Intramolecular occlusion of the diacylglycerol-binding site in the C ₁ domain of munc13-1

Protein kinase C (PKC) isozymes and other receptors of diacylglycerol (DAG) bind to this widespread second messenger through their C₁ domains. These alternative DAG receptors include munc13-1, a large neuronal protein that is crucial for DAG-dependent augmentation of neurotransmitter release. Whereas the structures of several PKC C₁ domains have been determined and have been shown to require little conformational changes for ligand binding, it is unclear whether the C₁ domains from other DAG receptors contain specific structural features with key functional significance. To gain insight into this question, we have determined the three-dimensional structure in solution of the munc13-1 C₁ domain using NMR spectroscopy. The overall structure includes two β-sheets, a short C-terminal α-helix, and two Zn²⁺-binding sites, resembling the structures of PKC C ₁ domains. However, the munc13-1 C₁ domain exhibits striking structural differences with the PKC C₁ domains in the ligand-binding site. These differences result in occlusion of the binding site of the munc13-1 C₁ domain by a conserved tryptophan side chain that in PKCs adopts a completely different orientation. As a consequence, the munc13-1 C₁ domain requires a considerable conformational change for ligand binding. This structural distinction is expected to decrease the DAG affinity of munc13-1 compared to that of PKCs, and is likely to be critical for munc13-1 function. On the basis of these results, we propose that augmentation of neurotransmitter release may be activated at higher DAG levels than PKCs as a potential mechanism for uncoupling augmentation of release from the multitude of other signaling processes mediated by DAG.