CHAPTER 4 STRUCTURE OF αGlyBP……………………………………... 73-123
4.3 RESULTS
4.3.3 N-terminal domain of αGlyBP dictates the open and closed conformations… 104
of hydrogen bonds as compared to trehalose, it still retains the ability to replace and occupy the Glc1 position of trehalose at the subsite B. To assess the cause, a comparison of αGlyBP_WT•GLC was drawn with disaccharide α-glycosides (i.e. trehalose, sucrose, maltose and palatinose)-bound structures. Interestingly, at subsite B the Glc1 unit of all the disaccharide α-glycosides is absolutely superimposable on glucose and forms similar number of hydrogen bonds (Figure 4.7A and Table B.1). Furthermore, analysis of the
active site of the structural homolog Atu4361 protein bound to four different type of ligands viz. glycerol (PDB ID: 4QSE), sucrose (PDB ID: 4QSD), maltose (PDB ID:
4QSC) and maltotriose (PDB ID: 4QRZ) reveals a similar mode of binding. Despite the vast diversity of carbohydrates, both αGlyBP and Atu4361 proteins are specific to α- glycosides and conserves a similar binding mode. Hence, to further infer the cause of conserved binding mode for diverse ligands in both the proteins, glycerol-bound Atu4361 protein was compared with sugar-bound structures. Since, glycerol is the smallest ligand among the three, it can occupy any position in the active site. However, akin to glucose in αGlyBP_WT•GLC complex, all three hydroxyl group and carbon backbone of glycerol is well overlaid upon the O2, O3 and O4 oxygen atoms of α-glycosides (sucrose, maltose and maltotriose) (Figure 4.7B). In-depth investigation of the active site of Atu4361 reveals that the three hydroxyl groups of glycerol strongly interact with the residues Asp89, Asn142, Asp298 and Arg367 (Numbering according to Atu4361 protein).
Notably, out of four interacting residues, three (Asn142, Asp298, and Arg367) are from the hinge region and one (Asp89) from the NTD, whereas no residue from the CTD is involved in the interaction (Figure 4.7C). Based on this analysis, the hinge region can be considered as the first interacting site for the ligand and Asp89 from NTD as the first residue responsible for the domain closure while no involvement of CTD residues suggest that CTD does not participate in domain closure upon ligand binding.
Furthermore, analysis for conservation of NTD and hinge residues in αGlyBP as well as its structural homologs suggests that the subsite B (i.e. Glc1 binding site) is the initial ligand binding site as it possess all the four residues i.e. domain closure (Asp70) and hinge (Asp118, hinge1; Gly286, hinge2 and Arg356, hinge3) residues and thus conserves the similar binding mode for Glc1 unit in αGlyBP and for glycerol in Atu4361 protein (Figure 4.7C and 4.7D).
Figure 4.7. Conservation of structural determinant in the subsite B for ligand binding. (A) Overlay of αGlyBP bound to trehalose (yellow), sucrose (blue), maltose (grey), palatinose (green) and glucose (violet). (B) Overlay of Atu4361 protein bound to glycerol (green), sucrose (blue), maltose (grey) and maltotriose (magenta). (C) Active- site comparison of αGlyBP and Atu4361 protein (glycerol bound). Three hinge residues (Asn142, Asp298, and Arg367) and NTD residue (Asp89) interacting with glycerol (ball- and-stick model in green) via hydrogen bonding (dotted lines) are represented in cyan and orange, respectively. The highly conserved hinge residues (Asp118, Gly286 and Arg356) and NTD residue (Asp70) of αGlyBP occupying a similar position are shown with grey line. (D) Structure-based sequence alignment of αGlyBP with trehalose/maltose-binding protein (PDB ID: 1EU8; UniProt ID: Q7LYW7), extracellular solute-binding protein family 1 (PDB ID: 5CI5; UniProt ID:A8F7X5), acarbose/maltose-binding protein GacH (PDB ID: 3K00; UniProt ID: B0B0V1), ABC-type sugar transporter (PDB ID: 4QSE;
UniProt ID: A9CGI0) and sugar ABC transporter (PDB ID: 5IAI; UniProt ID: B9JM84) using the program PROMALS3D (Pei and Grishin, 2014) followed by further rendering using online web tool ESPript 3.0 (Gouet et al., 2003). The accession codes for the PDB and UniProt IDs are provided in the parenthesis. For the figure clarity, only a partial alignment has been shown here. Conservation of domain closure (Asp70) and hinge residues (Asp118, Gly286, and Arg356) in all homologous proteins are highlighted in orange and cyan, respectively.
