BACKGROUND Network motifs, recurring subnetwork patterns, provide significant insight into the biological networks which are believed to govern cellular processes. METHODS We present a comparative network motif experimental approach, which helps to explain complex biological phenomena and increases the understanding of biological functions at the molecular level by exploring evolutionary design principles of network motifs. RESULTS Using this framework to analyze the SM (Sec1/Munc18)-SNARE (N-ethylmaleimide-sensitive factor activating protein receptor) system in exocytic membrane fusion in yeast and neurons, we find that the SM-SNARE network motifs of yeast and neurons show distinct dynamical behaviors. We identify the closed binding mode of neuronal SM (Munc18-1) and SNARE (syntaxin-1) as the key factor leading to mechanistic divergence of membrane fusion systems in yeast and neurons. We also predict that it underlies the conflicting observations in SM overexpression experiments. Furthermore, hypothesis-driven lipid mixing assays validated the prediction. CONCLUSIONS Therefore this study provides a new method to solve the discrepancies and to generalize the functional role of SM proteins.