CHAPTER 5 STRUCTURE OF βGlyBP……………………………………… 124-183
5.3 RESULTS
5.3.6 Structural determinants distinguishing between the α- and β-glycosides…
(Holm and Rosenstrom, 2010) yielded subcluster D-I SBPs bound to α-glycosides with high Z-score rather than to β-glycosides (Table C.3). Notably, among these homologs, the maltose-binding protein MalE3 (α-glycoside bound, PDB ID: 6DTQ) is annotated as a
“putative β-glucosides ABC transporter, substrate-binding protein” in the UniProtKB database (The UniProt Consortium, 2019), anticipating the shared structural homology between α- and β-glycosides-binding proteins. Since these structural data provide only about their multi-specificity and not about their ligand selection, we sought to perform an in-depth investigation to elucidate the mechanism(s) of glycosidic linkage selection and substrate preferences by the subcluster D-I SBPs. For this, structures of subcluster D-I SBPs α-glycoside-binding protein or αGlyBP (PDB ID: 6J9Y, Chandravanshi et al., 2020) and βGlyBP (PDB ID: 7C66, this study) from the bacterium T. thermophilus HB8 were compared as both the proteins strictly maintain their ligand preferences. A structural superimposition of αGlyBP_WT•MAL (α-glycoside bound, α-1,4; PDB ID: 6J9Y) and βGlyBP_WT•CEL2 (β-glycoside bound, β-1,4; PDB ID: 7C66) demonstrates that two structures are almost identical with a rmsd of 0.5 Å (Figure 5.9A). Interestingly, the binding mode of the nonreducing end (Glc1) is also reminiscent of αGlyBP. A comparison of the active site residues of the proteins αGlyBP and βGlyBP coordinating with the respective Glc1 unit of MAL and CEL2 exhibits that the active site regions (NTD, hinge and CTD) are conserved in both the proteins (Figure 5.9B-5.9D). Moreover, the conserved active site residues of the protein αGlyBP/βGlyBP Asp118/Glu117 and Gly286/Gly297 (hinge) and
Asp70/Thr66 (NTD) anchor the Glc1 unit of α- and β-glycosides (Figure 5.9E). These observations designate that the nonreducing end (Glc1) facilitates the initial ligand binding, however, may not govern the selectivity between the α- and β-glycosides.
Thus, to find out the structural factor(s) determining the stereo-chemical selectivity for α- and β-glycosides, the Glc1 unit of α-glycosides (MAL) was manually modeled on to that of the β-glycoside (CEL2) bound to the protein βGlyBP. The result reveals that owing to different glycosidic linkages, Glc2 unit of the α-glycosides alter its position with respect to the Glc2 unit of the β-glycoside and thus causes a steric clash with the residue Trp41 of the protein βGlyBP (Figure 5.9F). Thus, it can be proposed that the residue Trp41 of βGlyBP maintains the stereo-chemical selection and distinguishes between its cognate and non- cognate ligands.
Figure 5.9. Structural determinant(s) for ligand selection. (A) Structural superimposition of the proteins αGlyBP (pink, PDB ID: 6J9Y) and βGlyBP (blue and cyan, PDB ID: 7C66) bound to MAL (pink) and CEL2 (yellow), respectively. The bound ligands are shown as spheres while proteins are as ribbon model. (B and C) Active site comparison of the proteins αGlyBP and βGlyBP, respectively, where the conserved NTD, hinge region and CTD are shown in blue, orange and cyan, respectively. (D) Superimposition of MAL (pink) and CEL2 (yellow) occupying a similar spatial position at the active site with a slight shift in their configuration due to different glycosidic linkages. (E) The comparison of the
active site residues of the proteins αGlyBP and βGlyBP from NTD (blue) and hinge (orange) that interact with the nonreducing end (Glc1) of MAL (pink) and CEL2 (yellow).
(F) A close-up view of the active site of the protein βGlyBP with the modeled MAL (pink) in place of CEL2 (yellow). It depicts the steric clash between the Glc2 unit of MAL and the residue Trp41 (blue line).
To further substantiate this proposed ligand selection mechanism, structures of multi- saccharides-binding proteins such as MalE (PDB ID: 6DTU), GacH (PDB ID: 3K00), CpMnBP1 (PDB ID: 4R9G), BlAXBP (PDB ID: 3ZKK) and MBP (PDB ID: 4MBP) from the subcluster D-I SBPs were investigated. Superimposition of the protein βGlyBP with these proteins reveals that irrespective of the ligands type, all carbohydrates occupy a similar spatial position (Figure 5.10A). However, the orientation of β-glycoside bound to the protein βGlyBP is found to be in an opposite direction to that of α-glycoside bound to the protein MalE (Figure 5.10B and 5.10C). Thus, unlike the α-glycosides which occupy subsites A, B, C and D in the active site of MalE, β-glycoside fills four subsites -A, -B, A and B in the active site of protein βGlyBP (Figure 5.10D). To find out the cause of the occupancy of α- and β-glycosides in an opposite orientation, active sites of the two proteins βGlyBP (CEL4 bound, PDB ID: 7C68) and MalE (Maltotetraose or MTT bound, PDB ID:
6DTU) were investigated. Result reveals that the residues from the four secondary structural elements loop1 (L1), helix1 (H1), helix2 (H2) and helix3 (H3) drive the orientation of ligands (Figure 5.10E and 5.10F). A similar feature could also be observed in other subcluster D-I SBPs (Figure C.6).
Figure 5.10. Structural determinant(s) guiding the orientations of carbohydrates. (A) Structural superposition of the protein βGlyBP (blue and cyan, PDB ID: 7C68) and the subcluster D-I SBPs MalE (PDB ID: 6DTU), GacH (PDB ID: 3K00), CpMnBP1 (PDB ID:
4R9G), BlAXBP (PDB ID: 3ZKK) and MBP (PDB ID: 4MBP) in grey shows their conserved topology and the ligand binding sites. (B) Comparison of the orientation of the bound β-glycoside (CEL4, pink) to the protein βGlyBP and α-glycoside (MTT, green) to the protein MalE. Each Glc unit is numbered sequentially (1,2,....), considering the nonreducing end as the 1st Glc unit. (C) A schematic representation of α- and β-glycoside binding in an upward (top) and downward direction (bottom), respectively. Four subdomains of the proteins are labeled as N1, N2, C1 and C2 and the Glc units are numbered sequentially (1 to 4). (D) The four subsites (-A, -B, A and B) of βGlyBP (top) bound to a β-glycoside (CEL4, pink) and that (A, B, C and D) of αGlyBP (bottom) bound to an α-glycoside (MTT, green) are partitioned by a dotted vertical line. (E and F) Structural
determinants loop (L1) and helices (H1-H3) guiding the orientation of β-glycoside (CEL4, pink) and α-glycoside (MTT, green), respectively, are shown here. The amino acid residues from the structural elements (L1 and H1-H3) and carbohydrates are represented as dots and ball-and-stick model, respectively. The arrow denotes the directionality of the orientation of the bound ligand.