Dynamics Simulations Of CD44 Dimerization Modulated By Palmitoylated Modifications And Membrane Micreenvironments

Cluster-of-Differentiation-44（CD44）is a type I single transmembrane cell surface adhesion protein which playing an important role in the physiological pathology of cell migration and cancer invasion.In specific membrane environments,the homodimerization of CD44 plays a key role in signal transduction and tumor progression.Homodimerization of CD44 depends on cysteine in juxta-membrane to form a disulfide bond.It is worth noting that S-palmitoylation modification can occur at the relevant cysteine sites at the same time.Recent studies have found that palmitoylation modifications can enhance the affinity of CD44 and lipid raft domains,resulting in the spatial displacement of CD44 membrane domain and reducing the binding activity to extracellular ligands,which will inhibit cell proliferation and cancerization process.The specific membrane molecule phosphatidylinositol-4,5-biphosphate（PIP2）competes with palmitoylation and reduces the affinity of the CD44 to the lipid raft domains which change the membrane domain distribution of palmitoylated CD44.Specific membrane molecules can affect the self-assembly ability of membrane proteins and the spatial fluidity in specific membrane domains,therefore in-depth elucidation of palmitoylation modifications and specific membrane microenvironment are important for exploring the underlying molecular mechanisms of CD44 dimerization and tumor or related diseases treatment.Traditional experimental methods are difficult to provide accurate dynamic research vision on the transfer and effect information of proteins at atomic levels.Molecular dynamics simulation,especially the high-throughput kinetic simulation method based on Martini coarse-grained force field has been widely used in the study of membrane protein interactions.This study constructed four protein systems based on Martini coarse-grained force field,including CD44 wild type（CD44-WT）,CD44 modified with palmitoyl chains at the 286^thcysteine（Pal-286）,CD44 modified with palmitoyl chains at the 295^th cysteine（Pal-295）and two cysteine sites mutated into alanine site（C286AC295A）.Two membrane systems,the phospholipid dual molecular layer of dipalmitoyl-phosphatidylcholine（DPPC）and binary-phase membrane containing the lipid raft,are used to explore the homodimerization and conformations of CD44 in different membranes.And the connection among palmitoylation,PIP2 and lipid raft in mediate cultivation of protein.Followings are the main results:Fristly,in the DPPC single-phase membrane,the palmitoylation modification will impair the dimerization of CD44.The CD44 depends on the transmembrane domain 286^thcysteine to form the homodimer.CD44-WT can be quickly gathered within 1.0μs,forming a right-handed 25°conformation.Compared to CD44-WT,the dimeric conformation of Pal-286 did not change,but the dependent amino acid residue changes.The Pal-295 dimer is consistent with the binding mode of CD44-WT,and the dimer is converted into a right-handed 35°conformation.After cysteine mutation into alanine,the CD44 aggregation is suppressed.It shows an important role of cysteine in the dimerization.Secondly,in the DPPC single-phase membrane,PIP2 lipid molecules promote the dimerization of CD44.The CD44-WT can be rapidly aggregated in 0.5μs,and the dimer forms a 25°right-handed conformation.Each of the Pal-286 and Pal-295 can be rapidly accumulated within 1.0μs,and the Pal-286 dimer forms a conformational mode of the right-handed 30°,and the dependent residue sequence is transformed.There are two types of Pal-295 dimer constructive modes:the right-handed 25°and left-handed 20°,the dimer dependent the amino acid region transitions.Thirdly,in the binary-phase membrane containing lipid raft,the dimerization capacity of CD44-WT is be impaired.The Pal-295 is unable to form homodimerization in the lipid raft.CD44-WT can achieve aggregation after 3.7μs,forming a stable single right-handed 25°conformation.The addition of PIP2 accelerates the occurrence of CD44-WT aggregation,and can release Pal-295 anchored in the Lo domain to achieve dimerization.The dimer of the CD44-WT exhibits a second-level conformation of the right-handed 25°conformation and the left-handed 10°.The crossing angle distribution of the Pal-295 dimeric is disordered.The above-mentioned research can help to better understand the effect of CD44palmitoylation on controllable dimerization in different membrane microenvironments,and also provide a theoretical basis for future research on targeted drugs.