Consensus structure prediction of A. thaliana’s MCTP4 structure using prediction tools and coarse grained simulations of transmembrane domain dynamics

Multiple C2 Domains and Transmembrane region Proteins (MCTPs) in plants have been identified as important functional and structural components of plasmodesmata cytoplasmic bridges, which are vital for cell-cell communication. MCTPs are endoplasmic reticulum (ER)-associated proteins which contain three to four C2 domains and two transmembrane regions. In this study, we created structural models of Arabidopsis MCTP4 ER-anchor transmembrane region (TMR) domain using several prediction methods based on deep learning (DL). This region, critical for driving ER association, presents a complex domain organization and remains largely unknown. Our study demonstrates that using a single deep-learning method to predict the structure of membrane proteins can be challenging. Our models presented three different conformations for the MCTP4 structure, provided by different deep learning methods, indicating the potential complexity of the protein’s conformational landscape. We then used physics-based molecular dynamics simulations to explore the behaviour of the TMR of MCTPs within the lipid bilayer.We found that the TMR of MCTP4 is not rigid but can adopt multiple conformations. The membrane-embedded region contains two helical pairs: HP1 (TM1–TM2) and HP2 (TM3–TM4). Deep learning predictions revealed three distinct types of inter-helical contact interfaces: ESMFold, AlphaFold-Multimer, trRosetta, and RoseTTAFold consistently predicted a TM2–TM3 contact; AlphaFold2 did not predict any contact between these two helical pairs, while OmegaFold instead suggested a TM1–TM4 interface. Our physics-based coarse-grained simulations not only confirmed the contacts predicted by these models but also revealed a broader conformational landscape. In particular, structural clustering identified five distinct conformational clusters, with additional and more extensive inter-helical contacts not captured by the deep learning predictions. These findings underscore the complexity of predicting protein structures. We learned that combining different methods, such as deep learning and simulations, enhances our understanding of complex proteins.