CHAPTER 1-INTRODUCTION………………………………………………... 1-21
1.1.5 ABC transporter architecture
L-arabinose porter 3.A.1.2.2 AraFGH of E.
coli
L-arabinose, D- fucose, D-
galactose
1ABE
Glucose/ galactose
porter 3.A.1.2.3 MglABC of E.
coli Glucose/galactose 2FVY D-allose porter 3.A.1.2.6 AlsABC of E.
coli D-allose 1GUB
* Information of TCID was retrieved from Transporter Classification Database (TCDB, Saier et al., 2016).
Figure 1.4. The overall topology of ABC importer and exporter. (A and B) Domain architecture of ABC importer e.g. maltose ABC transporter (PDB ID:
4KHZ) and exporter e.g. Sav1866 (PDB ID: 2HYD), respectively. The ABC transporter subunits TMDs, NBSs and SBP are represented as blue, green and yellow, respectively. Schematic representation of SBP organization in (C) Gram-negative bacteria and (D) Gram-positive bacteria and archaea (right). Respective free-floating and lipid anchored SBPs are shown in yellow.
1.1.5.1 Transmembrane domains (TMDs)
TMDs are embedded in the membrane with the core structure containing 10-12 transmembrane (TM) helices, which form a central translocation pathway. The number of TM helices varies in ABC exporters and importers. The central channel of exporters comprises of 12 TM helices, while in importers, this number varies between 10 and 20 (Hollenstein et al., 2007). Depending upon ABC transporters Types (I, II and III), the TMDs folds vary. Also, the TMD fold of Type III importer is related to ABC exporters (Rees et al., 2009). Type I ABC transporter contains six transmembrane (TM) helices per TMD subunit (Parcej et al., 2007). For example, TMD subunit of moldydate ABC transporter (ModB, PDB ID: 3D31) and maltose ABC transporter (MalF and MalG, PDB ID: 4KHZ) contains six to eight TM helices and organized into homo-and hetero-dimer association (Oldham et al., 2007; Gerber et al., 2008). Similarly, Type II transporters such as vitamin B12 ABC transporter (PDB ID: 1L7V) comprises a higher number of TM helices with 10 to 12 α-helices per TMD subunit (Locher et al., 2002). In contrast, in Type III transporters (e.g. ECF
transporter, Sav1866 and P- glycoprotein ABC transporter) TM helices from six helices, transverse the plasma membrane and extended into the cytoplasm (Figure 1.5) (Dawson and Locher, 2006).
TMDs are generally found in two confirmations (1) open towards the periplasmic site (outward open) and (2) open towards the cytoplasmic site (inward open) (Wilkens, 2015). TMDs lack conservation at the primary structure level, thus allowing the transport of various substrates. Inner TM helices provide binding sites for substrate or may form a hydrophobic cavity (Korkhov et al., 2012; Oldham et al., 2013). For example, maltose ABC importer from E. coli (Type I fold) contains a site for three sugar rings. During translocation, the reducing end of sugar interacts with the TM helices residues (Oldham et al., 2013). Like Type I importers, ABC exporters also contain substrate-binding sites, but with more number of interaction sites. For instance, P-glycoprotein, a multidrug exporter, contains multiple drug-binding sites leading to the efflux of various drugs (Loo et al., 2003; Aller et al., 2009).
Figure 1.5. Schematic representation of the transmembrane domain (TMD) fold. A canonical transmembrane domain (TMD) fold of (D) Type I, (E) Type II and (F) Type III ABC transporters. Distribution of TM helices per TMD subunit in a different class of ABC transporter are numbered and color-coded according to TMD subunit.
