Structural and Conformational Analysis of the TMEM176b Ion Channel

The TMEM176B has been characterized as a non-selective monovalent cation channel involved in the regulation of intracellular ionic homeostasis. TMEM176B is known to inhibit activation of the NLRP3 inflammasome by modulating cytosolic ion concentrations, and silencing of the Tmem176b gene has been reported to enhance innate antitumor immunity through caspase-1/IL-1β signaling. Moreover, recent studies suggest that the Ala134Thr variant in the Tmem176b gene may have a protective effect in the prognosis of colorectal cancer. This study aims to investigate the structural dynamics and metastable states of TMEM176B, with particular emphasis on understanding how different oligomeric states, pH conditions, and point mutations modulate the flexibility of the channel and may influence its functional activity. Starting from AlphaFold3-predicted structures, we combined Normal Mode Analysis (NMA), Molecular Dynamics (MD), and Molecular Dynamics with Excited Normal Modes (MDeNM) to investigate the conformational dynamics of TMEM176B under different oligomeric states and pH conditions. The effects of mutations were evaluated through RMSF analysis of the sampled conformational ensembles. For the tetrameric model at pH 4.0, MDeNM simulations were performed for the wild-type and A134T mutant proteins, and the resulting ensembles were analyzed by mapping intra- and inter-chain contacts, followed by feature selection to identify the most informative descriptors of the conformational landscape. The results suggest that the tetrameric state may represent the functional conformation of TMEM176B. Furthermore, acidic pH conditions appear to promote conformational changes consistent with increased channel activity. Feature selection identified contacts involving residues previously associated with gain- and loss-of-function mutations, in agreement with unpublished experimental evidence. TICA analysis indicated that 16 components were sufficient to capture approximately 80% of the cumulative kinetic variance, while projection of the conformations onto the space defined by the leading tICs revealed a progressive shift with increasing excitation intensity, suggesting the occurrence of a continuous conformational transition across the excitations.