Fig 1.
Comparison of β-prism domains from VCC, Bap1, and RbmC.
(A) Crystal structure of the heptameric pore state of VCC [62] showing β-prism domains in orange and D617 (essential for carbohydrate binding) indicated by magenta spheres. (B) Schematic showing approximate locations of β-prism domains (shaded orange) in VCC, Bap1, and RbmC. Red box in Bap1 indicates the approximate location of the 58 amino acid insertion of unknown function. (C) Sequence alignment produced using MEGA7 [63] and ESPript v. 3.0 (http://espript.ibcp.fr) [64] of four V. cholerae β-prism domains. Identical residues are shaded red whereas similar residues are indicated by red type. The second RbmC β-prism domain contains a five-amino acid insertion near the glycan-binding pocket (shaded blue). VCC D617 is marked by a purple asterisk. Structure figures made using the PyMOL Molecular Graphics System, Version 1.8 (Schrödinger, LLC).
Fig 2.
Glycan chip results for RbmC1 and RbmC2.
Glycan chip data is represented in relative fluorescence units with the standard deviation of four replicates indicated by red error bars for (A) RbmC1 and (B) RbmC2. The top glycan hits are shown in schematic representation based on the key (inset). Fragments of complex N-linked glycans make up most positive hits.
Fig 3.
Binding affinities for various glycans were determined using ITC (blue) or intrinsic tryptophan fluorescence spectroscopy (red). Errors are reported as 95% confidence levels (for ITC data) or the standard error of the mean (for fluorescence data). Dashes represent experiments not carried out due to lack of feasibility. Carbohydrates are represented as described in Fig 2A. *Asialofetuin is a glycoprotein containing three primary glycosylation sites, which contain a mixture of bi- and tri-antennary glycans [35]. Data were fit assuming three sites per glycoprotein, but due to the heterogeneity of glycosylation these numbers may represent an overestimation of the binding affinity. The asialofetuin schematic shows the predominant tri-antennary glycan.
Fig 4.
Crystal structures of RbmC2 with mannotriose and GlcNAc-Man bound.
(A) Crystal structure of the isolated RbmC2 β-prism domain with mannotriose (1,3-α-1,6-α-D-mannotriose) shown in stick representation. The mannotriose core in a typical complex N-glycan is boxed (lower left, see key in Fig 2A). A simulated annealing OMIT electron density map contoured to 2.5 σ for the ligand is displayed in blue mesh, duplicated and extracted from the overall structure for clarity (lower right). (B) Stereo view of residues that directly interact with the mannotriose ligand. Putative hydrogen bonds are displayed as blue dotted lines and van der Waals contacts are denoted with a semi-transparent surface representation. Within the mannotriose ligand (green sticks), the attachment point of connecting saccharides in an intact complex N-glycan are colored yellow. A water molecule (B-factor = 4.18 Å2) (blue sphere) is held in a tetrahedral coordination sphere involving N871, W948, and two hydroxyl groups from the mannotriose glycan fragment. (C) Same as in A, but showing the GlcNAc-Man-bound structure, solved to 1.8 Å resolution. (D) Same as in B, but for the GlcNAc-Man structure.
Table 1.
X-ray and refinement statistics.
Fig 5.
Structure/function implications of glycan binding.
(A) The effect of selected mutations on the hemolytic activity of VCC is displayed as the ratio of the concentration of half-activity (HD50) for each mutant relative to wild-type toxin. (B) Crystal structure of the VCC β-prism domain with the composite GlcNAcMan3 model superimposed. The effects of mutations described in (A) are color coded and displayed on the protein surface. The inset box shows a closeup of the D617, L707, W706 core in a similar orientation. (C) Model for complex N-glycan binding based on the RbmC2 mannotriose structure, GlcNAc-Man structure, and mutagenesis data. The pentasaccharide core is shown boxed on a schematic representation of a complex N-glycan. Yellow arrows indicate the point of attachment of the subsequent two galactosyl moieties in a typical glycan and blue arrow denotes the attachment point of the double N-acetylglucosamine stem that attaches the glycan to asparagine residues on cell-surface proteins. The RbmC2 PVQGT loop insertion is colored orange and the surface of W706 colored yellow.
Fig 6.
Binding of RbmC1 and RbmC2 to mammalian cells.
(A) Fluorescence microscopy of defibrinated rabbit whole blood incubated with the RbmC2-GFPUV fusion. Wild-type or a D853A point mutation (with significantly reduced glycan-binding activity) are shown. Images include GFP, brightfield, and merged channels for constructs at 2.5 μM and 0.5 μM concentrations. Each WT/mutant pair was placed on an identical brightness scale, but WT and D853A are on different brightness scales. The white scale bar (lower right on the merged image) represents 10 microns. (B) Same as in A, but with WT RbmC1 and the D539A point mutation (purple asterisk on Fig 1C).