results have been disappointing. Recently various RNAi

RNAI

RNAI is a non-coding RNA that is an antisense repressor of the replication of some E. coli plasmids, including ColE1. Plasmid replication is usually initiated by RNAII, which acts as a primer by binding to its template DNA. The complementary RNAI binds RNAII prohibiting it from its initiation role...

strategies have been applied to reverse MDR in different tumor models and this technology is effective in reversing ABC-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.

Physiological role

In addition to conferring MDR in tumor cells, ABC 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, ABC 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 ABC transporters themselves or affect the transport of other drugs. The latter scenario can lead to drug-drug interactions

Drug interaction

A drug interaction is a situation in which a substance affects the activity of a drug, i.e. the effects are increased or decreased, or they produce a new effect that neither produces on its own. Typically, interaction between drugs come to mind...

, sometimes resulting in altered effects of the drugs.http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8371

Methods to characterize ABC transporter interactions

There are a number of assay types that allow the detection of ABC transporter interactions with endogenous and xenobiotic compounds. The complexity of assay range from relatively simple membrane assays like vesicular transport assay, ATPase assay

ATPase assay

The ATPase assay is a membrane assay that indirectly measures the activity of efflux transporters. ATP Binding Cassette or efflux transporters mediate the transport of substrates across cell membranes against a concentration gradient. ATP cleavage is tightly linked to substrate translocation, as...

to more complex cell based assays up to intricate in vivo detection methodologies.

Membrane assays

The vesicular transport assay detects the translocation of molecules by ABC transporters. 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 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 ABCB11 (BSEP

ABCB11

ATP-binding cassette, sub-family B member 11 also known as ABCB11 is a protein which in humans is encoded by the ABCB11 gene.- Function :...

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

Calcein

Calcein, also known as fluorexon, fluorescein complex, is a fluorescent dye with an excitation and emission wavelengths of 495/515 nm, respectively. Calcein also self-quenches even at concentrations below 100mM. It is used as a complexometric indicator for titration of calcium ions with EDTA,...

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 be hydrolyzed 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.

Human subfamilies

There are 48 known ABC transporters present in humans, which are classified into seven families by the Human Genome Organization.
|-
| ABCG >

Family	Members	Function	>\|- \| ABCA	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.	ABCA12 ABCA12 ATP-binding cassette sub-family A member 12 also known as ATP-binding cassette transporter 12 is a protein that in humans is encoded by the ABCA12 gene.... ABCA1 ABCA1 ATP-binding cassette transporter ABCA1 , also known as the cholesterol efflux regulatory protein is a protein which in humans is encoded by the ABCA1 gene... >- \| ABCB	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.	ABCB5 ABCB5 ATP-binding cassette sub-family B member 5 also known as P-glycoprotein ABCB5 is a plasma membrane-spanning protein that in humans is encoded by the ABCB5 gene... >- \| ABCC	Consists of 12 full transporters.	Used in ion transport, cell-surface receptors, toxin secretion. Includes the CFTR protein, which causes cystic fibrosis Cystic fibrosis Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine... when deficient.	ABCC6 ABCC6 Multidrug resistance-associated protein 6 also known as ATP-binding cassette sub-family C member 6 and multi-specific organic anion transporter E is a protein that in humans is encoded by the ABCC6 gene... >- \| ABCD	Consists of 4 half transporters	Are all used in peroxisome Peroxisome Peroxisomes are organelles found in virtually all eukaryotic cells. They are involved in the catabolism of very long chain fatty acids, branched chain fatty acids, D-amino acids, polyamines, and biosynthesis of plasmalogens, etherphospholipids critical for the normal function of mammalian brains... s.	ABCD1 ABCD1 ABCD1 is a protein that transfers fatty acids into peroxisomes.The protein encoded by this gene is a member of the superfamily of ATP-binding cassette transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are divided into seven distinct... >- \| ABCE/ABCF	Consists of 1 ABCE and 3 ABCF proteins.	These are not actually transporters but merely ATP-binding domains that were derived from the ABC family, but without the transmembrane domains. These proteins mainly regulate protein synthesis or expression.	ABCE:RLI RLI RLI is an essential and highly conserved protein that is required for both eukaryotic translation initiation as well as ribosome biogenesis. The most studied homologues are Rli1p in yeast and Pixie in Drosophila.-Structure:...
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.	ABCG2 ABCG1-

A full list of human ABC transporters can be found at http://nutrigene.4t.com/humanabc.htm.

