The dynamics of membrane proteins that are well-folded in water and become functional after self-insertion into cell membranes is not well understood. Herein we report on single-molecule monitoring of membrane association dynamics of the necroptosis executioner MLKL. We observed that, upon landing, the N-terminal region (NTR) of MLKL anchors onto the surface with an oblique angle and then is immersed in the membrane. The anchoring end does not insert into the membrane, but the opposite end does. The protein is not static, switching slowly between water-exposed and membrane-embedded conformations. The results suggest a mechanism for the activation and function of MLKL in which exposure of H4 is critical for MLKL to adsorb on the membrane, and the brace helix H6 regulates MLKL rather than inhibits it. Our findings provide deeper insights into membrane association and function regulation of MLKL and would have impacts on biotechnological applications.
Keywords: MLKL; conformation transitions; membrane proteins; regulation; self-insertion.