ATP-binding cassette transporters (referred to as ABC) are essential membrane-embedded proteins that are ubiquitously present in all cells. These facilitate the import of external nutrients, such as sugars and amino acids, into the cell as well as the removal of toxic compounds, including antibiotics and chemotherapeutics, out of the cell (1,2). To drive the transport of these different molecules, ABC proteins utilize the energy of ATP hydrolysis (1). Collectively, ABC transporters comprise one of the largest superfamilies of integral membrane proteins present in eukaryotes and prokaryotes. In its biological active conformation, an ABC transporter is typically made up of four functional units (1), namely: two transmembrane domains (TMD) containing six transmembrane helices (TMH) each as well as two nucleotide-binding (NBD) domains that bind and hydrolyze ATP (left panel of figure 1 adapted from 2). The TMDs confer substrate specificity as they contain the substrate-binding site and its translocation pathway (1). The NBDs are strictly conserved unlike the TMDs that are highly heterogenous, which allows ABC proteins to recognize a wide variety of substrate molecules. Both NBDs and two TMDs with 12 TMHs can be located either on a single polypeptide chain or on two separate polypeptides containing a single copy of the NBD and one TMD with six TMHs each, resulting in a monomeric or (hetero) dimeric transporter. If all four functional domains are located on a single polypeptide chain the ABC protein represents a full transporter, whereas if two of these domains are located on a single polypeptide the protein is known as a half transporter (1,2).
The human proteome contains 48 ABC transporters that, unlike bacterial ABC transporters, mainly function as exporters, pumping their substrates against a concentration gradient (1). Human ABC proteins are divided over seven subfamilies based on their phylogenetic relationship (3). These are known as ABC superfamily A through G. The ABCA subfamily encompasses 12 transporters, while the ABCB subfamily contains 11 proteins. The ABCC subfamily comprises 12 transporters with different functionalities, ranging from ion transport to cell surface receptor and secretion of toxic compounds. The ABCDD subgroup contains 4 half transporters that are localized to the peroxisome and facilitate fatty acid transport in these organelles. The ABCE and ABCF subfamilies comprise NBD-containing proteins without a clear transport function. The human ABCG subfamily is made up of four half transporters with a reversed topology e.g. an N-terminally located NBD and TMD at the C-terminus. Members of this group are involved in the transport of cholesterol and other sterols. A subset of human ABC transporters function as efflux pumps for toxic molecules and these therefore comprise a crucial defense system to protect tissues against endogenous and exogenous toxicity (2,4). These ABC proteins are, moreover, also expressed in many human tumors where they are probably responsible for conferring chemoresistance (2,4). For example, ABCB1 (Pgp) is a prototypical member of the B subfamily and represents a multidrug transporter that is responsible for clinical resistance to chemotherapy. Its detailed structure was recently solved at a resolution of 3.4 Å (5), which is shown in surface representation in the right panel of figure 3. This reveals that ABCB1 possesses an architecture commonly observed in ABC proteins with two cytosolic NBDs (in orange) and two TMDs (in blue) made up of 12 TMHs. In addition to ABCB1, ABCG2 (Breast Cancer Resistance Protein) is another human multidrug transporter that secretes antitumor drugs from cancer cells, thereby contributing to the development chemoresistance (4). This protein belongs to subfamily G of ABC proteins and comprises, therefore, a half transporter with a reversed topology as described above (3). Detailed structures of human ABCG2 in its apo state and in complex with structurally different chemotherapeutics were presented recently (6). Here, I will discuss these novel structures as well as their current mechanistic and functional understanding.
