CONFORMATIONAL CHARACTERIZATION OF TMEM176B ION CHANNEL FOR DIFFERENT OLIGOMERIC STATES AND UNDER DISTINCT PH CONDITIONS

This study investigates the structural dynamics and metastable states of the transmembrane protein TMEM176B, with a focus on its role as a non-selective monovalent cation channel. TMEM176B is known to inhibit the NLRP3 inflammasome by regulating cytosolic ionic concentrations, and silencing of the Tmem176b gene has been shown to enhance caspase-1/IL-1β–mediated antitumor immunity. Starting from structural models predicted by AlphaFold, we employed an integrative approach combining Normal Mode Analysis (NMA) and Molecular Dynamics (MD) simulations to investigate the flexibility and conformational transitions of TMEM176B across different oligomeric states and pH conditions. Our goal was to propose a plausible native model that reflects the protein's functional dynamics. NMA results revealed that the tetrameric form exhibits greater global structural flexibility, particularly at pH 4.0, as indicated by elevated RMSF values. This pH-dependent increase in mobility was also observed in the dimeric and trimeric forms, suggesting a consistent effect of acidic environments on conformational behavior. Energy minimization along selected normal modes identified regions with enhanced dynamic coupling, further supporting this trend. Principal Component Analysis (PCA) showed a clear separation between the initial structures and more open conformational states, especially under low pH conditions. Interatomic distances measured from visually selected residues corroborated the structural shifts associated with specific vibrational modes. Based on these insights, we selected representative normal modes to initiate Molecular Dynamics with excited Normal Modes (MDeNM) simulations. Preliminary results revealed conformational changes consistent with ion channel-like activity in the tetramer, including coordinated helical twisting. In ongoing Steered Molecular Dynamics (SMD) simulations with constant velocity pulling, early findings suggest a preferential conduction pathway for sodium over potassium ions in the extracellular-to-intracellular direction. Together, these results provide novel insights into the structural mechanisms of TMEM176B and its potential role in immune regulation, while also indicating promising directions for future functional and pharmacological studies.