Gonococcal Mimitope Vaccine Candidate Forms a Beta-Hairpin Turn and Binds Hydrophobically to a Therapeutic Monoclonal Antibody

The spread of multidrug-resistant strains of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, represents a global health emergency. Therefore, the development of a safe and effective vaccine against gonorrhea is urgently needed. In previous studies, murine monoclonal antibody (mAb) 2C7 was raised against gonococcal lipooligosaccharide (LOS). mAb 2C7 elicits complement-dependent bactericidal activity against gonococci, and its glycan epitope is expressed by almost every clinical isolate. Furthermore, we identified a peptide, cyclic peptide 2 (CP2) that mimicked the 2C7 LOS epitope, elicited bactericidal antibodies in mice, and actively protected in a mouse vaginal colonization model. In this study, we performed structural analyses of mAb 2C7 and its complex with the CP2 peptide by X-ray crystallography, NMR spectroscopy, and molecular dynamics (MD) simulations. The crystal structure of Fab 2C7 bound to CP2 showed that the peptide adopted a beta-hairpin conformation and bound the Fab primarily through hydrophobic interactions. We employed NMR spectroscopy and MD simulations to map the 2C7 epitope and identify the bioactive conformation of CP2. We also used small-angle X-ray scattering (SAXS) and native mass spectrometry to obtain further information about the shape and assembly state of the complex. Collectively, our new structural information suggests strategies for humanizing mAb 2C7 as a therapeutic against gonococcal infection and for optimizing peptide CP2 as a vaccine antigen.


Figure S3 .
Figure S3.WaterLOGSY (WL), CPMG and 1D NH chemical shift perturbation NMR experiments on the mixture of mAb 2C7 with peptide CP2.A, WL experiment.In WL spectra, the change in the peak phase passing from the free to the bound state is indicative of binding.The positive signals in WL correspond to the peptide protons most involved in the binding with mAb that are also mediated by surrounding water molecules.The red arrows indicate the signals that remain negative in the bound state; and that do not interact with the antibody.B, CPMG experiments.Comparing the CPMG experiments of the peptide alone and in the mixture with the antibody, a slight decrease of signals in the bound state and differences in the T2 values are detected with respect to the free state.The decrease of transverse relaxation time T 2 of CP2 signals upon binding was indicative of complex formation.C, 1D NH chemical shift perturbation.Because of the rapid exchange regime, we observed broadened CP2 ligand resonances (or reduction of signal intensity) and a few chemical shift perturbations, which was further evidence for binding of the CP2 peptide to the 2C7 antibody.Upon mAb-CP2 interaction, changes in the chemical and magnetic environment at the peptide protein interfaces are induced, hence affecting the chemical shifts of the nuclei in this region.

Figure S4 .Figure S5 .Figure S6 .
Figure S4.NMR analysis of the mixture between mAb2C7 and TCMP2.A, Pulsed-field gradient (PFG)-NMR measurements of the TCMP2 in absence and presence of mAb2C7 to determine the diffusion coefficients of the molecules (top).B and C, STD and WL NMR experiments show the molecular binding between mAb2C7 and TCMP2.

Figure S7 .Figure S8 .Figure S9 .Figure S12 .
Figure S7.Structural details of Fab-peptide complex.A, Superposition of Fab alone and Fab-peptide complex.Fab 2C7 alone is shown in light green (VH) and aqua (VL) and the Fab from the complex is shown in dark green and blue (VH and VL, respectively).The superposition was based on the variable region domains.B, Ordered water molecules bridging Fab 2C7 and CP2.Seven ordered water molecules were observed making putative H bonds with both Fab and peptide chain F (shown in Figure), and six for peptide chain E (not shown).Of these, two water molecules were seen bridging the same atoms in both copies of the asymmetric unit of the crystal (chain A/C: Asn57 ND2 to F/E: Gly10 O, and A/B: Asn76 ND2 to F/E: Asp7 OD2).Distances in the two independent copies are given in TableS4.

Table S3 .
Analysis of residues interacting between Fab 2C7 and peptide CP2 a

2 ) b Solvation energy (D i G) b H-bond
b the two values refer to first chain/second chain (i.e., A/C, B/D, or F/E)

Table S5 .
Restraints used in the structure calculation of CP2