Insights into 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 galactocerebrosidase (GALC). Disease-causing muta- ease cannot easily be predicted from the genotype at the GALC tions have been identified throughout the GALC . However, (13–15). As with many other lysosomal storage diseases, a molecular understanding of the effect of these 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 . 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 (CAZy) Furthermore, our structure provides the atomic framework for family 59 (GH59). The overall fold comprises three the design of such drugs. domains: a central triosephosphate (TIM) barrel, a β-sandwich domain, and a lectin domain (Fig. 1). The first 24 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 , 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 (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). degradation Data deposition: The atomic coordinates and structure factors have been deposited in the requires the combined action of water-soluble hydrolases and , 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 of the galac- loop that wraps over the top of the TIM barrel. A cysteine residue tosyl moiety from 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- to 2.4-Å resolution (Fig. 2B and Table S1). main of GALC possesses a similar fold and calcium- 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) 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 - calcium ion (gray sphere), and glycosylation moieties (violet sticks) are (yellow sticks) containing 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. Downloaded by guest on October 2, 2021 The overall fold of GALC is unchanged upon galactose binding be involved via these different mechanisms. Indeed, it has been ¼ 0 17 641 α (rmsd . Å over C atoms), the core of the binding shown for GlcCerase that both sapA and sapC can activate the SEE COMMENTARY pocket being formed by the long loops on the C-terminal face enzyme and that they mediate their activating effects by of the TIM barrel as has been observed for other enzymes con- binding to distinct sites on the enzyme (26). It has been noted taining TIM barrels. However, in our structure we see that loops previously that saposins possess a highly negatively charged sur- from both the β-sandwich and lectin domains also contribute to face (27) and that in the case of sapC this needs to be partially the substrate-binding pocket (Fig. 2C). The position and orienta- neutralized to trigger membrane binding (28). Our structure tion of galactose in the active site is unambiguous as shown by the reveals that the surface charge of GALC at pH 4.8, equivalent characteristic shape of the difference density calculated before to the acidified lysosome, is þ10 e− with a very large, highly po- inclusion of galactose in the model (Fig. 2D). The interactions sitively charged patch surrounding the substrate-binding site and atomic distances between atoms of galactose and GALC (Fig. S7). As this patch is near to the substrate-binding pocket identify E258 as the active site nucleophile and E182 as the pro- and possesses a surface charge that would be compatible with ton donor (Fig. 2E). The average distance between the carboxyl sapC or negatively charged lipid binding, it is likely to be the site oxygens of these catalytic residues is 5.0 Å, consistent with the of their association. Interestingly, human GlcCerase, which also retaining mechanism of enzymatic hydrolysis binds sapC, possesses a similar surface charge distribution but in which the product retains the same stereochemistry as the without conserving the residues that confer this charge (Fig. S7). substrate (16). In addition to the catalytic glutamates E182 and E258, several Disease-Associated Mutations of GALC. Mutations have been iden- residues contribute to the formation of the substrate-binding tified in the GALC gene that affect gene splicing and mRNA pocket and form hydrogen bonds with the bound galactose stability or cause deletions, frameshifts, and missense mutations molecule (Fig. S3). The position and orientation of galactose in (5, 13–15, 29–34). A large proportion of the missense mutations the GALC active site differs from that seen in other galactose- in GALC that cause Krabbe disease are likely to result in protein containing hydrolase structures, such as acid α-galactosidase mistargeting or premature degradation (33). Mistargeting could (Fig. S4), but is similar to the position of glucose analogues in occur at several stages in the GALC processing pathway, includ- (GlcCerase, also known as acid β-glucosi- ing failure to transit from the ER to Golgi due to misfolding, and dase), the hydrolase responsible for cleaving glucose-containing blockage of trafficking to the lysosome due to altered binding to (17, 18). Our structure reveals how GALC specifi- the M6P receptor. In addition, if mutations affect the binding of cally recognizes galactose-containing lipids. Galactose and glu- GALC to essential activating factors in the lysosome (including cose differ only in the position of a hydroxyl group at one saposins) then, despite being correctly processed and localized, carbon (C4, Fig. 2F). In the galactose-bound structure of GALC, GALC may not be able to access substrate for efficient glyco- this hydroxyl forms a hydrogen bond with T93: a residue with sphingolipid cleavage. unusual backbone dihedral angles (φ ¼ 139°, ψ ¼ −48°) that The structure of GALC provides detailed insight into the role are maintained in the absence of galactose, indicating this residue of many disease-associated mutations. Nearly 70% of the mis- confers specificity rather than undergoing an induced fit. Glucose sense mutations that result in Krabbe disease involve the modi- is not compatible with the substrate-binding pocket as the posi- fication of residues that are buried within the structure of the tion of its hydroxyl group would clash with residue W291. The enzyme (Table S2). These mutations will lead to instability or mis- conformation of W291 is stabilized by a hydrogen bond with folding of GALC resulting in the premature degradation of the the backbone carbonyl of G47. Interestingly, other lysosomal gly- enzyme. Examples of such mutations are not restricted to the cosyl hydrolases, including GlcCerase and β-glucuronidase, con- core TIM barrel domain but can be found in each domain of serve a tryptophan residue at this position but in a different GALC. Mutations that are likely to result in severe misfolding conformation that is incompatible with the structure of GALC include E114K and S257F in the TIM barrel, L364R and (Fig. S5). The binding of galactose is further stabilized by hydro- W410G in the β-sandwich domain, and G537R and L629R in gen bonding to R380 at the tip of the long loop that stretches the lectin domain (Fig. 3 and Table S2). Previous work has shown from the β-sandwich domain. This loop is secured in position that GALC possessing this last , L629R, does indeed ac- by the disulfide bond formed between C271 in the TIM barrel cumulate in the ER and is not secreted by cells or detected in the and C378 in the loop. The position of the side chain of R380 al- lysosome (33). Misfolding or destabilizing mutations of GALC do ters slightly upon galactose binding to accommodate the ligand. not necessarily result in an enzyme that is catalytically inactive. Several disease-associated mutations that are completely buried Discussion in the structure have been shown to retain residual enzyme activ- Substrate Binding. The galactose-bound structure of GALC re- ity (Table S2) (14, 15, 33, 35, 36). In those cases where the mu- veals that the active site pocket accommodates only the galactose tated form of GALC is trapped in the ER but still capable of moiety and does not have space for the lipid tails that are present cleaving substrate the use of pharmacological chaperone therapy in the natural substrates (Fig. 2 A and C). Analysis of the loops (PCT) would be clinically relevant. PCT involves the use of small surrounding the active site suggests that GALC is unlikely to molecules that stabilize the enzyme structure allowing it to escape undergo a conformational change similar to that described the ER, thus correcting the trafficking defect. Pharmacological for GlcCerase in order to accommodate substrate (Fig. S6) chaperones have been identified for similar lysosomal storage dis- (18, 19). The scissile bond points out toward the surface of eases such as Fabry, Gaucher, and Pompe diseases and are now the protein but in order to gain access to the membrane-em- being translated into clinical applications (37).

