Proteins and protein-protein interactions (PPI) are the key mediators driving biological processes and functions. Understanding relation between structural and thermodynamic aspects of protein interactions gives us insight into the nature of interactions and paves the way for prediction of protein interactions from genomic data. The presented thesis focuses on antibody-antigen and toxin-antitoxin interactions, as two representative families of complexes with interactions between two globular proteins or between intrinsically disordered proteins (IDP) and its globular target.
In the first part of the thesis we present a comprehensive structural characterization of nanobody (camelid antibody fragments)–antigen complexes [1]. We discovered that nanobodies bind to structured, rigid, concave and conserved protein surfaces on the structure of antigen. Very often the binding is accompanied by additional interactions mediated by residues outside the conventional CDR regions (“non-CDR” contacts). Such non-CDR contacts can therefore importantly influence the binding energetics and specificity. Comparison with the conventional antibody-antigen interactions revealed a pronounced hydrophobic character of the nanobody-antigen interactions, which in light of other identified properties suggests that nanobody-antigen are in many respects very similar to general PPI interactions. The observed structural characteristics explain how and why nanobodies exhibit relatively high interaction affinities as observed in the thermodynamic dataset of nanobody-antigen interactions.
Interactions of intrinsically disordered proteins were studied on the model system from HigAB toxin:antitoxin module, where the intrinsically disordered fragment of the HigA antitoxin binds its globular target HigB toxin. Binding of IDP to globular target is coupled with folding of IDP into α-helical structure, which complicates the thermodynamic interpretation of the process. Herein, we present a novel experimental method that enables separation of standard thermodynamic properties into binding and folding contributions. The combination of spectroscopic (CD spectroscopy) and thermodynamic (ITC) methods with a theoretical description of helix-coil transition was used to integrate the structural and thermodynamic informations. The separation of folding and binding contributions explains the molecular forces that drive interactions where folding is coupled to binding. Presented method is general and may be applied to other systems where IDP folds to α-helix upon binding.
|
