Structural Biochemistry/Membrane Proteins/ATP-Binding Cassette Proteins
ATP-Binding Cassette Proteins
ATP-binding cassette transporters, also known as ABC transporters, are transmembrane proteins that utilize the energy of 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, including metabolic products, lipids, and sterols. Medically, ATP-binding cassette (ABC)transporters contribute to the resistance of multi-drug to cytotoxic drugs. Proteins are classified as ABC transporters based on the sequence and organization of their ATP-binding cassette domains.
ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.
ABC transporters utilize the energy of ATP hydrolysis to transport various substrates across cellular membranes. They are divided into three main functional groups. In prokaryotes, importers mediate the intake of nutrients to the cell. Ion, amino acids, peptides, sugars and other molecules that are mostly hydrophilic are the substrates that can be transported. The area of the membrane around the ABC transporter protects hydrophilic substrates from the lipids within the membrane bilayer, providing a pathway across the cell membrane. Eukaryotes do not have any importers. Instead, they have exporters, or effluxers, which are also present in prokaryotes. Exporters function as pumps that remove toxins and drugs from the cell. The third subgroup of ABC proteins do not function as transporters, but are more involved in the process of translation and DNA repair.
Prokaryotic ABC Proteins
Bacterial ABC transporters are necessary in cell viability, virulence, and pathogenicity. For instance, the iron ABC uptake systems are very important effectors of virulence. Siderophores are used by the pathogens to scavenge for iron that is in complex with high-affinity iron-binding proteins called erythrocytes. Erythrocytes are high-affinity iron-complexing molecules that the bacteria secretes and is reabsorbed into iron-siderophore complexes. Bacterial ABC transporters are also essential in cell survival. This is because they function as protein systems that act against any undesirable change that may occur in the cell. One example of this is seen when osmosensing ABC transporters that mediate the uptake of solutes is activated in order to counteract a potentially lethal increase in osmotic strength. In addition to functioning in transport, there are some bacterial ABC proteins that are involved in the regulation of several physiological processes.
Eukaryotic ABC Proteins
Most eukaryotic ABC transporters are effluxers, but there are some that are not directly in the transporting of substrates. For example, in the cystic fibrosis transmembrane regulator (CFTR) and in the sulfonylurea receptor (SUR), ATP hydrolysis is used to regulate the opening and closing of the ion channels carried by the ABC protein itself or other proteins. ABC transporters in humans are involved in quite a few diseases that are a result of polymorphisms in ABC genes rather than the complete loss of function of single ABC proteins. Some of these diseases include Mendelian diseases and complex genetic disorders such as cystic fibrosis, immune deficiencies, Stargardt disease, Tangier disease, Dubin-Johnson syndrome, and many others.
NKA a P-type ATPase
NKA, which stands for Na+,K+ -ATPase, is a sodium pump that uses energy to transport ions across the plasma membrane. The ATP hydrolysis energy is used to transport the sodium and potassium, generating electrochemical gradients. NKA falls under the P-type group, which is a protein complex in the form of αβϒ. The α-subunit is the actual pump that transports the sodium and potassium, and contains the three nucleotide-bindings. It contains M1-M10, which are the ten transmembrane segments. It also contains the three cytoplasmic domains which are: the nucleotide-binding (N-) domain, the phosphorylation (P-) domain, and the actuator (A-) domain. The β-subunit is responsible of transporting the potassium ions and making sure that the α-subunit is inserted correctly in the plasma membrane. The β-subunit contains one membrane-anchoring helix, disulfide bridges, and a extracellular domain. Then, the ϒ-subunit regulates the pumping activity of the sodium pump in the tissue.
The interaction between agrin signaling and the ion sodium-potassium pump is due to the bimodular mechanism. Two homologous sites of agrin are cleaved by neurotrypsin at the synapse. This affects to both of the pumping and the signaling function of the NKA. This causes the C-terminal 22 (C22) to restrain the ion pumping function of NKA. Then, agrin binds to the C-terminal. Due to this, the membrane can affect the activity and insertion since the β-subunit is displace. To know if the α-subunit or the β-subunit will dominate, based it on the binding affinity or the concentration.
Structure and mechanism
ATP-binding cassette transporters constitute a large superfamily of integral membrane proteins that includes both importers and exporters. In recent years, several structures of complete ABC transporters have been determined by X-ray crystallography. These structures suggest a mechanism by which binding and hydrolysis of ATP by the cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains and therefore which side of the membrane the translocation pathway is exposed to. A basic, conserved two-state mechanism can explain active transport of both ABC importers and ABC exporters, but various questions remain unresolved. In this article, I will review some of the crystal structures and the mechanistic insight gained from them. Future challenges for a better understanding of the mechanism of ABC transporters will be outlined.
