Peptides from coronin 1 inhibit fusion by balancing the activation barrier and hemifusion stability

One of the crucial processes in the lifespan of the eukaryotes is membrane fusion, which mediates various important cellular events. The process is utilized by enveloped viruses to enter the host cells and cause viral infection. The study of the stability of fusion intermediates and the kinetics of evolution of states becomes crucial to understand the membrane fusion mechanism in detail. In this work, we have studied the temperature-dependent fusion in the absence and presence of two coronin 1-derived tryptophan-aspartic acid repeat-containing inhibitory peptides, AG-22 and mTG-23, to unravel the inhibitory mechanism of these peptides. The time courses of lipid mixing (LM), content mixing (CM), and content leakage (CL) were carried out in the absence and presence of both peptides separately at four different temperatures and globally fitted data using a two-step sequential model of fusion to obtain the probability of CM at the intermediate state (thermodynamic property), rate constants, and activation thermodynamics of each step. We further monitored the peptide-induced change in depth-dependent membrane organization and dynamics by exploiting steady-state and time-resolved fluorescence techniques. Our findings demonstrate that both peptides enhance the rate of hemifusion and pore formation by reducing the activation barrier. However, they inhibit CM by reducing the thermal fluctuation of lipids by increasing the acyl chain order, leading to a decrease in the probability of CM at the hemifusion state. Therefore, the inhibitory property of these peptides is a result of a balance between the thermodynamic stability of the hemifusion state and reducing the activation barrier for the formation of hemifusion and pore opening. Taken together, our results suggest the triumph of thermodynamic stability over the favorable activation energy toward the inhibitory ability of AG-22 and mTG-23.