bedded substrate, hydrolases require saposins (saps) Within the ER-Golgi complex GALC undergoes modification BIOCHEMISTRY (20). GALC-mediated degradation of galactolipids requires the by the addition of glycans to specific asparagine residues. This activity of sapA (21–23) and possibly also sapC (21, 22, 24). N-linked glycosylation is essential for the correct trafficking of Two mechanisms of action for saposins have been postulated. GALC including binding to the M6P receptor (36). A common The first suggests saposins extract the lipids from bilayers to form mutation found in Krabbe disease patients of Arab ancestry is water-soluble lipid–protein complexes that present the substrate D528N (5), which has been shown to introduce a new glycosyla- to the appropriate enzyme (11). The second involves the binding tion site into GALC (33). The mutant GALC protein is hypergly- of enzyme at the bilayer surface where saposin molecules facil- cosylated, not efficiently taken up by cells and not present in the itate the access to substrate by distorting the bilayer (12, 25). Dur- lysosome (33). D528 lies on a loop of the lectin domain with the ing GALC activation it is possible that both sapA and sapC will side chain buried in the interface with the TIM barrel (Fig. 3).

Deane et al. PNAS ∣ September 13, 2011 ∣ vol. 108 ∣ no. 37 ∣ 15171 Downloaded by guest on October 2, 2021 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.

