Abstract
Susceptibility to endosomal degradation is a decisive contribution to a protein's immunogenicity. It is assumed that the processing kinetics of structured proteins are inherently linked to their probability of local unfolding. In this study, we quantify the impact of endosomal acidification on the conformational stability of the major timothy grass pollen allergen Phl p 6. We use state of the art sampling approaches in combination with constant pH MD techniques to profile pH-dependent local unfolding events in atomistic detail. Integrating our findings into the current view on type 1 allergic sensitization, we characterize local protein dynamics in the context of proteolytic degradation at neutral and acidic pH for the wild type protein and point mutants with varying proteolytic stability. We analyze extensive simulation data using Markov state models and retrieve highly reliable thermodynamic and kinetic information at varying pH levels. Thereby we capture the impact of endolysosomal acidification on the structure and dynamics of the Phl p 6 mutants. We find that upon protonation at lower pH values, the conformational flexibilities in key areas of the wild type protein, i.e., T-cell epitopes and early proteolytic cleavage sites, increase significantly. A decrease of the pH even leads to local unfolding in otherwise stable secondary structure elements, which is a prerequisite for proteolytic cleavage. This effect is even more pronounced in the destabilized mutant, while no unfolding was observed for the stabilized mutant. In summary, we report detailed structural models which rationalize the experimentally observed cleavage pattern during endosomal acidification.
Highlights
Immunoglobulin E (IgE)-mediated allergies are one of the most prominent health issues in western countries, with more than 20% of the population being affected (Valenta, 2002; Thalhamer et al, 2010; Valenta et al, 2010; Curin et al, 2018)
We use a combination of double boost aMD and constant pH simulations to profile local unfolding events of the Phl p 6 WT and two point-mutants at pH values ranging from 4.0 to 7.0
To compare the structural variations found in the simulations, we projected each simulation into a principal component analysis (PCA) space, constructed from the combined trajectories of all systems at all pH values
Summary
Immunoglobulin E (IgE)-mediated allergies are one of the most prominent health issues in western countries, with more than 20% of the population being affected (Valenta, 2002; Thalhamer et al, 2010; Valenta et al, 2010; Curin et al, 2018). Over 40% of the patients suffer from plant pollen induced allergy, rendering it the most common allergy type in industrialized countries (Asam et al, 2015). Despite the extensive research efforts which have been undertaken in this field, it is still unclear why some proteins induce an allergic immune response in predisposed individuals. It was reported repeatedly that slightest distinctions in sequence and/or structure suffice to shift the immune reaction from an allergic to a protective one (Scheurer et al, 2015; Verhoeckx et al, 2019). It is known that high similarity in sequence and/or structure to an allergen protein does not necessitate a similar immune response (Mitropoulou et al, 2018; Eichhorn et al, 2019; Seutter Von Loetzen et al, 2019; Tscheppe et al, 2020).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
