**2. Architecture of ABCG2**

ABCG2 belongs to the ABCG subfamily, the members of which, in addition to sequence homology, exhibit considerable structural similarities. To our recent knowledge, the minimal structure of a functional ABC transporter is composed of two cytoplasmic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). The usual arrangement of domains in the core protein is as follows: TMD1-NBD1-TMD2-NBD2. Contrary to the canonical ABC transporters, members of the ABCG subfamily possess only one NBD and one TMD; thus, they are called half-transporters. Moreover, the domain order in ABCG proteins is reversed, i.e., NBD is localized N-terminally to TMD. This reverse domain arrangement could be one of reasons for the sensitivity of ABCG2 to tagging at the C-terminus [9], contrary to many other ABC transporters, which can regularly be tagged Cterminally. To form a functional complex, ABCG proteins, like other ABC half-transporters, assemble into either homo- or heterodimers. While ABCG2 solely forms homodimers, ABCG5 and ABCG8 are obligate heterodimers. In contrast, two other members of the subfamily, ABCG1 and ABCG4 can form both homo- and heterodimers [10].

Membrane topology models of ABCG2 suggest six membrane-spanning helices, a relatively short C-terminal tail, and short loops between the transmembrane helices (TMHs) except for the last extracellular loop (EL3) between TMH5 and TMH6 [11]. N-glycosylation on asparagine 596 located in the EL3 loop has also been demonstrated [12,13]. Although initial reports suggested that glycosylation at N596 was not essential for proper expression, localization, and function, subsequent studies demonstrated N-glycosylation to be an important checkpoint determining the stability and intracellular trafficking of the transporter [14,15]. There are twelve cysteine residues in ABCG2, but only three of them are positioned in an oxidative milieu, and thus capable of forming disulfide bonds. All three of these cysteines are located in the EL3 loop, and while an intramolecular disulfide bond is established between C592 and C608, an intermolecular disulfide bridge is formed between the two halves of the homodimer at C603. Whereas the latter disulfide bond is not required for proper trafficking and function of ABCG2 [16–18], the C592-C608 intramolecular disulfide bond represents another critical checkpoint for protein folding and trafficking [15,18].

Although the X-ray structures of isolated NBDs have been available since the late nineties, the first high-resolution structures of full-length ABC transporters were only published in 2006 and 2007 [19,20]. In the last decade, the spread of cryogenic electron

microscopy (cryo-EM) and the substantial progress in crystallography have given a boost to our understanding of ABC protein structures. However, homology modeling was not quite applicable to ABCG proteins, as the members of this family are rather distinct from other 'classical' ABC transporters, such as the P-glycoprotein (MDR1/ABCB1) or the CFTR/ABCC7. The appearance of the first high-resolution structure of an ABCG protein, i.e., that of the heterodimeric sterol transporter ABCG5/ABCG8, was therefore a breakthrough [21], fueling extensive homology modeling of ABCG2 [22–24]. Subsequently, several ABCG2 structures based on cryo-EM analyses have been published [25–28]. The structural characteristics of ACBG2, obtained from these cryo-EM studies and from parallel molecular dynamic stimulations, are recently reviewed in [29,30].

There are some distinguishing structural features in the ACBG2 as compared to the full-length ABC transporters. In general, the structure of ABCG2 is more compact, the NBDs are positioned close to the TMDs. A similar compact arrangement was observed for the ABCG5/ABCG8 crystal structure [21], which resembles the architecture of BtuCD-like bacterial importers, rather than that of MDR1-like transporters. This originates from the relatively short transmembrane helices, possessing no cytosolic extension unlike the helices in the classical ABC proteins, which create a sort of spacer between the TMDs and NBDs. In the MDR1-like proteins, two of the elongated four pairs of helices cross over and bind to the opposite NBD, while the other two pairs interact with the ipsilateral NBD. The interfacings to NBDs are realized by small, so-called coupling helices at the cytosolic tip of the elongated TMHs [19]. In contrast, there is only one coupling helix in each half of the ABCG2 dimer (between TMH2 and TMH3), which does not cross over to the other half. However, an amphipathic helix, called the connecting helix, linked to only TMH1 and reclining against the membrane bilayer, provides an additional TMD-NBD interface in ABCG2.

Another distinct feature of the structure of ABCG2, as well as of ABCG5-ABCG5, is the relatively closed conformation in the absence of ATP. In the classical ABC transporters in this 'apo' form (without ATP), the NBDs are located far from one another, and consequently, the intracellular parts of TMHs also remain apart, forming a large central cavity, the main substrate-binding pocket, which is widely open to the cytoplasm [19]. The presence of a similar cavity at the cytoplasmic side of ABCG2 (cavity 1) has been reported by cryo-EM studies using an anti-ABCG2 antibody to reduce flexibility in the structure [25–27]. Nevertheless, the NBDs, and consequently the intracellular parts of TMHs, are closer to one another in ABCG2 than in MDR-like transporters, resulting in a more compact structure even in the absence of ATP. Residues in this central cavity were shown to be essential not only for transport function but also for biogenesis [24]. Interestingly, a study using no anti-ABCG2 antibodies for structure stabilization reported the lack of cavity 1 [28]. An additional prominent feature in the inward-facing structure (apo form) of ABCG2 is the hydrophobic di-leucine valve (L554 and L555) separating the central substrate-binding pocket from an additional cavity (cavity 2) located toward the extracellular part of ABCG2 [25,26]. Experiments supplemented by molecular dynamic simulations demonstrated an essential role for this di-leucine plug in the transport function [31]. Putting together the different structures in the absence and presence of ATP and/or substrates, MDR1-like proteins seem to alternate between a widely open inward-facing and a fairly open outward-facing conformations, whereas the translocation of substrates through ABCG2 via cavities 1 and 2 rather involves a peristaltic-like movement.

With regard to the NBDs, both sequences and structures are fairly conserved. The two composite ATP-binding pockets are constituted by two separate NBDs in a head-to-tail orientation, i.e., one ATP molecule binds to the Walker A and B motifs of one NBD and to the ABC signature sequence of the other NBD. Unlike in full-length ABC transporters, the cytoplasmic part of the homodimeric ABCG2 is composed of two identical halves, but otherwise the ABC-folds in ABCG2 are structurally similar to that of the classical ABC transporters. It is worth noting that a phenylalanine at position 142 in ABCG2 interacts with the connecting helix, representing a key residue in TMD-NBD interface assembly and

a critical checkpoint for protein folding and function [32,33]. Interestingly, this amino acid is analogous to F508 in CFTR/ABCC7, the mutation of which is responsible for diminished trafficking of CFTR, and ultimately the cystic fibrosis (CF) phenotype.
