The Interaction Research Between Moesin-ferm Protein?calmodulin(cam) And L-selectin Peptide And Its Co-crystallization

Ectodomain shedding,in which many proteins undergo regulated proteolysis at a site close to the cell membrane,is an important step in many signaling pathways.Alteration in the shedding step can lead to cardiovascular diseases,cancer and neurologic diseases. As a member of the selectin family, L-selectin is one of the best characterized ectodomain shedding substrates and its shedding process mainly rely on the regulation of intracellular areas. The study found that moesin-FERM and calmodulin(CaM) jointly associate with the cytoplasmic domain of L-selectin in the cell to modulate the function and ectodomain shedding of L-selectin.The molecular mechanisms underlying the across-the-membrane regulation of ectodomain shedding remain unclear.The two peptide molecules named AFII24 and RRL17 aimed at simulating the function of L-selectin were designed according to the sequence of L-selectin transmembrane domain and ectodomain.The secondary structure of the peptides were analyzed by CD experiment. At the same time, we successfully constructed the recombinant plasmids of moesin-FERM protein and calmodulin (CaM). Through the optimization of expression and purification conditions, we obtained the high concentration and high purity moesin-FERM protein and calmodulin(CaM).The binding constant (KD=558.65nM) of the peptide AFII24 and moesin-FREM protein was obtained by ITC experiment. The binding mechanism of the peptide AFII24 to calmodulin (CaM) is the result of entropy change and enthalpy change.While peptide RRL17 did not bind to either moesin-FERM protein or calmodulin (CaM). Furthermore, the interaction between the three was demonstrated by fluorescence experiment and GST-pull down experiment.The complex crystal of moesin-FERM and peptide AFII24 were screened by co-crystallization method.We obtained a crystallization condition but no diffraction data was obtained. Subsequently, by the"Linker" method, the recombinant plasmids of FERM-Linker-AFII24(F-L-P) and AFII24-Linker-FERM (P-L-F) were successfully constructed and we obtained the high concentration and high purity F-L-P protein.Through crystal screening and the optimization of crystal conditions, we obtained the complex crystal, which laid a foundation for the follow-up study. Unfortunately, for the diffraction of F-L-P protein is not good enough to meet the requirement of the structure analysis, the interaction mechanism of the complex remains to be further studied.