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

Abstract

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 1

Antibody 2C7 and TMCP2 complexes were detected by native mass spectrometry (MS). (A) Deconvoluted spectrum of the SEC-MS analysis performed on a sample containing mAb 2C7 (violet) and TMCP2 (green) in a 1:4 molar ratio. (B) Deconvoluted spectrum of the SEC-MS analysis performed on Fab 2C7 (blue) and TMCP2 in a 1:4 molar ratio.

Figure 2

STD NMR spectroscopy. (A) STD NMR (red) and off-resonance (black) spectra of the mixture of mAb and CP2. Specific binding is observed due to the differences in the relative intensities and multiplicities of several signals, as highlighted in the aromatic and aliphatic regions. (B) Relative STD intensities for NH protons. The relative degree of saturation for the individual protons is normalized to the largest STD signal, the Phe12 HN, set to 100%, to evaluate the STD effects. (C) Titration of CP2 into a solution of mAb to calculate K_D via STD NMR.

Figure 3

SAXS experiments. (A) Pair correlation function, P(r). (B) Fitted SAXS profiles were obtained from DAMMIN ab initio reconstruction. (C) Three-dimensional (3D) model obtained for Fab 2C7 alone. (D) 3D model obtained for Fab 2C7 bound to TMCP2 tetramer.

Figure 4

Crystal structures of Fab and the Fab-peptide complex. (A) Fab 2C7 alone (chains A and B). The heavy chain is shown in green, and the light chain is shown in blue. Constant region domains are labeled C_H and C_L, respectively, and variable region domains are labeled V_H and V_L. (B) Similar view of Fab 2C7 (chains A and B) in a complex with cyclic peptide 2 (CP2; chain F). CP2 is shown as a surface rendering, colored by atom type (C, gray; N, blue, O, red). (C) View of the variable regions and bound peptide, with a shake-omit electron density map (mFo-DFc) calculated without peptide atoms (contour level 2.5 sigma). CP2 residues 1, 17, and 18 were not visible in the electron density map. (D) Shake-omit map showing a closer view of peptide CP2 from the Fab 2C7 complex. (E) View similar to panel C with CP2 shown in putty representation. The diameter and color of the tube represent the thermal- (B-) factors. Larger diameter and red coloring indicate higher B-factors and smaller diameter and blue color depicts lower B-factors. (F) Hydrogen (H) bonds in the beta-hairpin structure of CP2. The two copies of CP2 (chain F, red; chain E, gray) are superimposed, and the backbone hydrogen bonding distances between nonhydrogen atoms are shown. Chain F had clear electron density for residues 2–16 and chain E had density for residues 4–14. Measurements are based on chain F.

Figure 5

trNOESY-derived bioactive conformation of CP2 colored according to the STD effects (see legend). (A–C) Different renderings of CP2 in its bioactive conformation. (D,E) Different renderings of Fab 2C7–CP2 complex.

Figure 6

Interacting amino acid residues of the Fab 2C7–CP2 peptide complex. (A) Interacting residues observed in the MD minimized structure. The Fab heavy chain is shown in green, the light chain is shown in aqua, and CP2 is shown in stick representation colored by atom. (B) H-bond interactions between the Fab heavy chain and peptide as seen in the crystal structure. The coloring is same as in panel A. (C) Residues involved in hydrophobic interactions between Fab and peptide. The interacting residues from the crystal and MD minimized structures are given in Table 2.