Human Saposin B Ligand Binding and Presentation to α-Galactosidase A

Abstract

Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by -galactosidase A. However, the manner by which SapB stimulates -galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to -galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates -galactosidase A cleavage of Gb3-NBD in vitro. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactions between SapB and -galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to -galactosidase A. These findings establish general principles for molecular recognition in saposin:hydrolase complexes and highlight the utility of NBD reporter lipids in saposin biochemistry and structural biology.