Insights into the structural dynamics and free energy landscapes of Danio rerio RAB1A and RAB1B: a comparative molecular dynamics simulation approach.

CONTEXT: RAB1A and RAB1B are highly conserved small GTPases that regulate endoplasmic reticulum-to-Golgi vesicle trafficking and are implicated in neurodegenerative diseases and cancer. Despite the increasing use of Danio rerio as a vertebrate model organism, the structural dynamics of zebrafish Rab1 isoforms remain uncharacterized. In this study, we performed the first comparative computational investigation of zebrafish RAB1A and RAB1B in their GDP- and GTP-bound conformational states. Sequence and structural analyses demonstrated a high degree of conservation between the two isoforms while revealing a characteristic Tyr-to-Phe substitution within the RAB1B Switch I region. Long-timescale molecular dynamics simulations showed that GDP-bound complexes exhibited greater structural stability, reduced residue fluctuations, and more compact conformations than the corresponding GTP-bound systems. These findings provide molecular insights into the functional dynamics of zebrafish Rab1 isoforms and support the use of Danio rerio as a translational model for Rab1-related biological processes and disease mechanisms. METHODS: The amino acid sequences of zebrafish RAB1A and RAB1B were analyzed using MUSCLE, PRATT, the Conserved Domain Database, and ExPASy ProtParam. Three-dimensional structures were generated by homology modeling with SWISS-MODEL using experimentally determined human Rab1 structures as templates and were validated using PROCHECK, ERRAT, Verify3D, ProSA-web, and QMEANDisCo. All-atom molecular dynamics simulations were performed in duplicate for 1 µs using GROMACS 2024.1 with the CHARMM36m force field and TIP3P explicit water model under periodic boundary conditions. Trajectory analyses included backbone root-mean-square deviation, root-mean-square fluctuation, radius of gyration, principal component analysis, dynamic cross-correlation matrix analysis, and free energy landscape calculations. Structural visualization and analyses were performed using PyMOL, Visual Molecular Dynamics (VMD), and the Bio3D package in R.