Structure and Zeatin Binding of the Peach Allergen Pru p 1.

Peach (Prunus persica) is among the fruits most frequently reported to cause food allergies. Allergic reactions commonly result from previous sensitization to the birch pollen allergen Bet v 1, followed by immunological cross-reactivity of IgE antibodies to structurally related proteins in peach. In this study, we present the three-dimensional NMR solution structure of the cross-reactive peach allergen Pru p 1 (isoform Pru p 1.0101). This 17.5 kDa protein adopts the canonical Bet v 1 fold, composed of a seven-stranded ß-sheet and three α-helices enclosing an internal cavity. In Pru p 1, the inner surface of the cavity contains an array of hydroxyl-bearing amino acids surrounded by a hydrophobic patch, constituting a docking site for amphiphilic molecules. NMR-guided docking of the cytokinin molecule zeatin to the internal cavity of Pru p 1 provides a structure-based rationale for the effect that zeatin binding has on the protein's RNase activity.

Profiling selectivity of chagasin mutants towards cysteine proteases cruzain or cathepsin L through molecular dynamics simulations.

Chagasin, an endogenous cysteine protease inhibitor from Trypanosoma cruzi, can control the activity of the parasitic cruzain and its homologous human cathepsin L. While chagasin inhibits both enzymes with similar potency, mutations have different effects on binding to these enzymes. Mutants T31A and T31A/T32A bind well to cathepsin L, but their affinity for cruzain drops ∼40 to 140-fold. On the other hand, the mutant W93A binds well to cruzain, but it loses potency against cathepsin L. Here, we employed molecular dynamics simulations to understand the selectivity in inhibition of cruzain or cathepsin L by chagasin mutants W93A, T31A, and T31A/T32A. Our results allowed profiling the nonbonded interactions in the interfaces of each mutant with these cysteine proteases. Additionally, we observed differences in the binding conformation of the chagasin loops L2 and L6 of the W93A mutant, favoring interactions with cruzain and reducing interactions with cathepsin L. These differences are associated with a partial dissociation of the W93A-cathepsin L complex, providing a likely cause for the selectivity of the mutant W93A towards cruzain.Communicated by Ramaswamy H. Sarma.

In silico Design of Phl p 6 Variants With Altered Fold-Stability Significantly Impacts Antigen Processing, Immunogenicity and Immune Polarization.

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.