Structural and energetic basis of interactions among globular and intrinsically disordered proteins

Proteini in medproteinske interakcije so ključne za vrsto bioloških funkcij in procesov, zato ima njihov študij osrednjo vlogo pri razumevanju življenjskih procesov na molekularni ravni. Razumevanje strukturnih in termodinamskih osnov medproteinskih interakcij nam omogoča vpogled v naravno interakcij in odpira možnosti napovedovanja proteinskih interakcij na podlagi genomskih sekvenc. V magistrskem delu smo se posvetili interakcijam protitelo-antigen in antitoksin-toksin, kot reprezentativnima družinama kompleksov z interakcijami med dvema globularnima proteinoma oziroma med intrinzično neurejenim proteinom (IDP) in globularnim proteinom. V prvem delu smo skušali ovrednotiti značilnosti interakcij med nanotelesi (fragmenti kamelidnih protiteles) z antigeni [1]. Ugotovili smo, da nanotelesa prepoznajo bolj strukturirane, rigidne, evolucijsko ohranjene, konkavno ukrivljene površine proteina, ki se lahko nahajajo globlje v proteinski strukturi. Tak način vezanja pogosto spremljajo interakcije zunaj klasičnih prepoznavnih zank CDR (non-CDR kontakti), zato lahko taki kontakti pomembno vplivajo na termodinamiko in specifičnost interakcije. Primerjava z interakcijo klasičnih protiteles je pokazala, da imajo interakcije med nanotelesi in antigeni izrazito hidrofoben značaj, kar poleg ostalih ugotovitev kaže na to, da je interakcija bolj podobna splošnim interakcijam protein-protein. Ugotovitve strukturnih analiz smo povezali z dostopnimi termodinamskimi podatki ter skušali pojasniti, zakaj so opažene afinitete interakcije nanotelo:antigen izrazito visoke. Interakcijo intrinzično neurejenih proteinov (IDP) smo študiral s pomočjo vezanja intrinzično neurejenega fragmenta antitoksina HigA z globularnim toksinom HigB iz toksin-antitoksin modula HigAB. IDP ob vezavi na toksin zavzame strukturo α-vijačnice, kar otežuje termodinamsko interpretacijo, saj sta procesa vezanja in zvitja sklopljena. V magistrskem delu smo uspeli s kombinacijo spektroskopskih (CD spektroskopija) in termodinamskih (ITC) eksperimentalnih tehnik ter teorijskega opisa prehoda vijačnica-naključni klobčič modelno povezati strukturno in termodinamsko informacijo. Slednje nam je omogočilo eksperimentalno razčlenitev standardnih termodinamskih prispevkov vezanja in zvitja. Z rezultati smo na molekularni ravni razložili gonilne sile interakcije med intrinzično neurejenim proteinom in globularnim partnerjem. Razvita metoda je splošna in omogoča razčlenitev termodinamskih prispevkov v primerih, ko se IDP ob vezanju zvije v α-vijačnico.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