To further affirm the importance of domain closure residue (Asp70) and hinge residues (Asp118, Gly286 and Arg356) in ligand binding, all of them were mutated to alanine except Gly286 as its backbone atom (N) participates in ligand binding. All the mutant proteins were subjected to crystallization in similar condition; however, the crystals of only αGlyBP_R356A mutant protein could be obtained, albeit in a different space group
P21. Interestingly, the overall structure of αGlyBP_R356A mutant protein reveals the open conformation of αGlyBP in an unliganded state. Topologically, αGlyBP_R356A mutant protein (unbound structure) is identical to that of a αGlyBP_WT•TRE (sugar- bound structure) with an RMSD of 4.2 Å. Overlaying the NTD and CTD of the open and closed conformations using the web server DynDom (Hayward and Berendsen, 1998) showed an RMSD of 1.23 Å and 0.68 Å, respectively, illustrating a larger conformational change for NTD than CTD. Furthermore, the distance between Cα atom of Lys31 (helix α1) and Ala59 (helix α2) in both the open and closed conformations remain constant (~31 Å) indicating that NTD undergoes a rigid translation movement (1.3 Å) (Figure B.1).
Calculation of rotational angle of NTD movement by DynDom server shows that change in the torsion angle of hinges allows a rotation of 41.9°. A similar rigid movement of NTD is also identified in the structural homologs such as Atu4361 protein (unbound, PDB ID: 4RJZ and sugar-bound, PDB ID: 4QRZ) and acarbose/maltose-binding protein GacH (unbound, PDB ID: 3K01 and sugar-bound, PDB ID: 3K02), where the distance (~30 Å) between helices α1 and α2 in their respective NTD remains unaltered while the degree of rotation angles (29.5º, 33.4º and 41.9º) increases as the ligand size (GacH receptor- pentasaccharide, Atu4361 protein-trisaccharide and αGlyBP-disaccharide, respectively) decreases (Figure 4.8A-4.8C).
Interestingly, analysis for domain closure mechanism based on DynDom server prediction suggests that upon ligand binding NTD shows a larger conformation change than the CTD which is in accordance with the previously reported asymmetric domain movement mechanism (Pandey et al., 2016). However, based on above mentioned analysis, we propose an extension for asymmetric domain movement mechanism, where only NTD participates in domain closure while CTD plays no role. Moreover, despite the available information for various ligand binding, the molecular details of the mechanism are still underexplored. Hence to delineate the atomic details of domain closure, the molecular organization of active-site residues were compared in both the open and closed conformations. In the unliganded form, the hinge 2 residue Gly286 interacts with residue Asp70 from the NTD via Val71 as well as with residue Asp118 and Arg356 from hinge 1 and hinge 3 region, respectively. Upon ligand binding, the
Glc1 unit interacts with the hinge residues which pulls the domain closure residue Asp70 via Val71 subsequently compelling the NTD to move towards CTD (Figure 4.8D). In addition, further characterization of αGlyBP_D70A mutant protein was done through measurement of the energetic contribution of Asp70 in binding to α-glycosides using ITC experiments considering the active site to be free from endogenously bound ligand due to mutation. Strikingly, a D70A mutation led to a ~210-fold reduction in the binding affinity of trehalose as compared to that of αGlyB_WT protein. This observation suggests that although the Glc1 unit of all α-glycosides binds to the hinge region at subsite B with a negative enthalpy change, mutation of domain closure residue Asp70 to alanine inhibits the NTD to undergo an open-to-closed transition (Figure B.2 and Table 4.9).
