Abstract by Valeria Calvaresi

Membrane proteins constitute a large family of protein involved in numerous biological and cell functions. They are important drug targets or are themselves drugs (biopharmaceuticals), although their structural characterization still poses significant challenges. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) has recently emerged as a powerful tool for the interrogation of conformational dynamics of proteins in their native environment. In this PhD thesis, HDX-MS was applied for the analysis of pharmaceutically relevant membrane proteins. Two species were studied: Neisserial adhesin A (NadA), one of the main antigens of the anti- meningococcal vaccine 4CMenB, and the major stress-inducible human 70-kDa heat shock protein (Hsp70), drug candidate against lysosomal storage disorders. One of the main challenges of the HDX-MS analysis of membrane proteins is the large amount of lipids present in the sample. By integrating size-exclusion chromatography (SEC) into the standard HDX-MS workflow, a novel platform for rapid removal of lipids from protein-lipid systems was developed. Our SEC-MS method also showed to facilitate the HDX-MS analysis of other complex protein states, as highly disulfide-bonded species, proteins in complex pharmaceutical formulations, or associated to large protein ligands. The SEC-HDX-MS platform was applied to elucidate the conformational dynamics of a membrane-embedded form of NadA, revealing that the adhesin adopts a more folded conformation in meningococci, in situ, compared to the vaccine soluble form. By leveraging HDX data, a structural model of NadA antigen was also proposed, and a comprehensive elucidation of its in-solution features as homotrimer performed. Additionally, the conformational signature of NadA association to two of its main interactors, Siglec-5 and Hsp90, was investigated, probing their binding sites on the head domain of the adhesin. Hsp70 interacts with the lysosomal membrane and its association to the anionic phospholipid BMP is crucial for reverting lysosomal storage diseases. Using liposomes as membrane-mimetic systems, we mapped Hsp70 interaction with BMP and other main lipid components of lysosomal bilayers. The detrimental effect of lipids on the LC-MS analysis was mitigated by optimizing the HDX quench buffer. Our results show that the nucleotide binding domain of the chaperone is the primary orchestrator of BMP interaction, whereas association to positively charged lipids occurs at the level of the substrate binding domain. By the study of an inactive mutant form, we also revealed important molecular details of Hsp70 protective effect. As a whole, those findings indicate that HDX-MS analysis of pharmacologically active proteins provide structural insights that can be useful for the design of optimized biopharmaceuticals.