Single-Molecule Monitoring of Membrane Association of the Necroptosis Executioner MLKL with Discernible Anchoring and Insertion Dynamics

The dynamics of membrane proteins that are well-foldedin waterand become functional after self-insertion into cell membranes isnot well understood. Herein we report on single-molecule monitoringof membrane association dynamics of the necroptosis executioner MLKL.We observed that, upon landing, the N-terminal region (NTR) of MLKLanchors onto the surface with an oblique angle and then is immersedin the membrane. The anchoring end does not insert into the membrane,but the opposite end does. The protein is not static, switching slowlybetween water-exposed and membrane-embedded conformations. The resultssuggest a mechanism for the activation and function of MLKL in whichexposure of H4 is critical for MLKL to adsorb on the membrane, andthe brace helix H6 regulates MLKL rather than inhibits it. Our findingsprovide deeper insights into membrane association and function regulationof MLKL and would have impacts on biotechnological applications.