Figure 4.8. Domain movement upon ligand binding. (A-C) Superimposition of unliganded (αGlyBP_R356A-cyan, PDB ID: 6JAL; Atu4361-lime, PDB ID: 4RJZ and GacH-magenta, PDB ID: 3K01) with ligand-bound structures (αGlyBP_WT•TRE-blue, PDB ID: 6J9W; Atu4361-green, PDB ID: 4QRZ and GacH-purple, PDB ID: 3K02). The bound ligand molecule is shown as yellow sphere. Open and closed structures are superimposed at the CTD residues. Rigid movement of NTD is depicted by highlighting the helix α1 (open conformation) and α1' (closed conformation) in red along with their respective angle of movement. (D) A schematic and atomic details of change in the interaction in the presence (left panel) and absence of ligand (right panel) during domain movement.
4.3.4. Mutation of active-site residues alter ligand specificity
Since mutation of hinge 3 residue Arg356 into alanine led to the open conformation of αGlyBP, the energetic contribution of αGlyBP_R356A mutant protein for the disaccharide α-glycosides binding was also determined. Similar to αGlyBP_D70A mutant protein, mutation of Arg356 into alanine leads to a decrease in the binding affinity for trehalose. However, it did not completely lose the binding activity as it showed interaction with all α-glycosides in the ITC experiments. The binding affinity (Kd) of αGlyBP_R356A for disaccharide α-glycosides are found to be in the range of
~1-16 µM, which is in agreement with previous reports for other SBPs (Berntsson et al., 2010). Thermodynamically, similar to αGlyBP_D70A mutant protein, αGlyBP_R356A mutant protein also exhibited a higher affinity for maltose (Kd: 1.61 µM) than trehalose (Kd: 5.95 µM). The binding isotherm for all disaccharide α-glycosides and glucose with αGlyBP_R356A mutant protein are driven by a negative enthalpy change while a positive enthalpy change is observed for maltose (Figure 4.9A, Figure B.3 and Table 4.9). According to the structural data, the three-dimensional structure of all the αGlyBP_R356A mutant protein complexes (αGlyBP_R356A•TRE, αGlyBP_R356A•MAL, αGlyBP_R356A•SUC, αGlyBP_R356A•PAL and αGlyBP_R356A•GLC) are similar to αGlyBP_WT complex structures with an RMSD value of ≤ 0.1 Å for Cα atoms. No differences, except for the loss of hydrogen bonding of O3 and O4 atoms with Arg356 residue, in hydrogen-bonding pattern of the WT and mutant complexes were found. However, in-depth investigation of αGlyBP_R356A mutant protein complexes reveals that in sucrose, O3 and O4 atoms of Glc1 forms two water-mediated interactions with Leu287 (hinge residue) and Glu174 (CTD residue) and thus compensate for the loss of interaction with Nε1 and Nε2 atoms of Arg356 due to mutation. Similar to sucrose, the binding of other disaccharide α-glycosides and glucose also restores the hydrogen bonding with the O4 atom, (Figure 4.9B-4.9F). This observation for restoration of hydrogen bonding correspond well with thermodynamic data, where water-mediated interaction plays the role of hinge 3 residue and thus favors the binding of disaccharide α-glycosides and glucose with αGlyBP_R356A mutant protein.
Figure 4.9. Binding of disaccharide α-glycosides with αGlyBP_R356A mutant protein. (A) Thermodynamic profile of ligand binding with αGlyBP_R356A mutant protein, where the change in enthalpy (ΔH, green) and entropy (TΔS, blue) and the Gibbs free energy of binding (ΔG, red) for each ligand are represented as histogram in kcal mol-
1. The histograms show that the binding of disaccharide α-glycosides with αGlyBP_R356A mutant is more entropically favourable excluding sucrose which shows the enthalpically favourable binding profile. (B-F) Active site of αGlyBP_R356A mutant protein bound to sucrose (blue), trehalose (yellow), maltose (grey), palatinose (green) and glucose (violet), respectively. In all the complexes, the position of Arg356 is occupied by water (W1, red sphere) molecule, which forms water-mediated (W1) interaction (dotted lines) with Glu174 represented in green line model. In the sucrose complex structure, another water (W2) forms water-mediated interaction (dotted lines) with hinge residue Leu284 shown in cyan line model.
4.3.5. Calcium ion (Ca2+) imparts the role of hinge 1 residue in conferring stability