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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.

Siglec-10 is an inhibitory I-type lectin selectively recognizing sialoglycans exposed on cell surfaces, involved in several patho-physiological processes. The key role Siglec-10 plays in the regulation of immune cell functions has made it a potential target for the development of immunotherapeutics against a broad range of diseases. However, the crystal structure of the protein has not been resolved for the time being and the atomic description of Siglec-10 interactions with complex glycans has not been previously unraveled. We present here the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. We used combined spectroscopic, computational and biophysical approaches to dissect glycans' epitope mapping and conformation upon binding in order to afford a description of the 3D complexes. Our outcomes provide a structural perspective for the rational design and development of high-affinity ligands to control the receptor functionality.

CD22 (Siglec-2) is a B-cell surface inhibitory protein capable of selectively recognising sialylated glycans, thus dampening autoimmune responses against self-antigens. Here we have characterised the dynamic recognition of complex-type N-glycans by human CD22 by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide new molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results could enable the development of additional therapeutics capable of modulating the activity of h-CD22 in autoimmune diseases and malignancies derived from B-cells.

All living cells are covered by a layer of glycans at the interface between the environment and the cell membrane, capable of mediating cellular behavior, including critical mechanisms in immunoregulation and pathological processes. The molecular recognition of glycoconjugates from several proteins triggers a plethora of biological functions, especially in the infection process, immune response, and inflammation. Within this frame, interactions between glycans and their binding partners at molecular level have been studied, using a multidisciplinary approach of advanced NMR techniques, including ligand- and protein-based approaches, in combination with biophysical and computational methodologies, such as docking and molecular dynamic simulations. Siglecs (Sialic acid-binding immunoglobulin-type lectins) exploit a major application in the immune system regulation, recognizing glycans containing sialic acid. Indeed, in their cytoplasmic region, Siglecs contain one or multiple tyrosine-based signaling motifs that trigger cellular signaling, inhibiting the immune cell activation. In this thesis, the molecular bindings of different inhibitory Siglecs, in particular Siglec-2 and -7, containing cytosolic immunoreceptor tyrosine-based inhibition motifs (ITIMs), have been investigated with several glycoconjugates. Although the inhibition of immune system plays a fundamental role in some aberrant events, such as the over-reaction of response against self-molecules that often leads to produce autoimmune diseases, it is worth knowing that many pathogens have evolved the ability to cover their surfaces of sialic acids, subverting the immune system and dampening the host immune recognition. Thus, Siglecs have been studied as attractive targets for the design of therapeutic agents, such as antibodies or glycomimetics, for the treatment of inflammatory, autoimmune, and infectious diseases. In the case of Siglec-2, or CD22, the binding mode with complex-type N-glycans has been asses