Insights into Krabbe disease from structures of galactocerebrosidase

Abstract

Krabbe disease is a devastating neurodegenerative disease characterized by widespread demyelination that is caused by defects in the enzyme galactocerebrosidase (GALC). Disease-causing mutations have been identified throughout the GALC gene. However, a molecular understanding of the effect of these mutations has been hampered by the lack of structural data for this enzyme. Here we present the crystal structures of GALC and the GALC-product complex, revealing a novel domain architecture with a previously uncharacterized lectin domain not observed in other hydrolases. All three domains of GALC contribute residues to the substrate-binding pocket, and disease-causing mutations are widely distributed throughout the protein. Our structures provide an essential insight into the diverse effects of pathogenic mutations on GALC function in human Krabbe variants and a compelling explanation for the severity of many mutations associated with fatal infantile disease. The localization of disease-associated mutations in the structure of GALC will facilitate identification of those patients that would be responsive to pharmacological chaperone therapies. Furthermore, our structure provides the atomic framework for the design of such drugs.

Fig. 1.

Structure of GALC shown in two orthogonal views. Ribbon diagram of GALC colored by domain: β-sandwich (red), TIM barrel (blue), linker (orange), and lectin domain (green). The disulfide bond (yellow spheres), calcium ion (gray sphere), and glycosylation moieties (violet sticks) are shown. The N and C termini are marked by labeled circles (Top).

Fig. 2.

Substrate-binding site of GALC. (A) Schematic diagram of the GALC substrate galactocerebroside. (B) Ribbon diagram of GALC illustrating bound galactose (pink sticks) with domains colored as in Fig. 1. (C) Surface representation of GALC zoomed in on the galactose-binding site, colored and oriented as in B. (D) Unbiased difference (F_O-F_C) electron density (green mesh) corresponding to galactose bound to GALC. (E) Active site of GALC illustrating the relevant atomic distances (purple dashed lines) between the bound galactose (pink sticks) and the proposed catalytic residues: the nucleophile (E258, blue sticks) and the proton donor (E182, blue sticks). (F) Substrate specificity for galactose- (pink sticks) rather than glucose- (yellow sticks) containing glycolipids is conferred by W291 and T93 (blue sticks).

Fig. 3.

Disease-associated mutations of GALC. A selection of residues that are mutated in Krabbe disease are shown as sticks (beige) on the structure of GALC. For each residue a relevant view is provided (Insets) illustrating the surrounding region of the structure that would be perturbed by the mutation. Because of the insertion of a residue in the human sequence at position 507, residues 528 and 629 correspond to 527 and 628, respectively, in the mouse structure.