Role of ABC transporters in multidrug resistant cancer
Since its introduction into the clinic in the early 1940s, chemotherapy has revolutionized cancer treatment and has evolved to the current standard therapy for many cancers. However, the first cases of ineffective chemotherapy with a single agent were soon reported probably owing to acquired resistance by tumor cells (7). This directly spurred the development of combination chemotherapy in the 1960s, which is a therapeutic approach based on the simultaneous use of multiple anticancer drugs with different cellular targets (7). Despite the use of combination chemotherapy, many tumor cells now possess a multidrug resistant phenotype e.g. acquired resistance to different structurally unrelated antitumor compounds (2,4). Consequently, this represents a serious problem for treating chemotherapy-resistant cancers. Multidrug resistance can be obtained through different mechanisms, although the ability to remove toxic compounds from the cell represents a very powerful one. With regards to this it should be noted that several lines of evidence have implicated a subset of 15 ABC transporters in conferring potential resistance to chemotherapeutics (2). Nevertheless, the most prominent multipledrug resistance-related transporters are ABCB1 (Pgp), ABCC1 and ABCG2. These ABC proteins are overexpressed in variety of tumors and actively secrete anticancer drugs from tumor cells (2,4). ABCB1 was first identified in 1976 as a factor that rendered tumor cells insensitive to chemotherapeutics (8). The human gene was cloned in 1986 and characterization of the gene product confirmed that it concerned an ABC transporter (9,10). ABCB1 belongs to the B subfamily and comprises a full transporter with two NBDs and two TMDs as shown in figure 1. This protein is constitutively expressed in the plasma membrane of epithelial cells that form a barrier to seal off body cavities such as the intestine, kidney, liver and blood-brain-barrier. ABCC1 was the second transporter implicated in multidrug resistance (2,4). It was identified in 1992 as an ABC transporter that is overexpressed in a chemotherapeutic-resistant cancer cell line (11) and based on its homology with ABCB1 it was suggested that ABCC1 also functions as a multidrug efflux pump. ABCC1 is a member of the C subfamily and represents a full transporter with five functional domains, namely: two NBDs and 17 TMHs in three TMDs (3,12). Its topology therefore does not resemble that of a typical ABC protein which comprises four functional domains (two NBDs and two TMDs). The function of the N-terminal TMD is not well understood. ABCG2 comprises the third transporter with an established role as resistance protein (2,4). Like ABCB1, ABCG2 is expressed in the plasma membrane of epithelial cells that form tissue barriers such as the blood-brain-barrier. Physiologically, these transporters probably protect sensitive tissues from toxic molecules.
The substrates of these ABC proteins are typically amphipathic lipid-soluble compounds often with aromatic rings as shown in the right panel of figure 3. Imatinib is a 2-phenyl pyrimidine derivative that specifically inhibits a few tyrosine kinases. It is commonly used for the treatment of leukemia’s and although imatinib was reported to be a substrate of ABCG2, it was also found to block this transporter. Mitoxantrone is an anthracycline analog that displays cytotoxicity against cancer cells through inhibition of DNA topoisomerases. This compound is an established substrate of ABCG2 and, like imatinib, mitoxantrone is also used for the treatment of leukemias. SN38 is a pyranoindolizinoquinoline that also represents a DNA topoisomerase inhibitor that displays potent cytotoxic activity against tumor cells. It is mainly used for the treatment of colorectal cancer and similar to mitoxantrone, SN38 is excreted by ABCG2. Based hydrophobic character of most chemotherapeutics, it seems likely that these substrate molecules readily partition into the lipid bilayer. It has, therefore, been proposed that, in contrast to ABC transporters that function as a pump to remove polar substrates from the cytosol, multidrug transporters function as a vacuum cleaner for hydrophobic molecules present within the membrane (left panel of figure 2 adapted from 2). In this model, drugs that enter the cell form the extracellular side partition into the membrane and are removed by multidrug transporters without entering the cell. These drugs accumulate in the membrane up to 1000-fold and therefore the affinity of the transporter for its substrates can be relatively low.
Biochemical and structural features of ABCG2
Human ABCG2 belongs to the G subfamily of ABC transporters and comprises a half transporter with an inversed topology e.g. two functional domains, an N-terminal NBD and a C-terminal TMD made up of six TMHs, on a single polypeptide (3). The active transporter probably represents a dimeric protein with a molecular mass of about 144 kDa. Its gene was identified in a drug-selected model cell line by three independent groups in 1998 (13) and although ABCG2 has been detected in different tissues, the protein is mainly localized in healthy tissues to the plasma membrane of epithelial cells of the lung, gastrointestinal tract, brain, liver, gall bladder and testis. Physiologically, ABCG2 is probably part of a defense system that protects tissues against endogenous and exogenous toxicity. This notion is supported by the finding that ABCG2-deficient mice are more susceptible to certain toxic compounds (13). Additionally, ABCG2 is frequently overexpressed in different tumors, thereby contributing to clinically relevant chemotherapy resistance. It is therefore not surprising that many of ABCG2’s experimentally verified substrates comprise a broad spectrum of anticancer drugs, xenobiotics, antibiotics and toxins (13). Despite its important contribution in developing multidrug resistant cancer, attempts to produce clinically useful ABCG2 inhibitors have been unsuccessful. (4) Ko143, a nontoxic derivative of fungal toxin fumitremorgin C, is a potent inhibitor of ABCG2, although it also impairs the activity of ABCB1 and ABCC1. Owing to this broad selectivity, Ko143 is not suitable as specific ABCG2 inhibitor for the treatment of cancer (13). However, it can be expected that an appropriate modulation of its activity will likely improve the efficacy of anticancer drugs. A molecular understanding of ABCG2’s drug-binding pocket and catalytic mechanism is therefore crucial. With regards to this it should be noted that several detailed structures of ABCG2 are already available (14,15), while recently high-resolution structures of this protein in complex with different chemotherapeutics (imatinib, mitoxantrone and SN38) were reported (6). These structures are discussed below in more detail.
