CHAPTER 5 STRUCTURE OF βGlyBP……………………………………… 124-183
5.1 INTRODUCTION
ATP-binding cassette (ABC) transporters are the largest superfamily of proteins that facilitate the translocation of a diverse array of substrates across the plasma membrane using ATP as the energy source (Higgins, 1992). ABC transporters are classified into importers and exporters depending upon the direction (inside or outside of the cell, respectively) of the substrates being transported (Wilkens, 2015; Szollosi et al., 2018).
Although ABC exporters are ubiquitously distributed in all domains of life, ABC importers are reported only in prokaryotes and plants till date (Davidson et al., 2008; Lefevre and Boutry, 2018). Both the ABC importers and exporters share a typical architecture of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) which function as substrate translocator and energy generator from ATP hydrolysis, respectively. Unlike ABC exporters, ABC importers require an additional component known as substrate (or
solute)-binding protein (SBP) (van der Heide and Poolman, 2002; Marinelli et al., 2011;
Scheepers et al., 2016). SBPs capture their cognate ligands from the periplasmic or extracellular environment and deliver them to the TMDs for the subsequent translocation into the cell (Davidson et al., 1992). Irrespective of the types of the ligands, SBPs possess a conserved structural fold having two globular α/β domains with a central β-sheet flanked by α-helices (Berntsson et al., 2010). These two domains are linked by a flexible hinge region which allows the free rotation of these domains for substrate capturing via the
‘‘Venus Fly-trap’’ mechanism (Mao et al., 1982). Based on their topology, particularly of the hinge region, SBPs have been classified into seven different clusters, A-G (Berntsson et al., 2010; Scheepers et al., 2016).
Structural studies on SBPs have demonstrated that the hinge region facilitates the transition from an open to closed state upon ligand binding and controls the equilibration between these two states (Quiocho and Ledvina, 1996; Shilton et al., 1996). As the structural feature of the hinge region varies across the SBP clusters, the degree of domain movement from an open to closed state also differs (Begg et al., 2015; Chandravanshi et al., 2020).
Nevertheless, this degree of domain movement is independent of the size and type of ligands bound to the protein (Magnusson et al., 2004; Trakhanov et al., 2005; Pandey et al., 2016; Chandravanshi et al., 2020). Two basic models associated with the domain movement describing the ligand-binding mechanism have been proposed: (1) conformational selection and (2) induced-fit mechanism. In the conformational selection mechanism, ligands bind to a preformed closed-unliganded state, whereas, in the induced- fit mechanism, ligand binding triggers the domain movement to bring the closed conformational changes (de Boer et al., 2019a). Between the two mechanisms, most SBPs follow the latter i.e. the induced-fit mechanism as it enables the translocator (TMDs) to differentiate between the unliganded and liganded states (Doeven et al., 2008).
In the induced-fit mechanism, an essential intermediate (open-liganded) state is known to couple the two end (open and closed) states; it is a thermodynamically unfavorable and transient state (de Boer et al., 2019a). Owing to its transient nature, capturing its molecular details becomes difficult. Moreover, to obtain its mechanistic insights, details of ligand
binding as well as conformational dynamics of the intermediate state is inevitable.
Although an array of structural and biophysical data detailing the mechanistic insights into the induced-fit mechanism of SBPs have been reported (Skrynnikov et al., 2000;
Trakhanov et al., 2005; Silva et al., 2011; de Boer et al., 2019a, b), these relate to the initial (open) and/or the final (closed) states only. Moreover, the mechanisms for the ligand recognition and selection by SBPs are not well delineated. Although the selectivity of ABC importers that is shown to be governed by the conformational state(s) and ligand-release kinetics of SBPs is well accepted (de Boer et al., 2019b), a precise relationship between selective ligand binding and conformational dynamics of SBPs has not been well established till date.
Carbohydrate-specific SBPs are pertinent to understand this relationship due to the complexity of the substrate (i.e. carbohydrate) having varying length, anomeric configuration, glycosidic linkage and epimeric state (Hölemann and Seeberger, 2004;
Raich et al., 2016). Consequently, carbohydrate-specific SPBs attain different topologies and thus are classified into four distinct subclusters B-I, C-IV, D-I and cluster G specific to monosaccharides, linear oligosaccharides, linear, circular & branched oligosaccharides and polysaccharides, respectively (Fukamizo et al., 2019). Surprisingly, despite having different topologies, subclusters C-IV and D-I SBPs are designated to facilitate the uptake of linear β-glycosides (Cuneo et al., 2009b; Abe et al., 2018). Depending upon the glycosidic linkages, β-glycosides are categories as linear glucan such as β-1,2-glucan, β- 1,3-glucan (laminarin), β-1,4-glucan (cellulose), β-1,3/1,4-glucan (lichenan), and branched glucan such as β-1,3/1,4-glucan (calocyban) and β-1,3/1,6-glucan (lentinan) (Synytsya and Novak, 2014). These polysaccharides are further catabolized into shorter gluco- oligosaccharides such as sophoro- (SOPn; β-1,2), laminari- (LAMn; β-1,3), cello- (CELn;
β-1,4) and gentio-oligosaccharides (GENn; β-1,6), where n represents the number of glycosyl (Glc) unit or a degree of polymerization (DP). These gluco-oligosaccharides are the preferred substrates for β-glucosidases (Chuenchor et al., 2011). Although the understanding of the various gluco-oligosaccharides metabolism by β-glucosidases are well documented, their uptake through an ABC import system remains elusive.
Nevertheless, it can be speculated that multi-specificity of the ABC import system would
be essential to fulfill the demand of a broad range of substrates to β-glucosidases. In the previous in silico study, we suggested that a single ABC import system (ORF IDs:
TTHB082-TTHB086) of a thermophilic gram-negative bacterium Thermus thermophilus HB8 which is enough to uptake β-glycosides unlike α-glycosides for which multiple import systems (ORF IDs: TTHA0354-TTHA0356 and TTHA1650-TTHA1652) are required (Chandravanshi et al., 2019). However, this premise required further confirmatory evidences.
Thus, in this study, we report the crystal structures of the SBP (ORF ID: TTHB082) to accomplish the insights into the ligand binding and selection mechanism of β-glycosides.
Moreover, this study provides the first-ever structural data of an SBP bound to a variety of β-glycosides such as sophoro- (SOPn; β-1,2), laminari- (LAMn; β-1,3), cello- (CELn; β- 1,4) and gentio-oligosaccharides (GENn; β-1,6). Furthermore, the structural data corroborated the thermodynamic data establishing the SBP (ORF ID: TTHB082) as a β- glycosides-binding protein (βGlyBP). Using a combination of structural and thermodynamic data of the wild type and mutants, here we propose the ligand binding and selection mechanism of the protein βGlyBP.