The Structure and Function of Membrane-Integral Pyrophosphatases and a Mechanosensitive Ion Channel as Studied Through Computational Techniques

To study how membrane proteins perform their functions at a molecular level, and how this is modulated by interactions with lipids and the environment, I selected two protein families for simulation to provide new understanding and ideas. Membrane-integral pyrophosphatases (mPPases) couple pyrophosphate hydrolysis to cation translocation. Multi-scale simulations demonstrated anionic lipids binding at the mPPase interface and the distal ends of the protein. These sites were composed of positive residues on helices that are implicated in function and stability, suggesting that lipids are important for mPPase activity. The change in dynamics between catalytic states was investigated and, despite no evidence of asymmetry, several mechanistic insights were gained. Such as the mode of sodium ion binding through coordination by the D6.50/D16.46 pair to pass R5.50, and the mechanism of 5-6 loop closure by forming a hydrophobic interface with the 13-14 loop. Post-simulation structures were used for in silico chemical screening and the identification of compounds with improved performance. Sequential removal of the helical bundle units (HBUs) from mechanosensitive ion channel PIEZO1 uncovered the interplay between blade structure and the membrane. This revealed that the distal HBUs point downwards, in contrast to previous structural predictions, and maintain dome depth at ~5.4 nm. These data suggested the importance of the MPH and blade structure in controlling dome depth, dynamics and specific interactions with lipids. By investigating similar findings, such as specific protein-lipid interactions and protein dynamics, in dissimilar proteins, I have generated understanding of their structure, function and their modulation by the membrane, beyond what has been gained through other techniques. This may provide insights into comparable features in other proteins, which could be exploited for stability, functional and structural studies.