AI-BASED STRUCTURAL PREDICTION AND MOLECULAR DYNAMICS SIMULATION OF HEPATITIS B VIRUS X PROTEIN AND ITS INTERACTING PROTEIN (HBXIP) ISOFORMS WITHIN THE RAGULATOR COMPLEX

By 2024, 1.34 million deaths were attributed to hepatitis B and C worldwide. X protein of Hepatitis B (HBx) is associated with viral replication and carcinoma. HBx interaction with HBx Interacting Protein (HBXIP) deregulates HBXIP and inhibits its apoptotic activity. In mammals, HBXIP exists as two isoforms, composing the lysosomal Ragulator complex with p14/MP1, c7orf59, and p18. HBx may alter the subcellular localization of p14, c7orf59, and HBXIP, except in the presence of p18. However, the structural basis of HBx interaction and the role of HBXIP isoforms remain uncertain. Therefore, this project investigates the interaction between HBx and the short HBXIP isoform, and the role of the long HBXIP isoform, evaluating two AI-based prediction models, Boltz-2 and AlphaFold3, and applying All-Atom Molecular Dynamics (AA-MD) simulations. Using amino acid sequences, AlphaFold3 and Boltz-2 configurations — X-ray diffraction, molecular dynamics, and electron microscopy — were applied to model Ragulator. Predicted complexes were Ragulator with p18 for validation against the crystallographic structure, and Ragulator without p18: with HBx/short-HBXIP or with the long HBXIP isoform. Predicted structures were analyzed using PyMOL for comparison, RMSD (Root Mean Square Deviation) calculations, and neighborhood-distance analysis. The AA-MD simulations were performed with the Ragulator crystallographic structure (PDB ID: 6EHP), while other structures were generated using the X-ray diffraction and molecular dynamics configurations of Boltz-2, selected by PyMOL analysis. Without p18, structural mimicry between HBx or HBXIP and p18 was expected. Models containing the long HBXIP isoform exhibited structural and spatial similarities to p18, in Boltz-2 predictions. The HBx/HBXIP complex exhibited structural mimicry in all predictions using the 71-amino-acid HBx sequence. In contrast, the 100-amino-acid HBx sequence resulted in higher RMSD values. The similarity to the experimental reference, lower RMSD values, and great mimicry of p18 led us to select Boltz-2 predictions using the X-ray diffraction and molecular dynamics configurations. These findings provided structures for molecular dynamics simulations, analyzed using RMSD, RMSF (Root Mean Square Fluctuation), and MM/PBSA (Molecular Mechanics/Poisson-Boltzmann Surface Area) to investigate interaction mechanisms, structural stability, and mimicry. Therefore, this study begins to elucidate the interactions between HBx and the HBXIP isoforms in Ragulator complex.