Substrate‐binding proteins (SBPs) mediate ligand translocation and have been classified into seven clusters (A‐G). Although the substrate specificities of these clusters are known to some extent, their ligand‐binding mechanism(s) remain(s) incompletely understood. In this study, the list of SBPs belonging to different clusters was updated (764 SBPs) compared to the previously reported study (504 SBPs). Furthermore, a new cluster referred to as cluster H was identified. Results reveal that SBPs follow different ligand‐binding mechanisms. Intriguingly, the majority of the SBPs follow the ‘one domain movement’ rather than the well‐known ‘Venus Fly‐trap’ mechanism. Moreover, SBPs of a few clusters display subdomain conformational movement rather than the complete movement of the N‐ and C‐terminal domains.

Carbohydrate (or sugar) molecules are extremely diverse regarding their length, linkage and epimeric state. Selective acquisition of these molecules inside the cell is achieved by the substrate (or solute)-binding protein of ATP-binding cassette (ABC) transport system. However, the molecular mechanism underlying the selective transport of diverse carbohydrates remains unclear mainly owing to their structural complexity and stereochemistry. This study reports crystal structures of an a-glycoside-binding protein (aGlyBP, ORF ID: TTHA0356 from Thermus thermophilus HB8) in complex with disaccharide a-glycosides namely trehalose (a-1,1), sucrose (a-1,2), maltose (a-1,4), palatinose (a-1,6) and glucose within a resolution range of 1.6-2.0 A. Despite transporting multiple types of sugars, aGlyBP maintains its stereoselectivity for both glycosidic linkage as well as an epimeric hydroxyl group. Out of the two subsites identified in the active-site pocket, subsite B which accommodates the glucose and glycosyl unit of disaccharide a-glycosides is highly conserved. In addition, structural data confirms the paradoxical behavior of glucose, where it replaces the high-affinity ligand(s) (disaccharide a-glycosides) from the active site of the protein.Comparative assessment of open and closed conformations of aGlyBP along with mutagenic and thermodynamic studies identifies the hinge region as the first interaction site for the ligands. On the other hand, encapsulation of ligand inside the active site is achieved through the N-terminal domain (NTD) movement, whereas the C-terminal domain (CTD) of aGlyBP is identified to be rigid and postulated to be responsible for maintaining the interaction with the transmembrane domain (TMD) during substrate translocation.

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