Structural characterization of meningococcal vaccine antigen NadA and of its transcriptional regulator NadR in ligand-bound and free forms.

Liguori, Alessia (2016) Structural characterization of meningococcal vaccine antigen NadA and of its transcriptional regulator NadR in ligand-bound and free forms., [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biologia cellulare e molecolare, 28 Ciclo. DOI 10.6092/unibo/amsdottorato/7552.
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Serogroup B Neisseria meningitidis (MenB) is the cause of the invasive meningococcal disease (IMD). Bexsero is the first genome-derived vaccine against MenB. Neisserial adhesin A (NadA) is one of the three protein antigens included in Bexsero. The main aim of this work was to obtain detailed insights into the structure of vaccine NadA variant 3 (NadAv3) and into the molecular mechanisms governing its transcriptional regulation by NadR (Neisseria adhesin A Regulator). A deep understanding of nadA expression is important for understanding the contribution of NadA to vaccine-induced protection against meningococcal disease. NadA expression is regulated by the ligand-responsive transcriptional repressor NadR. The functional, biochemical and high-resolution structural characterization of NadR is presented in the first part of the thesis (Part One). These studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. In the second part of the thesis (Part Two), strategies involving both protein engineering and crystal manipulation to increase the likelihood of solving the crystal structure of NadAv3 are described. The first approach was the rational design of new constructs of NadAv3, based on the recently solved crystal structure of a close sequence variant (NadAv5). Then, a comprehensive set of biochemical, biophysical and structural techniques were applied to investigate all the generated NadAv3 constructs. The well-characterized trimeric NadAv3 constructs represented a set of high quality reagents which were validated as probes for functional studies and as a platform for continued attempts for protein crystallization. Mutagenesis studies and screenings to identify a new crystal form of NadAv3 were performed to improve crystal quality; structure determination is ongoing. The atomic resolution structure of NadAv3 will help to understand its biological role as both an adhesin and a vaccine antigen.

Tipologia del documento
Tesi di dottorato
Liguori, Alessia
Dottorato di ricerca
Scuola di dottorato
Scienze biologiche, biomediche e biotecnologiche
Settore disciplinare
Settore concorsuale
Parole chiave
Data di discussione
22 Aprile 2016

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