Abstract
Celiac disease (CD) is an autoimmune disorder triggered by pepsin-resistant, gluten-derived immunogenic peptides (GIPs) in genetically predisposed individuals. Enzyme therapy targeting GIPs has been suggested as a complementary practice to a gluten-free diet to help reduce the symptoms of CD. Here, we present the crystal structure of RmuAP1, a pepsin-like aspartic protease from Rhodotorula mucilaginosa, which effectively degrades the toxic 33-mer and 26-mer GIPs under postprandial gastric conditions (pH 3.0-6.0). RmuAP1 has a canonical fold characteristic of the aspartic protease subfamily A1; however, it features a distinct flap and a flexible loop structure. Compared to pepsin, RmuAP1 accommodates the tetrapeptides PQQP and PQPQ, motifs frequently repeated on GIPs, via an adaptable binding cleft. Molecular dynamics (MD) simulations have shown that RmuAP1 stably engages these ligands, maintaining both the catalytic water in position and a closed flap conformation, primarily through ligand-induced remodeling of the S1' pocket. In contrast, pepsin neither binds these ligands effectively nor achieves a catalytically competent conformation. Structural comparisons and dihedral analysis further support an induced-fit mechanism underlying RmuAP1's pocket remodeling. Together, this study clarifies the structural basis for RmuAP1 to hydrolyze GIPs, emphasizing the potential of RmuAP1 as a platform for developing enhanced oral peptidase for CD patients through protein engineering approaches.