Dynamic Conformational Changes in MUNC18 Prevent Syntaxin Binding

The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphoryalation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins.Author Summary: Protein phosphorylation plays a significant regulatory role in multi-component systems engaged in signal transduction or coordination of cellular processes, by activating or deactivating proteins. The potential of phosphorylation to induce substantial conformational changes in proteins, thereby changing their affinity to target proteins, has already been shown but the dynamics of the process is not fully elucidated. In the present study, we investigated, by molecular dynamics simulations, the dynamic conformational changes in munc18a, a protein that is crucial for neurotransmitter release and interacts tightly with the SNARE syntaxin-1. We further investigated the conformational changes that occur in munc18a when it is phosphorylated, reducing its affinity to syntaxin-1a. The results of the simulations suggest that there is a conformational flexibility of the syntaxin-unbounded munc18a that allows changes in the shape of the syntaxin-1a binding cavity. In silico insertion of phosphomimetic mutations into munc18a led to a reduction in the flexibility and closure of the syntaxin-binding site. We suggest that the reduced affinity of phosphorylated munc18a to syntaxin-1a stems from the difficulty of syntaxin-1a to bind to the munc18a closed-cavity conformation, induced by the PKC phosphorylation of munc18a.