Structure of apo ABCG2
Several structures of human ABCG2 have been reported both in the absence or presence of ATP as well as in complex with substrates or inhibitors. These reveal that ABCG2 adopts two structurally different conformations, namely: an inward and outward-facing state (14,15). In the first conformation, the substrate-binding site, located at the dimer interface of the TMDs, is open. In the outward facing conformation, ATP binding triggers dimerization of the NBDs and subsequent collapse of the substrate-binding site. Structures of ABCG2 in the apo state that were obtained recently are shown in figure 3 (6,14). To this end, the purified protein was reconstituted into nanodiscs and subjected to cryo-EM. The structure shown in the right panel (PDB 5NJ3) was established with the aid of a conformation-specific antibody fragment (14), while, in contrast, the structure depicted in the other panels (PDB 6VXF) was determined in the absence of antibody fragments (6). A surface representation of the transporter is given in the left panel with coloring according to hydrophobicity (polar residues in red and apolar residues in white), revealing a homodimeric complex made up of two major domains – TMD and NBD. The center and right panel show ABCG2 in ribbon representation at 3.5 and 3.8 Å resolution with the substrate-binding site situated between the two TMDs indicated by an asterisk. However, the conformation of both structures is markedly different. In the center panel, the TMDs adopt a closed conformation, while the NBSs are separated in the absence of ATP. Within the TMDs, the TMHs form a packed bundle near the cytosolic face of the membrane, while TMH5 (in white) is rotated in such a way that it seals the substrate-binding cavity (6). The structure displayed in the right panel reveals an overall architecture that is more compact when compared to other eukaryotic ABC transporters owing to shorter TMHs and intracellular loops. The dimerization interface of the two monomers is located between the opposing TMDs and comprises TMH2 and 5 (in orange). These are oriented in such a way that an inward-facing cavity is formed, while the NBDs are separated (14). EL3 is the longest extracellular loop that connects TMH5 and 6 and contains a single N-linked glycosylation site (N596) and two intramolecular disulfides at Cys592 and 608 in each monomer as well as an intermolecular disulfide between cys603 of each monomer (center panel). The N-linked glycosylation and intramolecular disulfides are important for stability of the protein.
The substrate-binding cavity is accessible from either the cytoplasm and inner leaflet of the plasma membrane, while at the top, half way across the membrane, two hydrophobic leucines (Leu534) of each monomer are located. These are shown in top the left panel of figure 4 as red spheres (top view of ABCG2) and form a plug that separates the substrate-binding cavity from a smaller one located below the E3 external loops (14). Phe545, that belongs to TMH5, is part of the substrate-binding site and was previously observed at the site of the binding cavity, facing the monomer to which it belongs (as red spheres in the upper right panel of figure 4). In the novel structure, however, this residue (as red spheres in the top right panel of figure 4) is rotated away such that it faces the opposite monomer (6). Moreover, five residues with sulfur-containing side chains (Cys438, Met523, Met541, Cys544 and Met548) are situated above and below Phe545 (bottom left panel of figure 4). These side chains are oriented towards the dimer interface. In addition to the rotation of TMH5, TMH2 also exhibits an altered conformation when compared to the previously compared structures. In the novel apo structure, TMH2 is partially unraveled, thereby reorienting several key residues that interact with substrates such as Phe439 and Asn436. Both of these are flipped outward from the substrate-binding cavity (6). Thus, in the absence of antibody fragments and inhibitors, apo ABCG2 adopts a novel conformation with the NBDs separated and substantial conformational rearrangements of the TMDs that close the substrate-binding cavity (6). Importantly, this closed state also occurs in vivo as revealed by disulfide crosslinking experiments used to probe the conformation of the transporter within a human cell line.
Structure of ABCG2 in complex with chemotherapeutics
To establish how ABCG2 interacts with anticancer drugs, its structure bound to chemically different chemotherapeutics – imatinib, mitoxantrone and SN38 - was assessed by cryo-EM. Imatinib probably serves as an inhibitor of ABCG2 through blocking ATP hydrolysis by preventing the formation of a nucleotide-bound outward facing conformation. Unlike imatinib, mitoxantrone and SN38 don’t inhibit the ATPase activity of ABCG2 and have therefore little effect on the conformational states of ABCG2. Figure 5 shows the structure in ribbon representation of ABCG2 bound to imatinib determined at a resolution of 4 Å (6). A single conformation was observed with the TMHs in an inward-open conformation (left panel). This structure is highly similar to previously determined structures of ABCG2 in complex with inhibitors and based on this it can be concluded that imatinib stabilizes the inward-facing conformation. The overall structure of the TMDs is presented in the right top panel and shows that one molecule of imatinib (as red spheres) is located in the substrate-binding cavity between the two ABCG2 monomers. Importantly, the substrate binding pocket is in an open inward-facing conformation. Imatinib is situated at the top of the binding pocket below the leucine plug and spans across the cavity. The bottom panel shows that imatinib is sandwiched between the aromatic side chains of Phe439 (in magenta sticks) of each monomer through π stacking. Imatinib, thus, acts as a wedge that forces ABCG2 into the inward-facing conformation and thereby preventing ATP hydrolysis.
