Mechanisms of neutralization of a human anti-α-toxin antibody

Abstract

MEDI4893 is a neutralizing human monoclonal antibody that targets α-toxin (AT) and is currently undergoing evaluation in the field of Staphylococcus aureus-mediated diseases. We have solved the crystal structure of MEDI4893 Fab bound to monomeric AT at a resolution of 2.56 Å and further characterized its epitope using various engineered AT variants. We have found that MEDI4893 recognizes a novel epitope in the so-called "rim" domain of AT and exerts its neutralizing effect through a dual mechanism. In particular, MEDI4893 not only sterically blocks binding of AT to its cell receptor but also prevents it from adopting a lytic heptameric trans-membrane conformation.

Keywords: Alpha Toxin; Antibody; Crystal Structure; Fab; Infectious Disease; Mutagenesis; Staphylococcus aureus (S. aureus).

FIGURE 1.

A, three-dimensional structure of the complex between monomeric AT (olive) and MEDI4893 Fab (HC (green)/LC (blue)). AT regions that undergo large conformational changes upon heptamerization (“latch” and stem; PDB code 7AHL) are shown in red. MEDI4893 Fab epitope comprises AT residues Asn-177–Arg-200 and Thr-261–Lys-271 in the rim (orange). B, AT binds in a crevice between MEDI4893 Fab HC (green) and LC (blue). Positive and negative electrostatic potentials are indicated in blue and red, respectively, and were calculated using APBS (28). This and all other figures were made using PyMOL (Schrödinger).

FIGURE 2.

Interface between MEDI4893 Fab HC (green) and AT (olive) (A) and MEDI4893 Fab LC (blue) and AT (olive) (B). Both chains of the Fab are in close contact with the rim of AT and create several hydrogen bonds (dotted lines) and one π-π stacking interaction between MEDI4893 Fab Trp-32 (LC) and AT Trp-187. AT residues in contact with MEDI4893 are shown in orange.

FIGURE 3.

A, superimposition of monomeric AT of this study (green) with pore-forming AT (light blue, PDB code 7AHL; Ref. 12). The latch in our monomeric AT is modeled starting residue 12 due to the absence of corresponding electron density map, whereas those residues are stabilized through interaction with neighboring molecules in the pore-forming state. The AT stem in the pore-forming conformation is extended to make the β-barrel together with six other protomers, whereas in our monomeric conformation, it is compactly folded into β-strands. B, superimposition of monomeric AT bound to MEDI4893 Fab (this study, green) with monomeric AT bound to mAb LTM14 (PDB code 4IDJ, Ref. , blue). Both Fab molecules bind to the same rim domain, though on opposite sides. Residues known to be critical for AT interaction with the cell surface receptor (13) are shown as red spheres.

FIGURE 4.

Stereographic representation of four out of seven AT protomers in a lytic pore (orange, blue, gray, and light blue; PDB code 7AHL; Ref. 12). AT·MEDI4893 Fab (this study, green) and AT·LTM14 Fab (PDB code 4IDJ, Ref. , red) complexes were superimposed with one of the pore AT protomers (blue). MEDI4893 Fab blocks the interaction with the other three AT protomers, thus preventing pore formation. In particular, (i) MEDI4893 Fab LC creates a steric hindrance with the neighboring AT protomer (orange) in the rim region, and (ii) two other AT protomers (gray and light blue) are restricted by portion of MEDI4893 Fab HC to extend their stem. LTM14 Fab does not appear to block heptamer formation.

FIGURE 5.

ProteOn sensorgrams for the binding of LC10 IgG to AT and select variants thereof. Assays were run as described under “Experimental Procedures.”

FIGURE 6.

AT regions identified as important for LC10 binding using KO variants. Amino acids 101–110 are shown in blue, 224–241 are shown in black, and 248–277 are shown in red.

FIGURE 7.

LC10 inhibits AT binding to THP-1 cell surface. LC10 (green) or negative control Arg-347 (purple) were mixed with biotin-conjugated AT_H35L, and added to THP1 cells. THP-1 cells were also incubated with AT_H35L alone (blue). The background consisted of THP1 cells alone (red). AT binding was measured by cytofluorimetry after addition of streptavidin-pacific blue.