Fig. 2. Stereospecificity of DAG binding by C1Bδ.

a Backbone superposition of 8 DAG-complexed C1Bδ chains of the AU (cyan, PDB ID: 7L92) onto the structure of apo C1Bδ (sienna, PDB ID: 7KND). The sidechain of Trp252 reorients towards the tips of membrane-binding β12 and β34 loops upon DAG binding. DAG adopts one of the two distinct binding modes: “sn-1” (b) or “sn-2” (c). The formation of the C1Bδ-DAG complex in bicelles is reported by the chemical shift perturbations (CSPs) of the amide ¹⁵NH (d) and methyl ¹³CH₃ (e) groups of C1Bδ. Asterisks denote residues whose resonances are broadened by chemical exchange in the apo-state. The insets show the response of individual residues to DAG binding through the expansions of the ¹⁵N–¹H and ¹³C–¹H HSQC spectral overlays of apo and DAG-complexed C1Bδ. f ¹H–¹H_N Thr242 and Gly253 strips from the 3D ¹⁵N-edited NOESY-TROSY spectrum of the C1Bδ-DAG-bicelle complex. The protein-to-DAG NOE pattern is consistent with the distances observed in the “sn-1” mode (Chain 5, light purple) but not the “sn-2” mode (Chain 7, green). All distances are in Å and color-coded in the “sn-1” complex to match the labels in the spectrum; “w” denotes water protons. The medium-range NOE that would be characteristic of the “sn-2” complex is shown in red.