Furthermore, structures of ABCG2 in complex with either mitoxantrone and SN38 were also obtained by cryo-EM (6). Two main conformations were observed, corresponding to the apo closed and inward-facing structures, implying that in the presence of mitoxantrone or SN38 a substantial amount of ABCG2 does not bind these substrates and remains in the closed conformation. The structure of ABCG2 bound to either mitoxantrone or SN38 was resolved at 3.6 and 4 Å, respectively. These are shown in figure 6 and reveal a conformation with an open, inward-facing substrate binding cavity. One molecule of mitoxantrone (as red spheres in the top panel of figure 6) or SN38 (as magenta spheres in the bottom panel of figure 6) is clearly visible within the substrate-binding pocket between the TMDs of both monomers. Similar to imatinib, mitoxantrone and SN38 are bound by π stacking interactions through t heir aromatic rings and the side chain of Phe439 (as orange sticks in the top and bottom panels in figure 6) located on the opposing monomers. These interactions were reported previously as well for substrates and inhibitors, implying that all substrates may diffuse from the cytosol or plasma membrane into the inward-facing substrate-binding pocket.
Mechanism of ABCG2-faclitated drug extrusion
Based on the data discussed above, the following model for substrate translocation by ABCG2 was proposed (figure 7 adapted from 6. ABCG2 is initially in the apo-closed conformation (1) with the TMHs collapsed and NBDs open. Binding of substrates or inhibitors induces a conformational change toward the inward-facing state where hydrophobic substrates are bound in the substrate-binding pocket (2). Subsequent binding of ATP triggers dimerization of the NBDs and collapse of the TMHs, thereby pushing the substrate through the leucine ring and into the extracellular space (3). Finally, ATP hydrolysis resets the transporter to the apo-closed state. In contrast to substrates, inhibitors that bind in the binding cavity prevent ATP binding/hydrolysis and the subsequent conformational change to the outward facing state. The structural data, moreover, revealed that all anticancer drugs tested where sandwiched between the aromatic side chains of Phe439 of both monomers. This raises the question why imatinib acts as a wedge and thereby prevents ATP hydrolysis? This can potentially be explained by ACG2’s higher affinity for imatinib as well as its larger molecular volume. Conceivably, ABCG2 selects its substrates by sensing whether these are able to induce a conformational shift from the apo-closed to the inward-open state.
ABC transporters are found throughout all biological kingdoms and represent one of the largest superfamilies of membrane-embedded transporters. These proteins drive essential transport processes that include the import of external nutrients, such as sugars and amino acids, into the cell as well as the removal of toxic compounds, including antibiotics and chemotherapeutics, out of the cell. The transport of these different molecules is powered by the energy that is released upon ATP hydrolysis. The general architecture of an ABC transporter comprises four functional domains viz. two TMDs containing six TMHs each as well as two NBDs that bind and hydrolyze ATP. Whereas the TMDs confer substrate specificity because they contain the substrate-binding site and the substrate translocation pathway. Humans contain 48 ABC transporters that are classified into seven subfamilies (A-G) based on their phylogenetic relationship. A subset of these transporters has been associated with the development of multidrug resistant cancers which makes these tumors difficult or impossible to treat. For example, ABCB1, ABCC1 and ABCG2 are able to confer resistance to a wide spectrum of chemotherapeutics. These transporters are expressed in different healthy tissues, that typically include epithelia that form a barrier to seal off body cavities such as the intestine, kidney, liver and blood-brain-barrier, and are probably part of a defense system that protects cells against endogenous and exogenous toxicity. In cancer cells, these ABC proteins are frequently overexpressed and actively secrete anticancer drugs. It can, therefore, be expected that an appropriate modulation of their activity will likely improve the efficacy of anticancer drugs. However, attempts to design clinically useful inhibitors with sufficient selectivity have as yet been unsuccessful, which is in part due to the lack of sufficiently detailed structural information. With regards to this it should be noted that several high-resolution structures of human ABCG2 are already available, while, recently, detailed structures of this protein in complex with chemically different anticancer drugs were reported. Together, these structures provide a profound molecular understanding of how chemotherapeutics interact with ABCG2 and unveil how this protein is able to extrude a variety of chemically unrelated substrates. It can be anticipated that these novel structures will aid in the design of specific ABCG2 inhibitors.
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