1. Volume 25, Issue 11, Pages 1667-1678.e4 (November 2017)
Structural Insights into VLR Fine Specificity for Blood Group Carbohydrates 
Bernard C. Collins, Robin J. Gunn, Tanya R. McKitrick, Richard D. Cummings, Max D. Cooper, Brantley R. Herrin, Ian A. Wilson 
Structure 
Volume 25, Issue 11, Pages e4 (November 2017)
DOI: /j.str
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2. Structure 2017 25, 1667-1678.e4DOI: (10.1016/j.str.2017.09.003)
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3. Figure 1 Crystal Structures of Monoclonal VLR Antibodies
(A) Schematic showing domain organization of the VLRs in this study. O13 and Tn4-22 VLRs here have an extra LRR compared with RBC36. SP, signal peptide; CP, connecting peptide.
(B) Alignment of VLR ectodomain amino acid sequences by LRR. Conserved residues are highlighted in yellow. Residues involved in direct interactions with ligands are underlined and marked by triangles (red triangles, if the interaction is common to all VLRs, or black triangles if the interaction is unique to a particular VLR). Based on alignment generated with ClustalO.
(C) Alignment of apo structures of RBC36 (cyan), O13 (green), and Tn4-22 (magenta).
Structure  , e4DOI: ( /j.str )
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4. Figure 2 Conserved Trp Residue in Glycan Binding
Location of the conserved Trp found in the extended loops of the LRRCTs. H-Trisaccharide-bound structures are shown and colored as in Figure 1C. For clarity, glycan ligands are not shown.
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5. Figure 3 VLR-Glycan Interactions
(A) Glycans used in structural studies. Monosaccharides that occupy the same positions in each structure are labeled with the same color. The red box highlights the acetamide group of H-trisaccharide that is not present in 2′-fucosyllactose (equivalent position of 2′-fucosyllactose is marked by a red dashed box). FUC, fucose; GLC, glucose; GAL, galactose; NAG, N-acetyl-glucosamine.
(B–F) Schematic showing the interactions of (B) RBC36 with H-trisaccharide, (C) RBC36 with 2′-fucosyllactose, (D) O13 with H-trisaccharide, (E) O13 with LNnT, and (F) Tn4-22 with H-trisaccharide. Modified from diagrams generated with MOE (Chemical Computing Group, 2016). Green dashed lines and arrows indicate hydrogen bonds; thick green dashed arrows indicate H-pi interactions. For clarity, residues and waters that do not interact directly with glycans were omitted.
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6. Figure 4 Detailed View of the VLR-Glycan Interactions
(A) Positions of overlapped glycan ligands in aligned structures. Colored as in Figures 1 and 2.
(B–E) Structures of (B) RBC36:H-trisaccharide, (C) RBC36:2′-fucosyllactose, (D) O13:H-trisaccharide, (E) O13:LNnT, and (F) Tn4-22:H-trisaccharide complexes shown as cartoon representations. Side chains involved in direct interactions are shown as sticks. Residue numbers shown in red refer to residues involved in VLR-glycan interactions that are common to all three VLRs. Green dashed lines indicate hydrogen bonds. Black dashed lines indicate H-pi interactions.
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7. Figure 5 Recognition of the Acetamide Group of H-Trisaccharide by O13
(A and B) Close-up view of the acetamide binding pocket of the O13:H-trisaccharide complex represented as (A) sticks and (B) a surface representation.
(C and D) The same views of the acetamide binding pocket of the RBC36:H-trisaccharide complex.
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8. Figure 6 Model of LNnT Bound to RBC36
RBC36 (ribbon) with LNnT (yellow sticks) modeled in the glycan binding pocket (top). Close-up view of the modeled fit shows predicted hydrogen bonds (green dashed lines) (bottom).
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9. Figure 7
Improved Specificity of an Engineered O13 VLR with Two Mutations
(A) Detailed view of the structural alignment of O13:LNnT complex (green) with apo RBC36 (cyan).
(B) Zoomed-out view of the O13 and RBC36 structural alignment highlighting the N-terminal regions of each VLR. Arrows indicate the distance between the backbone at O13 K36 and the backbone of the equivalent position in RBC36. Hydrogen bonds are colored magenta.
(C) Glycan binding affinity of RBC36, O13 wild-type, and O13 mutant, as determined by ITC (N.B., no binding).
(D) Raw ITC data (top of each panel) and binding isotherms (bottom of each panel) from ITC experiments with the O13 mutant and H-trisaccharide (left) or LNnT (right).
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