Importers

Carbohydrate Uptake Transporter-1 (CUT1)
Carbohydrate Uptake Transporter-2 (CUT2)
Polar Amino Acid Uptake Transporter (PAAT)
Peptide/Opine/Nickel Uptake Transporter (PepT)
Hydrophobic Amino Acid Uptake Transporter (HAAT)
Sulfate/Tungstate Uptake Transporter (SulT)
Phosphate Uptake Transporter (PhoT)
Molybdate Uptake Transporter (MolT)
Phosphonate Uptake Transporter (PhnT)
Ferric Iron Uptake Transporter (FeT)
Polyamine/Opine/Phosphonate Uptake Transporter (POPT)
Quaternary Amine Uptake Transporter (QAT)
Vitamin B₁₂ Uptake Transporter (B12T)
Iron Chelate Uptake Transporter (FeCT)
Manganese/Zinc/Iron Chelate Uptake Transporter (MZT)
Nitrate/Nitrite/Cyanate Uptake Transporter (NitT)
Taurine Uptake Transporter (TauT)
Cobalt Uptake Transporter (CoT)
Thiamin Uptake Transporter (ThiT)
Brachyspira Iron Transporter (BIT)
Siderophore-Fe3+ Uptake Transporter (SIUT)
Nickel Uptake Transporter (NiT)
Nickel/Cobalt Uptake Transporter (NiCoT)
Methionine Uptake Transporter (MUT)
Lipid Exporter (LipidE)

Exporters

Capsular Polysaccharide Exporter (CPSE)
Lipooligosaccharide Exporter (LOSE)
Lipopolysaccharide Exporter (LPSE)
Teichoic Acid Exporter (TAE)
Drug Exporter-1 (DrugE1)
Lipid Exporter (LipidE)
Putative Heme Exporter (HemeE)
β-Glucan Exporter (GlucanE)
Protein-1 Exporter (Prot1E)
Protein-2 Exporter (Prot2E)
Peptide-1 Exporter (Pep1E)
Peptide-2 Exporter (Pep2E)
Peptide-3 Exporter (Pep3E)
Probable Glycolipid Exporter (DevE)
Na+ Exporter (NatE)
Microcin B17 Exporter (McbE)
Drug Exporter-2 (DrugE2)
Microcin J25 Exporter (McjD)
Drug/Siderophore Exporter-3 (DrugE3)
(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 Exporter (CydDC-E)

Images

Many structures of water-soluble domains of ABC proteins have been produced in recent years.

External links

Classification of ABC transporters in TCDB
ABCdb Archaeal and Bacterial ABC Systems database, ABCdb
ABCdb
ABCdb is a database for the ATP-binding Cassette transportersencoded by completely sequenced archaealand bacterial genomes.- biological function of ABC systems :...

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Function

Prokaryotic ABC proteins

Eukaryotic ABC proteins

Structure

Transmembrane domain (TMD)

Nucleotide-binding domain (NBD)

ATP binding and hydrolysis

Mechanism of transport

ABC importers

Large ABC importers

Small ABC importers

Mechanism of transport for importers

ABC exporters

Human ABCB1/MDR1 P-glycoprotein

Sav1866

MsbA

Mechanism of transport for exporters

Role in multidrug resistance

Reversal of multidrug resistance

Physiological role

Methods to characterize ABC transporter interactions

Membrane assays

Whole cell based assays

Human subfamilies

Importers

Exporters

Images

See also

External links