CHAPTER 4 STRUCTURE OF αGlyBP……………………………………... 73-123
4.1 INTRODUCTION
Carbohydrates are crucial biological macromolecules which serve as a major source of carbon and energy in a variety of physiological functions. To accomplish these functions, carbohydrates are available in diverse forms. Nature provides diversity to carbohydrates by creating a different combination of the basic monosaccharide units (e.g. glucose, GLC and fructose, FRU) to form higher oligosaccharides and the glycosidic linkages which joins two carbohydrate molecules (e.g. α-1,4 and α-1,6 in starch; β-1,4 in cellulose) (Hölemann and Seeberger, 2004). Altogether, these results in a higher structural diversity of carbohydrates which makes them extremely complex in the context of their length, conformation, monosaccharide-ring constituent and α/β anomeric configuration (Raich et al., 2016). Due to this diversity and complexity, microorganisms adopt a diverse set of transporters for their uptake during cellular metabolism. Uptake of carbohydrates in a cell is executed by three classes of transporters viz. (1) primary active transporter, (2)
secondary transporter and (3) group translocator (Saier, 2000a). Out of which, ATP- binding cassette (ABC) transporter belonging to the primary active transporter is the largest superfamily for carbohydrate transport (Saier, 2000b). ABC transporters utilize ATP as an energy source to facilitate the transport of solute molecules across the membrane and are classified as ABC exporters and importers. Although, architecturally both ABC exporters and importers contain common subunits namely the transmembrane domain (TMD) and the nucleotide-binding domain (NBD), ABC importers possess an additional domain referred to as substrate-binding proteins (SBPs) (Rees et al., 2009;
Wilkens, 2015). Functionally, SBPs capture the substrates and bring it towards the TMDs for subsequent translocation and thus renders specificity and directionality to importers (Maqbool et al., 2015). Interestingly, ABC exporters are distributed in all the domains of life while ABC importers are present only in prokaryotes and recently reported in plants too (Kang et al., 2011; Kretzschmar et al., 2011).
Based on overall topology and ligand specificity, SBPs are classified into seven different clusters (viz. A-F) and further subdivided into various sub-clusters. Out of which, SBPs involved in transporting carbohydrates belong to the clusters B and D (particularly, sub- cluster D-I) (Scheepers et al., 2016). SBPs belonging to these clusters comprise of two α/β domains viz. N- and C-terminal domain (NTD and CTD, respectively) connected by a loop which acts as a hinge as well as the carbohydrate-binding site (Berntsson et al., 2010). In an unliganded state, both the domains (NTD and CTD) remain separated (i.e.
open conformation) and can freely rotate via the hinge region, whereas upon ligand binding they move asymmetrically closer to each other (i.e. closed conformation) and encapsulate the ligand between them (Pandey et al., 2016). This conformational change of SBP upon ligand binding is proposed as a ‘‘Venus Fly-trap’’ mechanism (Mao et al., 1982). Structurally, SBPs belonging to the sub-cluster D-I possess a common structural fold and can bind a diverse range of carbohydrates varying in size and chemical constituents. Earlier studies have reported the role of structural adaptation at the active- site pocket of SBPs to maintain the selectivity and specificity based on carbohydrate length (Cuneo et al., 2009a). This structural adaptation is governed by five secondary structural elements viz. two loops (L1 and L2) and three helices (H1, H2 and H3) that
modulate the occupancy of carbohydrate at the four subsites (A, B, C and D) of the active site (Cuneo et al., 2009a). Although, many SBPs have been structurally characterized for carbohydrate binding, the detailed enumeration of selective ligand binding mechanism associated with the vast carbohydrate diversity and complexity remains unfulfilled.
