pH-Induced Local Unfolding of the Phl p 6 Pollen Allergen From cpH-MD

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.

pH-dependent structural diversity of profilin allergens determines thermal stability

“…The applied technique of constant pH MD simulations allows us not only to investigate in protonation probabilities with high reliability, but also capture differences in pH-dependent conformational dynamics ( 12 , 53 , 54 ). For two of the studied allergen proteins we observe substantial structural rearrangements, i.e., unfolding of the α2 and α3 helix (see Figure 1 for the structure) at low pH in Amb a 8 and Bet v 2 ( Supplementary Figure S1 ).…”

“…Thus, understanding the mechanism of thermal degradation of allergens, and the structural consequences thereof is a crucial aspect to elucidate their role in the immune system ( 29 , 55 ). The structural and functional changes associated with the melting process of a protein are highly complex and still remain elusive ( 11 , 12 , 53 , 55 ). Here, we show that a decrease in thermal stability at varying pH levels is accompanied by an increase in flexibility, which is reflected in higher dihedral entropies ( Figure 5 ), B-factors ( Figure 6 ) and a broader conformational space ( Figure 3 ).…”

“…However, despite extensive research efforts, the reasons why some proteins cause an allergic immune response in individuals remains elusive (5,6). Additionally, it has been shown, that already small variants in sequence and or structure can trigger a completely different immune response (7)(8)(9)(10)(11)(12)(13). Thus, understanding or predicting the allergenic potential of a protein based on their sequence and structural similarity is still challenging.…”

“…However, with constant pH MD simulations we can explicitly model allergen dynamics local unfolding during endolysosomal acidification ( 12 ).…”

PEP-Patch: Electrostatics in Protein–Protein Recognition, Specificity, and Antibody Developability

“…Protein folding and thermal stability are also influenced by electrostatics. In particular, polar interactions are a major contributor to hydrogen bonding, and hydration of charged and polar amino acids has a profound impact on correct protein folding. − Due to protonation state changes, pH can influence protein stability and function. , Thus, understanding the role of electrostatics in protein function is crucial to advance, guide, and facilitate protein engineering and design.…”