Investigation Of The Influence Of Mechanical Signals On The Structure Of CD44/FERM Complex Via Molecular Dynamics Simulation

Clusters of differentiation 44(CD44)is a widely distributed plasma membrane adhesion molecule,especially highly expressed in tumor cells.The extracellular domain of CD44 can combine with hyaluronan(HA),while the intracellular domain can interact with ezrin/radixin/moesin(ERM).The combination of hyaluronan and CD44 mediates the initial adhesion and rapid rolling of tumor cells on endothelial cells.In the process of rolling,the interaction between hyaluronan and CD44 can generate the mechanical force,thus activating CD44,further enhancing the combination of CD44 and hyaluronan.At the same time,the activated CD44 intracellular domain will expose its phosphorylation sites,and then interact with FERM(protein 4.1-ezrin-radixin-moesin)domain of ERM proteins,initiating the downstream signaling cascade,eventually promoting the adhesion,migration and proliferation of tumor cells.In the process of intracellular signal transduction,the activation of spleen tyrosine kinase(Syk)is crucial step,which is usually activated by the combination with immunoreceptor tyrosine-based activation motif(ITAM),so the phosphorylation of phosphorylation sites on ITAM motif is critical for the activation of Syk.In recent years,some unconventional ITAM motif(ITAM-like)is proved to have a similar effect to the traditional ITAM motif,and even the phosphorylation of a single phosphorylation site on ITAM-like motif is sufficient to recruit Syk.There is such an ITAM-like motif on the ERM proteins,and there are two important phosphorylation sites on the motif,Y191 and Y205.In this paper,it was found at first that CD44/FERM structure was stable by observing CD44/FERM complex conformation and analyzing the interaction of interface residues both in static crystal structure and in equilibrium process.Meanwhile,ITAM-like motif,phosphorylation sites Y191 and Y205 were buried in FERM domain,which would hinder the phosphorylation of ERM proteins,the recruitment of Syk and subsequent cellular signal transduction.Since the cells in the blood flow is all affected by the shear stress and the force is a very significant physical signal,so we conjectured that after the combination of CD44 extracellular domain and HA,the mechanical signal produced by blood flow shear stress could induce the conformational changes of FERM domain through CD44 intracellular domain,thus promoting the exposure of ITAM-like motif and its phosphorylation sites,then facilitating the subsequent phosphorylation of ERM protein and the recruitment of Syk,eventually realizing the transduction of cellular signal.To verify the above conjecture,steered molecular dynamic simulation was applied to simulate the interaction between CD44 and FERM domain in the mechanical environment.The results showed that mechanical signal could induce the exposure of the ITAM-like motif and phosphorylation site Y205 by tracking and analyzing CD44/FERM complex conformational changes and the solvent-accessible surface area.This study revealed how the force regulated the activation of downstream cellular signal through CD44 intracellular domain for the first time and would be useful for further understanding the adhesion and migration pathway of tumor cells and the design of antitumor drugs.