1.1.5.2 Nucleotide-binding domains (NBDs)
NBDs are catalytic domains of ABC transporters and reside on the cytoplasmic phase of the transporter. Crystallographic structures of these domains reveal that bacterial and eukaryotic NBDs have a conserved fold similar to RecA domain. The comparative analysis of the crystal structures of ATP-bound NDBs and free forms (without ATP) suggests that the ATP binding to NBDs is required for its dimerization (Hung et al., 1998; Chen et al., 2003). Two ATP molecules are sandwiched between the dimer to form a ‘sandwich dimer’ (Kerr, 2002). In this dimeric arrangement, it contains several conserved motifs that are involved in the ATP binding (Schneider and Hunke, 1998;
Davidson and Chen, 2004). Two conserved motifs of NBDs known as ‘Walker-A or P-loop’ and ‘LSGGQ’ motifs help in binding ATP molecules (Figure 1.6). In addition, the ‘Walker B’ motif is required for the catalytic activity i.e. hydrolysis of ATP by NBDs. A glutamate residue of ‘Walker B’ motif plays a role of nucleophile. Another motif termed as ‘D loop’ mediates the interaction between TMDs and NBDs (Zaitseva et al., 2005; Ambudkar et al., 2006).
Figure 1.6. Domain architecture of the nucleotide-binding domain (NBD). (A) Dimeric conformation of NBD subunit of maltose ABC transporter (PDB ID: 3PUV).
Two protomers (chain A and B) are shown in light and dark green. ATP molecule bound at the interface of the dimer is shown in yellow ball-and stick-model. (B and C) A schematic representation of the ABC signature motif i.e. LSGGQ (light green) and Walker A (dark green) motifs interacting with ATP molecule. Hydrogen bonding between motif residues and ATP molecule are shown with dotted lines. Respective residue number of ABC signature motif is represented in the Figure 1.6C.
1.1.5.3 Substrate (solute)-binding proteins (SBPs)
Substrate (solute)-binding proteins (SBPs) are a class of proteins that is associated with membrane protein complexes for transport of substrates. SBPs are soluble proteins found in the periplasm of the Gram-negative bacteria, sometimes called as periplasmic-binding proteins (PBPs) (Berger and Heppel, 1974). Originally, SBPs were discovered as a component of prokaryotic ABC transporters (Berger, 1973;
Berger and Heppel, 1974; Tam and Saier, 1993; Quiocho and Ledvina, 1996;
Wilkinson, 2002). Later on, it has been found that besides ABC transporters, it is also associated with prokaryotic tripartite ATP-independent periplasmic (TRAP) transporters (Gonin et al., 2007; Mulligan et al., 2009). In order to import different types of nutrients, ABC importers uses different types of SBPs. Depending upon the substrate, different SBPs are named accordingly like sugar-binding protein, metal- binding proteins and amino acid-binding proteins. One of the well-characterized SBPs is a maltose-binding protein of maltose ABC transporter (MalE-MalFGK2) from E. coli (Orelle et al., 2008; Oldham and Chen, 2011). Topologically, all SBPs contains two α/β domains, in which β-sheets are flanked by α-helices. The two domains, N-and C-terminal domains (NTD and CTD) are linked by a ‘hinge region’, which is a site for ligand binding (Figure 1.7A). In a ligand unbound state (open conformation), these two domains are very flexible, allows the free rotation of NTD and CTD around the hinge region. Upon ligand binding, hinge region stabilizes the two domains (NTD and CTD) and attains the closed state of SBPs (Tang et al., 2007).
During open to closed state transition, both NTD and CTD move around the hinge region through a well-known ‘‘Venus Fly-trap’’ mechanism (Mao et al., 1982) (Figure 1.7B).
Figure 1.7. Structural topology of SBPs. (A) The tertiary structure of SBP (e.g. α- glycoside-binding protein, PDB ID: 6J9W) is shown as a ribbon model. The NTD and CTD are shown in orange and green, respectively. Hinge region connecting the NTD and CTD are represented in cyan. (B) Schematic model representation of the ‘‘Venus Fly-trap’’ mechanism. The conformational changes of the NTD (orange) and CTD (green) upon ligand (grey) binding are depicted with dotted lines.