Insights into Krabbe disease from SEE COMMENTARY structures of galactocerebrosidase Janet E. Deanea,1, Stephen C. Grahamb, Nee Na Kimc, Penelope E. Steinc, Rosamund McNairc, M. Begoña Cachón-Gonzálezc, Timothy M. Coxc, and Randy J. Reada,1 aDepartment of Haematology and bClinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0XY, United Kingdom; and cDepartment of Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved July 22, 2011 (received for review April 8, 2011) Krabbe disease is a devastating neurodegenerative disease charac- profile of patients is highly heterogeneous, often occurring in terized by widespread demyelination that is caused by defects in compound heterozygote patterns, such that the course of the dis- the enzyme galactocerebrosidase (GALC). Disease-causing muta- ease cannot easily be predicted from the genotype at the GALC tions have been identified throughout the GALC gene. However, locus (13–15). As with many other lysosomal storage diseases, a molecular understanding of the effect of these mutations has Krabbe patients with similar or identical genotypes can have var- been hampered by the lack of structural data for this enzyme. Here ied clinical presentations and course of their disease (4). we present the crystal structures of GALC and the GALC-product Here we present the structure of mouse GALC (83% identity complex, revealing a novel domain architecture with a previously with human GALC, Fig. S1) alone and in complex with D-galac- uncharacterized lectin domain not observed in other hydrolases. tose. These structures reveal the presence of a unique domain All three domains of GALC contribute residues to the substrate- arrangement, identify the nature of substrate binding and cataly- binding pocket, and disease-causing mutations are widely distrib- sis, and provide insight into the differing roles of mutations uted throughout the protein. Our structures provide an essential occurring in human Krabbe disease variants. insight into the diverse effects of pathogenic mutations on GALC function in human Krabbe variants and a compelling explanation Results for the severity of many mutations associated with fatal infantile Crystal Structure of GALC. The crystal structure of GALC refined to disease. The localization of disease-associated mutations in the 2.1-Å resolution contains one molecule per asymmetric unit com- structure of GALC will facilitate identification of those patients that prising residues 25 to 668 (Fig. 1 and Table S1); GALC is the would be responsive to pharmacological chaperone therapies. archetype of the carbohydrate-active enzymes (CAZy) glycoside Furthermore, our structure provides the atomic framework for hydrolase family 59 (GH59). The overall fold comprises three the design of such drugs. domains: a central triosephosphate isomerase (TIM) barrel, a β-sandwich domain, and a lectin domain (Fig. 1). The first 24 glycoside hydrolase family 59 ∣ glycosyl hydrolase residues of GALC encode the signal peptide for targeting to the ER that in our construct is replaced with the sequence required rabbe disease, also known as globoid-cell leukodystrophy, is for secretion by HEK293 cells and is cleaved during secretion Kan autosomal recessive neurodegenerative disorder caused (as occurs with the wild-type signal peptide in vivo). The structure by a deficiency of the lysosomal enzyme galactocerebrosidase of GALC reveals that residues 25–40 contribute two β-strands (GALC, EC 3.2.1.46) (1). Defects in GALC that result in reduc- to the β-sandwich domain before forming the ðβ∕αÞ8 TIM barrel tion of enzyme activity lead to the accumulation of the cytotoxic (residues 41–337) that lies at the center of the structure. Residues metabolite psychosine, resulting in demyelination due to the 338–452 then form the remainder of the β-sandwich domain. apoptosis of myelin-forming cells (2, 3). Most patients with Residues 453–471 lie across one face of the structure stretching Krabbe disease suffer from rapidly progressive leukoencephalo- from the β-sandwich to the lectin domain (residues 472–668). The pathy with onset before 6 mo of age and death occurring before interfaces formed between each of the domains of GALC are very 2 2 the age of 2 y (4). Rare attenuated variants of Krabbe disease large: 22% (1;770 Å ) and 15% (1;480 Å ) of the solvent acces- include late-infantile, juvenile, and adult presentations with dis- sible surface areas of the β-sandwich domain and the lectin ease severity and progression being highly variable. The preva- domain, respectively, are buried in their interfaces with the TIM lence of Krabbe disease is approximately 1 in 100,000 births, but barrel. Thus, the domains of GALC are not likely to move relative there is wide variation among countries. Two genetically homo- to each other. genous communities have an extremely high prevalence of infan- The central TIM barrel is composed of eight parallel β-strands tile Krabbe disease with about 1 in 100–150 live births (5). surrounded by α-helices. The connecting loops on the C-terminal Sequence characterization of the human GALC gene and the discovery of the naturally occurring Twitcher mouse model of the disease have contributed to the molecular understanding of Author contributions: J.E.D., P.E.S., M.B.C.