NadA3 Structures Reveal Undecad Coiled Coils and LOX1 Binding Regions Competed by Meningococcus B Vaccine-Elicited Human Antibodies

Abstract

Neisseria meningitidis serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion. Six major genetic variants of NadA exist and can be classified into immunologically distinct groups I and II. Knowledge of the crystal structure of the 4CMenB vaccine component NadA3 (group I) would improve understanding of its immunogenicity, folding, and functional properties and might aid antigen design. Here, X-ray crystallography, biochemical, and cellular studies were used to deeply characterize NadA3. The NadA3 crystal structure is reported; it revealed two unexpected regions of undecad coiled-coil motifs and other conformational differences from NadA5 (group II) not predicted by previous analyses. Structure-guided engineering was performed to increase NadA3 thermostability, and a second crystal structure confirmed the improved packing. Functional NadA3 residues mediating interactions with human receptor LOX-1 were identified. Also, for two protective vaccine-elicited human monoclonal antibodies (5D11, 12H11), we mapped key NadA3 epitopes. These vaccine-elicited human MAbs competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. The data presented provide a significant advance in the understanding of the structure, immunogenicity and function of NadA, one of the main antigens of the multicomponent meningococcus B vaccine.IMPORTANCE The bacterial microbe Neisseria meningitidis serogroup B (MenB) is a major cause of devastating meningococcal disease. An approved multicomponent vaccine, 4CMenB, protects against MenB. Neisserial adhesin A (NadA) is a key vaccine antigen and acts in host cell-pathogen interactions. We investigated the 4CMenB vaccine component NadA3 in order to improve the understanding of its immunogenicity, structure, and function and to aid antigen design. We report crystal structures of NadA3, revealing unexpected structural motifs, and other conformational differences from the NadA5 orthologue studied previously. We performed structure-based antigen design to engineer increased NadA3 thermostability. Functional NadA3 residues mediating interactions with the human receptor LOX-1 and vaccine-elicited human antibodies were identified. These antibodies competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. Our data provide a significant advance in the overall understanding of the 4CMenB vaccine antigen NadA.

Keywords: LOX-1 receptor; monoclonal antibody epitopes; neisserial adhesin A; three-dimensional structure; vaccine antigen.

FIG 1

Domain organization and biophysical characterization of NadA3 constructs. (A) Organization of NadA3 constructs; (B) combined data obtained from DSC experiments revealing mean melting temperature (n = 2) (left y axis, green) and from SEC-MALLS experiments showing the percentage trimer (right y axis, orange) for each fragment. L, leader.

FIG 2

NadA3 displays a head-on-stalk structure and electrostatic charge clusters. (A) Cartoon representation of NadA3 trimer. N- and C-terminal residues are labeled for one chain. A chloride ion (yellow) is buried in the head; wings are pink. Electron density maps lacked four N-terminal residues (₂₄ATND₂₇) presumably due to local disorder, and revealed four C-terminal residues (₁₇₁ASKH₁₇₄) derived from the hexahistidine tag linker. (B) Electrostatic surface representation of NadA3 (ranging from −3 kT/e [red] to +3 kT/e [blue]), calculated by APBS (adaptive Poisson-Boltzmann solver) methods (57). Artwork was prepared using Pymol (www.pymol.org).

FIG 3

Pairwise structure-based sequence alignment of NadA3 versus NadA5. (Above) Secondary-structure elements determined from crystal structures. Numbering corresponds to NadA3. Color code: bold black font on yellow background, sequence identity; red font on white background, sequence similarity; cyan background, contributes to positively charged apex in group I; magenta background, coordinates halide ions. (Below) Red and green ovals highlight heptad repeat positions a and d, respectively. Cyan ovals highlight position h in the undecad repeats. Orange ovals highlight residues forming a d-e or d-a layer within undecads. The figure was prepared using ESPript 3 (58).

FIG 4

Structure comparisons of NadA3 and NadA5. (A) Locations of 7-residue and 11-residue repeats on NadA3’s coiled coil. Cα atoms in positions a and d of canonical heptads are red and pale gray spheres, respectively. Black boxes highlight the two undecads. Buried undecad residues a and h are labeled, and Cα atoms are shown as red and cyan spheres, respectively. (B) Top-view trimeric helical ribbon representations of undecad V97 to K107, showing hydrophobic side chains packed into the core. Positions in the undecad are labeled a to k (top). Buried a and h residues are red and cyan sticks, respectively; the remaining Cα atoms are spheres. (C) Cross-sectional triangular layer (orange) in the coiled coil centered at the d (N100) and e (K101) positions of the undecad. Residues a and h are spheres, and d and e residues are sticks. (D) Topology-based superposition of the overall structures of NadA3 (blue) and NadA5 (yellow). The 7-Å difference between the wingtip positions is highlighted. The distance drawn between the wingtips was calculated for corresponding Cα atoms of E62 (NadA3) and E61 (NadA5).

FIG 5

Structure and analysis of stabilized NadA3 mutants. (A) Cartoon representation of superposed native NadA3 24–170 (blue) and the A33I I38L double mutant (green). (B, C) Magnifications of the core region to show the A33I and I38L mutations. Dashes show hydrophobic packing interfaces of residues 33 and 38 in the crystal structures of the native (B) and stabilized double mutant (C) proteins. The interface is more extensive in the double mutant. (D) Magnification of the head proximal to residue A39 (blue stick). Mutation of residue 39 to Val (magenta sticks, as predicted by the mutagenesis tool implemented in Pymol) may increase the extent of favorable hydrophobic packing by filling the cavity lined by adjacent side chains.

FIG 6

Bar plot summary of flow cytometry data for NadA3 proteins binding to mammalian CHO-K1 cells expressing LOX-1. The bar plot y axis shows the percentage of LOX-1-positive transfected CHO-K1 cells that were stained upon incubation with each distinct NadA protein. The NadA3 24–170 proteins carrying point mutations A33I, A33I I38L, or Y42A and the headless-stalk-only protein were heavily impaired for cell binding.

FIG 7

Sites of mutagenesis and NadA–LOX-1 interaction specificity. (A) Cartoon and surface representations of the trimeric-head region of NadA3, showing on one chain the side chain sticks for the subset of residues selected for mutation and on an adjacent chain the surface corresponding to residue A33′ (in pink). (B) SPR single-injection experiments showing binding of immobilized LOX-1 to injected NadA3 (orange line), but not to either NadA4 24–219 or NadA5 24–220 (black lines). Each protein (analyte) was injected in duplicate (n = 2); for clarity, one representative sensorgram is shown for each protein.

FIG 8

Affinity and selectivity of the NadA–LOX-1–humAb interactions. (A) SPR single-injection experiments, showing that both humAbs 5D11 and 12H11 bind to NadA3 but not to NadA4 24–219, NadA5 24–220, or the NadA3 E62A D63A double mutant. (B) SPR single-injection experiments. Injected NadA3 binds to immobilized LOX-1 but not if NadA is preincubated with humAb 12H11 or 5D11. Preincubation with humAb 7F11 (an anti-stalk humAb) does not abolish binding to LOX-1. Each protein (analyte) was injected in duplicate (n = 2); for clarity, one representative sensorgram is shown for each protein.