The role of ATG16L1 in Crohn's disease and the structural alteration mechanisms and functional consequences of the rs2241880 variant.

Background: ATG16L1 (Autophagy Related 16 Like 1) is a key regulatory protein in the autophagy pathway. Although previous studies have established a significant association between the ATG16L1 genotype and Crohn's disease (CD) susceptibility, the specific molecular mechanism of its high-frequency missense variant rs2241880 has yet to be systematically elucidated. Methods: In this study, we first confirmed the important role of ATG16L1 in CD pathogenesis through genome-wide association study analysis and Western blot, as well as qRT-PCR. Subsequently, high-precision structural prediction, protein model-based dynamic simulation, and AI model thermodynamic stability analysis were innovatively integrated. The thermal shift assay (TSA) was employed to validate the structural stability of the mutant, while the pull-down assay was used to examine its binding capacity with WIPI2b. Results: The results show that ATG16L1 plays a significant role in the pathogenesis of CD. The mutation causes the protein's overall conformation to become more compact, significantly increasing the rigidity of key functional regions, and enhancing the structural and thermodynamic stability, which in turn affects the cleavage efficiency of caspase-3 and the function of the WD40 domain. The results of the TSA experiment provided evidence for the computational findings. The pull-down assay confirmed that the binding capacity of the mutant to WIPI2b was significantly impaired. Conclusion: This finding not only provides the first molecular mechanism of the ATG16L1 T300A mutation, offering an important theoretical basis for understanding CD susceptibility differences, but also provides insights for precision medicine interventions and gene editing strategies.