9AXG

Human Saposin B Ligand Binding and Presentation to alpha-Galactosidase A.

(2024) bioRxiv 

• PubMed: 38617236 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1101/2024.04.04.584535
• Primary Citation of Related Structures:  
  9AVS, 9AXG


• PubMed Abstract: 
  

  Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-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 α-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 α-galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to α-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.

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Organizational Affiliation: 

Department of Biochemistry & Molecular Biology, Institute of Applied Life Sciences, University of Massachusetts, Amherst, Massachusetts 01003, USA.