-G., T.M.C., and R.J.R. designed research; J.E.D., – S.C.G., N.N.K., R.M., and M.B.C.-G. performed research; J.E.D., S.C.G., T.M.C., and R.J.R. Krabbe disease (6 9). GALC is produced and glycosylated in analyzed data; and J.E.D., S.C.G., N.N.K., P.E.S., M.B.C.-G., T.M.C., and R.J.R. wrote the endoplasmic reticulum (ER)-Golgi complex after which it the paper. is trafficked, via the mannose-6-phosphate (M6P) pathway, to The authors declare no conflict of interest. BIOCHEMISTRY the lysosome (10). GALC is essential for normal catabolism of This article is a PNAS Direct Submission. galactosphingolipids, including the principal lipid component Freely available online through the PNAS open access option. of myelin, galactocerebroside (10). Sphingolipid degradation Data deposition: The atomic coordinates and structure factors have been deposited in the requires the combined action of water-soluble hydrolases and Protein Data Bank, www.pdb.org (PDB ID codes 3zr5 and 3zr6). nonenzymatic sphingolipid activator proteins known as saposins See Commentary on page 15017. (11, 12). 1To whom correspondence may be addressed. E-mail: [email protected] or rjr27@cam. More than 70 mutations in the GALC gene have been asso- ac.uk. ciated with severe clinical phenotypes and these mutations are This article contains supporting information online at www.pnas.org/lookup/suppl/ distributed throughout the sequence of GALC. The mutational doi:10.1073/pnas.1105639108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105639108 PNAS ∣ September 13, 2011 ∣ vol. 108 ∣ no. 37 ∣ 15169–15173 Downloaded by guest on October 2, 2021 side of the barrel (proximal to the lectin domain) are generally glycosylation at additional predicted sites in the lectin domain longer than those on the opposite side of the barrel. The β-sand- (N585 and N629). wich domain comprises two twisted β-sheets with a similar topol- ogy to that seen in other glycosyl hydrolases except for a very long Substrate Specificity. GALC catalyses the hydrolysis of the galac- loop that wraps over the top of the TIM barrel. A cysteine residue tosyl moiety from glycosphingolipids such as galactocerebroside in this loop, C378, forms a disulfide bridge with C271 in the TIM and psychosine (Fig. 2A). To better understand the substrate barrel. A calcium ion is bound in the lectin domain of GALC in a binding of GALC, we solved the structure of the enzyme in com- pentagonal bipyramidal configuration (Fig. S2). The lectin do- plex with D-galactose to 2.4-Å resolution (Fig. 2B and Table S1). main of GALC possesses a similar fold and calcium-binding site to that of β-glucanase (PDB ID code 3d6e, rmsd ¼ 2.38 Å over 155 Cα atoms). However, the lectin domain of GALC does not possess the catalytic residues present in the β-glucanase family. The lectin domain of GALC also possesses structural similarity to galectin proteins (PDB ID code 2yv8, rmsd ¼ 2.45 Å over 129 Cα atoms) and other carbohydrate-binding lectins. Residues N284, N363, N387, and N542 displayed electron density for gly- cosylation moieties, and these have been modeled in the GALC structure (Fig. 1). No ordered electron density was observed for 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 F F and oriented as in B.(D) Unbiased difference ( O- 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 Fig. 1. Structure of GALC shown in two orthogonal views. Ribbon diagram the bound galactose (pink sticks) and the proposed catalytic residues: the of GALC colored by domain: β-sandwich (red), TIM barrel (blue), linker nucleophile (E258, blue sticks) and the proton donor (E182, blue sticks). (orange), and lectin domain (green). The disulfide bond (yellow spheres), (F) Substrate specificity for galactose- (pink sticks) rather than glucose- calcium ion (gray sphere), and glycosylation moieties (violet sticks) are (yellow sticks) containing glycolipids is conferred by W291 and T93 (blue shown. The N and C termini are marked by labeled circles (Top). sticks). 15170 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105639108 Deane et al.