The requirement of carbohydrate transporters varies with microbial habitat as it plays a pivotal role in the accumulation of diverse extracellular sugars inside the cells. Thermus thermophilus strains are halotolerant and reside in marine hot spring (Alarico et al., 2005). In order to survive the extreme environment, T. thermophilus utilizes a variety of carbohydrates including polysaccharides (e.g. starch and glycogen), oligosaccharides (e.g. α- and β-glucosides and galactosides) and monosaccharides (e.g. glucose, galactose and xylose) for its growth (Henne et al., 2004). In addition to these carbohydrates, the bacterium also accumulates compatible solutes such as trehalose and/or mannosylglycerate to maintain the halotolerancy (Santos and Da Costa, 2002). Trehalose is a disaccharide α-glycoside in which two glycosyl units (Glc1 and Glc2) are linked via an α-(1,1) glycosidic bond. For survival, T. thermophilus maintains the intracellular level of compatible solutes either by synthesis or by importing it through energy-dependent transport system such as ABC transporter (Silva et al., 2003). The ABC transport system of Thermus sp. for trehalose is identified to be multispecific in nature and is able to transport a wide range of substrates namely trehalose (TRE), maltose (MAL), sucrose (SUC), palatinose (PAL) and glucose (GLC) (Silva et al., 2005). Although, the trehalose transport system is identified as a multisubstrate transporter, the molecular basis of this multispecificity with preferential enumeration remains unclear. Currently, it is unknown whether this multispecific transport system possesses inherent selectivity or it transport substrates without any selection criteria. Henceforth, we hypothesize that the transport system exhibits specificity towards α-glycosides possessing various α-glycosidic linkages such as α-1,1 (e.g. trehalose, Glc1-(1,1)-Glc2), α-1,2 (e.g. sucrose, Glc1-(1,2)- Fru1), α-1,4 (e.g. maltose, Glc1-(1,4)-Glc2) and α-1,6 (e.g. palatinose, Glc1-(1,6)-Fru1).
Indeed, till date many ABC transport systems have been structurally described that transports α-(1,1)-, α-(1,4)- and α-(1,6)-glycosides from different microorganisms (Diez et al., 2001; Cuneo et al., 2009a; Ejby et al., 2016). However, the mechanism underlying
the transport of all types of α-glycosides through a single transport system still remains elusive. Furthermore, it remains unknown whether the transport system that recognizes the multiple disaccharide α-glycosides can also bind to other complex oligosaccharides that are decorated with α-glycosidic linkages. Also, whether the transport system that exist for multiple α-glycosides exhibits sufficient selectivity to discriminate from sugars that are composed of β-glycosidic linkages. The full evaluation of transport system is crucial to solve the enigma of a selective transport mechanism, where the full understanding of the physiological basis for the transport of multiple sugars through a single transport system is also essential.
Previously, we reported the genetic cluster involved in the transport and metabolism of α-glycosides involved in maintaining the intracellular trehalose level of T. thermophilus HB8 as well as discovered the physiological basis of transporting maltose and glucose through trehalose ABC transport system (Chandravanshi et al., 2019). In addition, we also discovered that the transport system possesses a selective mechanism based on the carbohydrate length and exhibit a significant preference for the disaccharides over higher oligosaccharide (Chandravanshi et al., 2019). Since, the closest homolog trehalose/maltose-binding protein (TMBP, ORF: TTC1627) from T. thermophilus HB27 has been biochemically characterized for the multisubstrate transport, however the molecular mechanism of multiple substrate translocation and stereoselectivity for α- glycosides remains elusive (Silva et al., 2005). In this study, we have determined the three-dimensional crystal structures of an SBP (ORF: TTHA0356 from Thermus thermophilus HB8) of an ABC transporter (ORFs: TTHA0354-TTHA0356) in open as well as closed conformations in complex with disaccharide α-glycosides (e.g. trehalose (α-1,1), sucrose (α-1,2), maltose (α-1,4), palatinose (α-1,6)) as well as with monosaccharide (e.g. glucose) and thus named it as “α-glycoside-binding protein or αGlyBP”. Information obtained from the structural and thermodynamic study of αGlyBP enabled us to postulate a selective mechanism for αGlyBP and also understand its ability to distinguish between α- and β-glycosides. In addition, structural characterization along with mutagenic studies provide a new prospect for the ligand-binding mechanism at atomic level which is in contrary to the well-established ‘‘Venus Fly-trap’’ mechanism.