**2. Structural Properties of ABCC6 Transporter**

The ATP-binding cassette (ABC) transporters are a well-known family and ubiquitously found in all living organisms. About 50 different types of ABC transporters have been recognized in humans, divided into seven subfamilies (ABCA to ABCG) based on their structure and genetic sequences. Among them we can find both half transporters, with only one transmembrane domain (TMD) and nucleotide-binding domain (NBD), and full transporters, with two TMDs and NBDs [9,10]. ABCC6 belongs to the ATP-binding cassette (ABC) transporter subfamily C and has been found to be highly expressed in basolateral plasma membrane of hepatocytes and, to some lesser extent, in the proximal tubules of the kidney [11].

The *ABCC6* gene has 31 exons and encodes a protein of 1503 amino acid residues, MRP6. It is made-up of two nucleotide-binding domains (NBD1 and NBD2) and two transmembrane domains (TMD1 and TMD2) with an additional auxiliary NH2-terminal transmembrane domain known as TMD0, which is connected with the canonical components through the cytoplasmic L0 loop [12,13]. The NBDs of ABC transporters consist of different conserved motifs which bind and hydrolyze ATP. Like in other transporters, ABCC6 NBDs domains have Walker A motif (P loop), Walker B motif (Mg2+ binding site), histidine loop (Switch region), signature motif (C loop), Q loop (between Walker A

and C-loop), and D loop (involved in the intermolecular interactions at the NBD dimmer interface). In the general process of dimerization, the Walker A/B motifs of one NBD are aligned with the C-loop of the other in a yin-yang fashion, forming a sandwich with two molecules of ATP in the middle, which are hydrolyzed in the catalytic cycle. Dimerization induces conformational changes in the TMDs, which are reversed by hydrolysis of ATP, leading to efflux of molecules [1,14,15]. In this situation, the ATP should be sandwiched between two sequence/structural motifs that are both rigorously conserved and form a stable homodimer [16]; this is not always possible with the human transporters, because one of the two NBDs is degenerate and the sequence of motifs which are responsible for hydrolysis of ATP is not identical or canonical [14].

In this context to elucidate the role of NBDs, different experiments were conducted on NBD1 of ABCC1/MRP1, which not only has a high level of homology with ABCC6/MRP6 but also shows a superposition of substrates [17]. Studies revealed that the NBDs of MRP1 are not functionally identical, as both the NBDs bind ATP but only NBD2 is involved in hydrolysis. Moreover, the ATP binding affinity of NBD1 of MRP1 is not completely dependent on NBD2 but the binding of ATP to NBD2 is highly dependent on NBD1 [18,19]. In respect of this, we have gone through by a series of experiments to identify the functional roles of NBDs, TMD0, and L0 loop of ABCC6 (Figure 2).

**Figure 2.** Topology of ABCC6 transporter [20].

α In order to evaluate the functioning of NBDs, we have first characterized the NBD1 of ABCC6/MRP6. In this experiment, by using the E748-A785 fragment of MRP6-NBD1, we have compared the helical structure and ATP binding properties of wild type and the R765Q mutated sequence, which is present in PXE patients. The study with circular dichroism analysis revealed that, in both the wild type and mutated (R765Q) NBD1, E748-A785 peptides adopted α-helical conformations, and the helical content was almost the same for both the peptides in aqueous solutions of trifluoroethanol (TFE). No differences in the length of helices have been found between the peptides in NMR spectroscopy. Moreover, Fluorescence Spectroscopy showed no significant difference in ATP binding capacity of both the peptides. These findings suggest that occurrence of PXE symptoms in R765Q mutated patients might be due to different kind of interactions [21].

In subsequent investigations, we have undergone two different experiments with NBD1 and NBD2, because mutational studies of PXE patients showed that domain-domain interaction is important for proper working of ABCC6 transporter and there is a functional

difference between both NBDs of MRP6 [22]. In the first study, we constructed the fulllength NBD1 (residues from Asp-627 to Leu-851) and short-length NBD1 (residues from Arg-648 to Thr-805) without some key residues; then differences in helical structure, ATP binding, and hydrolysis of both polypeptides were analyzed. Interestingly, both the polypeptides assumed predominantly α-helical conformation. However, only long-length NBD1 showed β-strand conformation, while short-length NBD1 showed higher helix content, which suggested that the sequences D627-H647 and T806-L851, which are only present in the long-length polypeptide, assume a β-strand conformation, similarly to the corresponding regions of MRP1. Although fluorescence quenching experiments revealed that both the polypeptides have the affinity to bind nucleotides (ATP and ADP), the longlength NBD1 showed a higher affinity to bind ATP than the short-length NBD. In addition, no hydrolytic activity was found in short-length NBD1 compared to long-length NBD1 during spectrophotometric analysis of ATPase activity. These findings suggest that shortlength NBD1 lacks of some essential residues, which are responsible for ATP hydrolysis. Whereas, long-length NBD1 form homodimer in the presence of ATP, which is the property of half-transporters and physiologically, it is not possible with the full-transporter where NBD1 interacts with NBD2 [12].

In order to explore how the ABCC6 transporter works in-vivo, we have constructed long-length (Thr-1252 to Val-1503) and short-length NBD2 (Val-1295 to Arg-1468), and we created homology models for homodimer of NBD1 and NBD2, and heterodimer of (NBD1-NBD2) [23]. Circular dichroism spectra of long-length NBD2 revealed that it is more structured and contains α-helical conformation. The nucleotide (ATP and ADP) binding affinity of NBD1 and NBD2 were found to be the same during fluorescence quenching analysis. The ATPase activity of both homo and heterodimer were different, as the amount of inorganic phosphate (Pi) produced by NBD2 was lesser then NBD1. Moreover, addition of NBD2 reduced the NBD1 hydrolytic activity. These findings suggest that NBD2 is well structured (it contains α-helix and β-strands) and binds the nucleotides efficiently, but the ATPase activity of NBD2 is lower compared to the NBD1; this reflects that NBD2 is not able to form a functionally active homodimer, as supported by in-silico structural analysis. In addition, decreased ATPase activity of combined NBD1-NBD2 compared to NBD1 alone suggests that NBD1 and NBD2 work together to form a stable heterodimer and function in a regulated manner, whereas NBD1 alone works in an uncontrolled manner. This can be justified by the in-vivo functioning of the transporter [24], where ATP is hydrolyzed after the binding of substrate on TMDs and leads to conformational changes in membrane domains that activate the NBDs.
