Gliadin peptide deamidation: effects on CXCR3 binding and signal transmission

Gluten is a complex molecule made of gliadin and glutenins. During digestion, gliadin is reduced into small peptides of about 20 amino acids enriched in glutamine and prolines. In the intestinal epithelium the CXCR3 (CX-Chemokine Receptor Type 3, a G-Protein Coupled Receptor - GPCR) binds two of the gliadin peptides (pep 111-130 QQQQQQQQQQQQILQQILQQ and pep 151-170 QVLQQSTYQLLQELCCQHLW). Moreover, it is involved in celiac disease (CD) development inducing an increase of intestinal permeability, resulting in zonulin release, which is induced and activated through the MyD88-pathway. Two of the three splicing variants of CXCR3 are involved in CD: CXCR3-A and CXCR3-B. Furthermore, it is known that gliadin peptides are modified by tissue transglutaminase (tTG) converting specific glutamine (Q) residues into glutamic acid (E): pep 111-130E QQQQQQQQQQQQILQQILQE and pep 151-170E QVLQESTYQLLQELCCQHLW. Evaluate differences between deamidated and non-modified gliadin peptides bound to both CXCR3 a and b isoforms The selected conformations resulting from the Autodock CG simulations (Table 1) correspond to the complexes with the lowest binding energy if considering peptide 111-130; instead, for peptide 151-170, the chosen conformations result the second in terms of binding energy and the first in terms of cluster numerosity, since the lowest binding energy conformation displays a backward orientation of the peptide. HADDOCK run (Table 2) returned up to 14 clusters for each protein-peptide docking simulation, displaying different score ranges between deamidated and normal peptides. Instead, regarding energy values they display overlapping results except for Cxcr3b-pep111 complexes. The model with the lowest score and/or the lowest binding energy (VdW, Electrostatic and Desolvation) from each run was selected for MD simulations. MD trajectories analysis suggested either a change in CXCR3 TM-helices or in C- ter flexibility when comparing non-modified peptides to deamidated ones. Therefore, what we expect to understand is the effect of the gliadin deamidation, first on binding to CXCR3, secondly on the signal transmission to the cytoplasmic domain. Accordingly, an H-bond analysis was performed on MD trajectories. Results displayed an uninterrupted H-bond network from the binding cavity to the C-ter helix in the CXCR3a/111-130 (Fig. 2a) and CXCR3b/111-130E (Fig. 2b) complexes. But, the two complexes differ for the receptor residues involved in peptide binding (in blue in Fig.2), since the deamidated peptide binds charged residues, while the non-modified interacts with non-charged residues. Likewise, also the resulting Hbond networks involve different residues. The other CXCR3 complexes display several H-bond network stops before the C-ter. Haddock docking simulations did not allow a discrimination between non-modified and deamidated peptides while Autodock CG application displays a preference for CXCR3 a or b isoforms by deamidated peptides 111-130 and 151-170, respectively. H-bond analysis let us evaluate the effects of deamidation on the binding mode and on the affinity with CXCR3 isoforms, concluding that deamidation actively influences the binding to the receptor. Furthermore, we did not observe any substantial differences in signal transduction for peptide 151-170 (Fig. 2c), while peptide 111-130 displayed two distinct networks for each isoform (Fig. 2a and 2b). Given that CXCR3 is a GPCR, so the C-ter is involved in the activation of the G-coupled protein, these results suggest pep 111-130 as favoured ligand than pep 151-170, both for CXCR3 a and b.