Single-molecule Technique To Study The Kinetics Of The Necroptosis Executioner MLKL

Necroptosis is a newly discovered form of programmed cell death that is independent of caspase activation.Loss of plasma membrane integrity and leakage of cellular contents are typical features of this process,and the rupture of plasma membrane by phosphorylated MLKL is the only execution pathway of necroptosis.Therefore,as the sole executioner of necroptosis,MLKL performs its functions properly,which is a crucial step in the successful completion of necroptosis.However,the mechanism by which MLKL interacts with phospholipid membranes as the functional execution unit to disrupt the integrity of plasma membrane remains unclear.It is of great scientific importance to investigate the interaction between MLKL and phospholipid membranes,to elucidate the molecular mechanisms by which MLKL oligomers cause cell membrane penetration,and to clarify the molecular events following MLKL activation and its translocation mechanisms.This thesis focuses on the kinetic process of conformational changes in protein molecules during the interaction between MLKL and phospholipid membranes,providing a scientific experimental and theoretical basis for exploring the molecular mechanism by which MLKL induces plasma membrane leakage.In this thesis,the kinetic processes of conformational changes in protein during the interaction between MLKL monomer and phospholipid membranes have been progressively investigated using the single-molecule SIFA technique.The results suggest that:Immediately after landing on the phospholipid membrane from aqueous solution,the MLKL monomer is obliquely anchored to the phospholipid membrane surface with the H4 helix as the anchor point,at which moment the protein is in the anchored state(Anchored).Then,the brace helix H6 of the MLKL detaches from its N-terminal 4HB domain,and the N-terminal 4HB domain is embedded into the phospholipid membrane,at which moment the protein is in the embedded state(Embedded).During the interaction between MLKL and the phospholipid membrane,the H4 helix,which is initially anchored to the phospholipid membrane,does not insert into the phospholipid membrane,but remains on the surface of the phospholipid membrane;and the brace helix H6 of the protein remains floating in the solution above the phospholipid membrane.During this process,the MLKL monomer is not stationary on the phospholipid membrane,but rather switch between two conformational states,the anchored and embedded states.Thus,we think that exposure of the H4 helix is critical for the translocation of MLKL to the phospholipid membrane during the performing its function,and that by modulating the interaction of the brace helix H6 with the 4HB domain,the conformation of the MLKL monomer on the phospholipid membrane can be regulated.Further,we investigated the kinetic process of conformational changes of the MLKL tetramers during the its interaction with phospholipid membranes and also established a structural model for the pore formation of the MLKL tetramers on phospholipid membranes.We found that after landing on the phospholipid membrane,similar to the monomer,the MLKL tetramer anchored obliquely to the phospholipid membrane surface through the H4 helix.Within a dozen seconds of the MLKL tetramer landing on the surface of the phospholipid membrane,a fraction of the proteins could already be inserted into the bottom of the phospholipid membrane,and each protein molecule in the MLKL tetramer is not perfectly synchronised in its insertion into the phospholipid membrane.The results show that when the interaction between the MLKL tetramer and the phospholipid membrane reaches relative equilibrium,the H1,H2 and H3 helices of the N-terminal 4HB domain of the MLKL tetramer are inserted essentially vertically into the bottom of the phospholipid membrane and act as the pore walls of the pores in the phospholipid membrane;the H4 helix lies below the headgroup of the phospholipid molecule,the H5 helix is attached to the surface of the phospholipid membrane and the H6 helix floats in solution above the phospholipid membrane.The results also indicate that the conformation of the MLKL tetramer on the phospholipid membrane is a dynamic equilibrium state.These results elucidate the molecular kinetic mechanism by which MLKL induces plasma membrane leakage and illustrate the conformational changes of MLKL tetramers as they pore-form on phospholipid membranes to perform their functions.In addition,we investigated the regulation process of the conformation of MLKL tetramer on phospholipid membranes by Monobody33(Mb33).GUVs leakage experiments showed that Mb33 could regulate the closure of pores formed on phospholipid membranes by MLKL tetramers,while the results of single-molecule SIFA revealed that Mb33 could regulate the conformational switch of the MLKL tetramer from the conformation that inserting into the bottom of the phospholipid membrane to the conformation that is closed to anchored state,which is responsible for the closure of the pores formed by the MLKL tetramer on the phospholipid membrane.This thesis systematically and clearly describes the details of the interaction of MLKL with phospholipid membranes,elucidates the molecular mechanisms by which MLKL performs its function of disrupting plasma membranes,and provides insight into the kinetic processes of MLKL tetramers in phospholipid membranes.The process by which Mb33 regulates the conformational changes of MLKL tetramers has also been investigated in depth,providing an effective scientific basis for the development of future drugs targeting pathogenesis due to MLKL and abnormal necroptosis.