Structural Superimposition

Abstact

The iminosugar N-butyldeoxynojirimycin (Miglustat) is clinically used for the inhibition of ceramide glucosyltransferase for treating Type 1 Gaucher and Niemann-Pick type C diseases. This drug also inhibits glycogen debranching enzyme (GDE), the enzyme responsible for terminal glycogen catabolism via coordinated glucotransferase and amylo-alpha-1,6-glucosidase (GC) activities, although the structural basis for inhibition has been undefined. Here, we report the crystal structure of Candida glabrata GDE in complex with Miglustat, revealing inhibitor engagement at the conserved GC domain in an area that was previously hypothesized to accommodate the alpha-1,6-linked glucose moiety of glycogen. Structure-guided mutagenesis demonstrates that alanine substitution of residues at the GC site abolishes Miglustat binding, functionally validating the pocket and defining the interaction hot spots. To assess the possible relevance of these observations to the human enzyme, in silico docking predicts that Miglustat binds to the human enzyme in a pose close, albeit not identical, to our structure. These findings provide an opportunity to determine the molecular basis of GDE-inhibitor recognition, rationalize reported off-target effects of Miglustat, and provide a template for designing iminosugar therapies with reduced off-target binding.