Mechanism of Transport
ABC transporters make use of the energy of ATP binding and/or hydrolysis to drive the conformational changes in the transmembrane domain (TMD) and consequently transports molecules. There are two types of ABC transporters, ABC importers and ABC exporters, that have a common mechanism in transporting substrates because they are similar in structure. The mechanism that describes the conformation changes associated with the binding of the 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 the transport. For ABC importers, the translocation is directed from the periplasm to the cytoplasm and so the outward-facing conformation will have higher binding affinity for the substrate. Contrastingly, 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 gives 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 and adenosine diphosphate (ADP). The switching between open and closed dimer conformations induces the conformation changes in TMD resulting in substrate translocation.
The general mechanism for the transport cycle of ABC transporters has not fully been clarified but substantial structural and biochemical data has been gathered to support a model in which ATP binding and hydrolysis is coupled to conformation changes in the transporter. The resting state of all ABC transporters has the NBDs in an open dimer configuration with a low affinity for ATP but a high affinity for substrate binding site. This open conformation contains a chamber that is accessible to the interior of the transporter. The transport cycle is initiated by the binding of the substrate to the high-affinity site on the TMDs, which induces the conformational changes in the NBDs and enhances the binding of ATP. Then, two molecules of ATP bind cooperatively to form the closed dimer configuration. The closed NBD dimer then induces a conformational change in the TMDs such that the TMD opens, which forms a chamber with an opening that is opposite that of the initial state. The affinity of the substrate to the TMD is then reduced, which releases the substrate. Following that is the hydrolysis of ATP and the sequential release of inorganic phosphate and ADP. The ADP restores the transporter to its basal configuration. Although this common mechanism has been suggested, the order of substrate binding, nucleotide binding and hydrolysis, and conformational changes as well as the interactions between the domains, is still being debated.
Recent studies have shown that ATP binding, rather than ATP hydrolysis, provides the principal energy input, or "power stroke", needed for transport. This may be because ATP binding triggers NBD dimerization and so the formation of the dimer may represent the "power stroke". Additionally, there are some transporters that have NBDs that do not have the similar abilities in binding and hydrolyzing ATP. Thus, the interface of the NBD dimer consists of two ATP binding pockets, suggesting that there is a concurrent function of the two NBDs in the transport cycle.
The transport mechanism for importers supports the alternating-access model. The importers' resting state is inward-facing, where the NBD dimer interface is held open by the TMDs and facing outward. When the closed, substrate-loaded binding protein attaches towards the periplasmic side of the transmembrane domains, the ATP binds and the NBD dimer closes. The resting state of the transporter then switches into an outward-facing conformation in which the TMDs have reoriented themselves so that they are able to receive the substrate from the binding protein. After the ATP has been hydrolyzed, the NBD dimer opens up and the substrate is released into the cytoplasm. The transporter then reverts back to its resting state upon the release of phosphate and ADP. The only inconsistency in this mechanism is that the conformation in its resting, nucleotide-free state is different from the expected outward-facing conformation. While this may be the case, the key point is that the NBD does not dimerize unless ATP and the binding protein is bound to the transporter.
ABC exporters have a transport mechanism that is consistent with both the alternating-access model and the ATP-switch model. In the apo states of exporters, the conformation is inward-facing while the TMDs and NBDs are relatively far apart to accommodate the amphiphilic or hydrophobic substrates. The binding of the substrate initiates the transport cycle. The binding of the ATP induces NBD dimerization and formation of the ATP sandwich, which then drives the conformational changes in the TMDs. The cavity where the substrate binds is lined with charged and polar residues that are most likely solvated, which creates an energetically unfavorable environment for hydrophobic substrates and an energetically favorable environment for the polar moieties in amphiphilic compounds or sugar groups in LPS. Since they hydrophobic compounds cannot be stable in the chamber environment for a long time, they "flip" into an energetically more favorable position within the outer membrane leaflet. The repacking of the helices will then switch the conformation to an outward-facing state. The hydrolysis of ATP can widen the periplasmic opening, pushing the substrate towards the outer leaflet of the lipid bilayer. The hydrolysis of a second ATP molecule releases phosphate, separating the NBDs, followed by the restoration of the resting state. This will open up the chamber towards the cytoplasm of the cell, readying the exporter for another cycle.
"How are ion pumps and agrin signaling integrated?" Tidow Henning,Aperia A., Nissen P.