Mutation to asparagine will not only alter the glycosylation state of sapA and sapC as activators (26, 39) and the positively charged of GALC but will destabilize this part of the structure. patch on the TIM barrel near the active site (Fig. S7). The lectin Some disease-causing mutations may not affect the residual fold is almost universally a carbohydrate recognition domain (40), enzyme activity, processing, or localization of GALC, but may suggesting that this region of GALC may play a role via carbo- interfere with binding to activating factors. Consistent with this hydrate binding. This activity may involve binding of glycosylated hypothesis, the disease-associated mutation E215K has been proteins during the processing and trafficking of GALC. In sup- shown to have a very mild effect on GALC enzymatic activity port of this, one family of lectins known as galectins, to which the (29), and our structure reveals that it is exposed on the surface GALC lectin domain possesses structural homology, has identi- of the TIM barrel (Fig. 3). This mutation confers an opposite fied roles in protein trafficking and sorting into specific vesicle charge on the same face as the substrate-binding pocket suggest- populations (41). It has been noted previously that GALC may ing that the mechanism of disease for this mutation will involve be taken up by neighboring cells via M6P-independent mechan- the perturbation of a binding face for an activating factor, such as isms (13). In cultured skin the uptake of GALC by un- a saposin, that is essential for efficient in vivo glycolipid metabo- transduced cells is inhibited less than 50% by M6P,indicating that lism. Similarly, residue P302 that is found on the surface of the uptake is not solely via the M6P receptor-mediated pathway. GALC very close to the substrate-binding pocket is mutated to It is possible that the lectin domain of GALC is responsible for in Krabbe disease, a mutation that would significantly conferring this additional uptake mechanism. However, we note alter the surface properties in this region (Fig. 3). that other lysosomal enzymes also undergo M6P-independent Perhaps the most unexpected observation from the structure of trafficking via mechanisms that do not require the presence of GALC is the contribution from the β-sandwich domain of a long a lectin domain (42). loop that forms an integral part of the substrate-binding site. R380 at the tip of this loop directly binds the galactose molecule Cleavage of GALC. Many studies of GALC have focused on the in the active site (Fig. 3). The importance of this residue for processing of GALC into 50- and 30-kDa fragments following up- GALC activity is confirmed by its mutation to tryptophan or leu- take into the lysosome (6, 10, 43, 44). The cleavage site lies within cine leading to severe infantile Krabbe disease (13, 38). a loop of the β-sandwich domain, far from the active site. Follow- ing cleavage it is extremely unlikely that the two fragments would Role of the Lectin Domain. A striking feature of the GALC structure dissociate given the very large buried surface area between them. is the presence of the lectin domain sitting over one face of the We therefore conclude that this cleavage event has no relevance TIM barrel. Although several lysosomal enzymes possess TIM for enzyme activity, consistent with the previous identification of barrel and β-sandwich domains similar to that seen in GALC, activity of the 80-kDa “precursor” (33). Thus, it is incorrect the presence of a lectin domain is unique. This domain contri- targeting, with the side effect of failure to encounter processing butes directly to the formation of the substrate-binding cleft enzymes, rather than lack of cleavage per se that is likely to be the (Fig. 2C) and the presence of several disease-causing mutations critical deficit in mutant forms of GALC that are not cleaved. in this domain highlights its importance in the normal processing The structure of GALC has provided essential insights into and activity of GALC (Fig. 3 and Table S2). It is likely that the the role of disease-associated mutations in the pathogenesis of lectin domain will be involved in processes that are not found in Krabbe disease. GALC is now seen to possess a unique domain related enzymes lacking this domain (18). For example, the lectin arrangement, including a previously uncharacterized lectin do- domain is unlikely to be involved in saposin binding as GlcCerase, main and a substrate-binding site that involves each of the three which does not possess the lectin domain, retains both the binding domains of the protein. This structure provides a rich framework

15172 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105639108 Deane et al. Downloaded by guest on October 2, 2021 for further understanding of Krabbe disease and the development crystals were soaked with 20 mM D-galactose for 45 min before data collec- tion. The structure was built using ARP/wARP (47) and COOT (48) and refined of potential therapeutics. SEE COMMENTARY using phenix.refine (49). Detailed methods, data collection, and refinement Materials and Methods statistics are provided in SI Materials and Methods. His-tagged mouse GALC was produced recombinantly in HEK293T cells and purified from the supernatant using cobalt-affinity chromatography. The ACKNOWLEDGMENTS. We thank the staff of beamline I03 at the Diamond protein was crystallized at 4 mg∕mL using sitting-drop vapor diffusion with Light Source. J.E.D. is funded by a Wellcome Trust grant (to R.J.R.). S.C.G. microseeding (45) against a reservoir of 0.2 M sodium acetate, 0.1 M sodium is an 1851 Research Fellow. N.N.K. was supported by a grant from the Jean cacodylate, 34% PEG 8000. Diffraction data were recorded at Diamond Light Shanks Foundation and the Cambridge Overseas Trust. M.B.C.-G. was funded Source beamline I03. The structure was solved by MIRAS using AutoSol (46) by the Hunter’s Hope Foundation and the Biomedical Research Centre of the with data from Mercury and Platinum derivatives. For the product complex National Institute of Health Research.

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