Fig 1.
Design and characterization of cap-stabilized RTX domain fragments containing blocks I–III.
(A) Design scheme of fusions between block III and block V cap fragment. RTX repeats from block III and V are represented in terms of their β-strands, numbered by RTX repeat within each RTX block, and their connecting segments, which are mostly Ca2+-binding turns but can contain loop insertions. For each variant, the strands and connecting segments from block III are shown in teal, and those from block V are shown in magenta. (B) Schematic of the ACT gene showing the catalytic domain (green), hydrophobic segment (brown), palmitoylation sites, RTX domain with RTX blocks (teal) and C-terminal cap (magenta). The scheme is shown for fusing the C-terminal cap to a fragment containing RTX blocks I-III, with block III and the C-terminal cap shown in 3D cartoon representation. (C) Size-exclusion chromatography elution profiles for purified cap fusion variants, with colors corresponding to the boxes in A. The dotted line represents the peak elution volume that is consistent with monomeric 123cap. (D) Differential scanning fluorimetry melting profiles for F3, F4, and F5, with data shown as the negative first derivative of the fluorescence intensity. Colors correspond to the boxes in A. (E) Surface plasmon resonance kinetic measurements of M2B10 and M1H5 Fab binding to RTX751 and 123cap. Measured signal for each Fab concentration is shown as a black line, and kinetic fits are shown as red lines. Fit kinetic parameters and equilibrium dissociation constants are shown for each interaction under the corresponding concentration series.
Fig 2.
Crystal structure of 123cap bound to the receptor-blocking antibodies M2B10 and M1H5.
(A) Overall structure of 123cap in complex with M2B10 and M1H5 Fabs. Ca2+ ions are shown as yellow spheres. (B) Structural alignment of the capping structure of 123cap with the WT capping structure from the crystal structure of block V (PDB ID: 5CVW), with the WT cap shown in magenta and the fused cap shown in teal. (C) Topology diagram of blocks I–III from 123cap, including the topology of linker 1 and linker 2. (D) Structure of L1, L2, L4 and the region from the capping structure with the same topological motif. Each is colored as an N to C rainbow (blue to red) to show the path of the mainchain.
Fig 3.
The ACT inter-block linkers are conserved modules.
(A) Sequence alignment of L1, L2, L3, and L4 from B. pertussis ACT. The locations of the core β-sheets are shown below the alignment, with the antiparallel β-strand shown with a black fill. Letters above the alignment denote the subsequent panels that highlight the specified residue. (B) Structural alignment of L1, L2 and L4 (L4 from PDB ID: 6SUS). (C) Conserved Ca2+-binding glutamate at the C-termini of L1, L2, and L4. (D) Conserved core tyrosine/phenylalanine at the N-termini of L1, L2, and L4. (E) Partially conserved buried lysine residue in L1 and L2.
Fig 4.
Interactions formed by M2B10 with linker 1 of the RTX domain.
Structures are shown as ribbon representation with select residues shown as sticks, with nitrogens colored blue, oxygens colored red, and sulfur colored yellow. Calcium is represented as yellow spheres.
Fig 5.
Interactions formed by M1H5 with linker 2 of the RTX domain.
Structures are shown as ribbon representation with select residues shown as sticks, with nitrogens colored blue, oxygens colored red, and sulfur colored yellow. Calcium is represented as yellow spheres.
Fig 6.
RTX751 and 123cap elicit similar neutralizing antibody responses.
(A) Mouse serum titers against RTX751, 123cap and ACT after immunizing and boosting with either RTX751 or 123cap and Freund’s adjuvant. Each data point represents one mouse, with titers measured by ELISA using the murine M1H5 antibody as a standard. The x-axis tick labels define the antigen of reactivity. (B) Toxin neutralization assay with 0.5 ug/mL ACT added to J774A.1 cells in the presence of a 30-fold molar excess of a neutralizing anti-ACT antibody (M1H5), non-neutralizing anti-ACT antibody (M1F5) or immune sera against 123cap or RTX751 (1:250 dilution, previously determined to be in the dose-response range; individual mice labeled #1, #2, #3). Each bar represents the mean of 2 independent assays, with error bars representing the standard error of the mean, and with each assay containing 3 technical replicates. The mean cell lysis value for sera resulting from RTX751 and 123cap immunization are shown as dashed red lines.