An artificial metallolyase with pliable 2-His-1-carboxylate facial triad for stereoselective Michael addition.

We repurposed the metal-binding site of a cupin superfamily protein into the 2-His-1-carboxylate facial triad, which is one of the common motifs in natural non-heme enzymes, to construct artificial metalloenzymes that can catalyze new-to-nature reactions. The Cu2+-H52A/H58E variant catalyzed the stereoselective Michael addition reaction and was found to bear a flexible metal-binding site in the high-resolution crystal structure. Furthermore, the H52A/H58E/F104W mutant accommodated a water molecule, which was supported by Glu58 and Trp104 residues via hydrogen bonding, presumably leading to high stereoselectivity. Thus, the 2-His-1-carboxylate facial triad was confirmed to be a versatile and promising metal-binding motif for abiological and canonical biological reactions.

Mib1 modulates dynamin 2 recruitment via Snx18 to promote Dll1 endocytosis for efficient Notch signaling.

Notch signaling regulates normal development and tissue homeostasis. Ligand endocytosis plays critical roles in Notch signaling activation. Endocytic proteins such as epsin and dynamin participate in Notch ligand activity by mediating Notch ligand endocytosis. The ubiquitin ligase Mib1 also plays essential roles in Notch signaling via Notch ligand ubiquitination. However, the molecular links between Mib1 and endocytic proteins have not been fully defined. Here, we show that Mib1 is involved in dynamin 2 recruitment to Dll1 and that Snx18, which interacts with dynamin 2, modestly regulates Dll1 endocytosis. Furthermore, the ubiquitin ligase activity of Mib1 is induced by Notch ligand-receptor interactions. Mib1 promotes the interaction between dynamin 2 and Snx18 in an ubiquitin ligase activity-dependent manner. These results suggest that Mib1 modulates dynamin recruitment by regulating the interaction between Snx18 and dynamin 2, thereby helping to ensure the efficient signaling activity of Notch ligands.