CrystallographicStructureofHuman -Hexosaminidase A ...mcfuser/2006... · Keywords: lysosomal...

doi:10.1016/j.jmb.2006.04.004 J. Mol. Biol. (2006) 359, 913–929

Crystallographic Structure of Human b-HexosaminidaseA: Interpretation of Tay-Sachs Mutations and Lossof GM2 Ganglioside Hydrolysis

M. Joanne Lemieux1, Brian L. Mark1, Maia M. Cherney1

Stephen G. Withers2, Don J. Mahuran3 and Michael N. G. James1*

1CIHR Group in ProteinStructure and Function,Department of BiochemistryUniversity of AlbertaEdmonton, Alta., CanadaT6G 2H7

2Chemistry DepartmentUniversity of British ColumbiaVancouver, BC, CanadaV6T 1Z1

3Department of LaboratoryMedicine and PathobiologySick Kids Hospital, 555University Avenue, Universityof Toronto, Toronto, Ont.Canada M5G 1X8

0022-2836/$ - see front matter q 2006 P

Present address: B. L. Mark, Depbiology, University of Manitoba, WR3T 2N2.Abbreviations used: Hex A, b-he

NANA,N-acetyl-neuraminic acid; NNAG, N-acetylglucosamine; GM2, G2,3)Gal-1,4 Glc-ceramide; TSD, Tay-adult/chronic form of Tay-Sachs disacute form of Tay-Sachs disease; SDISD, infantile/acute form of Sandhoadult/chronic form of Sandhoff dismethylumbelliferyl-b-N-acetylglucomethylumbelliferyl-7-(6-sulfo-2-aceglucopyranoside; GalNAc, N-acetyl2-acetamido-2-deoxynojirimycin; DNNBDNJ, n-butyl-DNJ; NGT, N-acetyline; ER, endoplasmic reticulum; WE-mail address of the correspond

[email protected]

Lysosomal b-hexosaminidase A (Hex A) is essential for the degradation ofGM2 gangliosides in the central and peripheral nervous system. Accumu-lation of GM2 leads to severely debilitating neurodegeneration associatedwith Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here,we present the X-ray crystallographic structure of Hex A to 2.8 A resolutionand the structure of Hex A in complex with NAG-thiazoline, (NGT) to3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown toact as a chemical chaperone that, to some extent, prevents misfolding of aHex A mutant associated with adult onset Tay Sachs disease and, as aresult, increases the residual activity of Hex A to a level above the criticalthreshold for disease. The crystal structure of Hex A reveals an abheterodimer, with each subunit having a functional active site. Only thea-subunit active site can hydrolyze GM2 gangliosides due to a flexible loopstructure that is removed post-translationally from b, and to the presence ofaAsn423 and aArg424. The loop structure is involved in binding the GM2

activator protein, while aArg424 is critical for binding the carboxylategroup of the N-acetyl-neuraminic acid residue of GM2. The b-subunit lacksthese key residues and has bAsp452 and bLeu453 in their place; theb-subunit therefore cleaves only neutral substrates efficiently. Mutations inthe a-subunit, associated with TSD, and those in the b-subunit, associatedwith SD are discussed. The effect of NGT binding in the active site of amutant Hex A and its effect on protein function is discussed.

q 2006 Published by Elsevier Ltd.

Keywords: lysosomal storage disorders; b-hexoasaminidase A; GM2ganglioside; Tay-Sachs disease; glycoside hydrolase
*Corresponding author
ublished by Elsevier Ltd.

artment of Micro-innipeg, MB, Canada

xosaminidase A;GT, NAG-thiazoline;alNAc-1,4(NeuAc-Sachs disease; ATSD,ease; ITSD, infantile/, Sandhoff disease;ff disease; ASD,ease; MUG, 4-saminide; MUGS, 4-tamido-2-deoxy)-b-D-galactosamine; AdNJ,J, deoxynojirimycin;lglucosamine-thiazo-T, wild-type.ing author:

Introduction

GM2 gangliosidosis is a family of three autosomalrecessive, lysosomal storage disorders characteri-zed by the intralysosomal accumulation of theacidic glycolipid GM2 ganglioside, primarily in thebrain and peripheral neural tissues.1 Deficiencies ofeither the a-subunit or the b-subunit of theheterodimeric b-hexosaminidase A (Hex A) protein,or the small monomeric GM2 activator protein (asubstrate-specific co-factor for Hex A) leads to thephenotypic neurodegeneration associated with thisfamily of devastating disorders; i.e. Tay-Sachsdisease (TSD), Sandhoff disease (SD) and the ABvariant form, respectively.2

Hex A is a member of the Family 20 glycosidehydrolases (glycosidase) (EC 3.2.1.52).3 It removesthe terminal non-reducing N-acetylgalactosamine

914 Crystallographic Structure of Human Hex A

(GalNAc) from the GM2 ganglioside. The a-subunitand b-subunit of human Hex A are encoded by theevolutionarily related genes HEXA and HEXB,respectively. The primary sequences of thesesubunits are approximately 60% identical. TheGM2 activator protein (GM2AP) encoded by GM2A,is a lipid transporter that removes GM2 from itsmembranous environment and presents it to Hex Afor hydrolysis.4

Hex A, as well as Hex B (a b-homodimeric Hexisozyme), can carry out the hydrolysis of b-linkedGalNAc and/or N-acetylglucosamine (GlcNAc)from substrates, such as the oligosaccharide moi-eties from proteins and neutral glycolipids, or fromcertain mucopolysaccharides. The hydrolysis of theGM2 ganglioside, which contains a negativelycharged sialic acid group, however, is carried outonly by the a-subunit of Hex A.5,6 The specificity ofthis reaction is made absolute by the mechanism bywhich the GM2AP–GM2 complex interacts with theHex A heterodimer.

The GM2 ganglioside, composed of GalNAcb(1-4)-[NANAa(2-3)-]-Galb(1-4)-Glc-ceramide, is pri-marily an intermediate in the synthesis anddegradation of the higher brain gangliosides, e.g.GM1 ganglioside. Gangliosides are degraded in thelysosomes in a stepwise manner by interdependentexo-glycosidases. A number of different geneticdisorders are the result of a deficiency of one ofthese exo-glycosidase or its co-factor, which pre-vents turnover of the remaining macromolecule.7,8

This results in the accumulation of partiallydegraded glycosphingolipid, e.g. GM2 ganglioside,primarily in neural tissue and resulting in neuro-degeneration.9,10

Mutations in HEXA, HEXB and GM2A genescausing GM2 gangliosidosis have been character-ized in detail,2 and include partial gene deletion,splicing mutations, nonsense mutations and mis-sense mutations. These mutations cause defects intranscription, translation, monomer folding and/ordimerization and, more rarely, in the catalyticfunction of Hex A. Different genotypes result indifferent clinical phenotypes, which generallycorrelate biochemically with the amount of residualHex A activity.11 The most common, severe andfatal form is the acute or infantile onset forms ofTay-Sachs disease (ITSD) or infantile Sandhoffdisease (ISD). ITSD and ISD are associated with atotal deficiency of Hex A activity. However, in ITSD,total Hex activity is nearly normal due to the stableHex B isozyme; whereas in ISD, total Hex activity isonly w3% of normal, due to the unstable Hex S (ana-homodimeric Hex isozyme). The less severe lateon-set forms of GM2-gangliosidosis, i.e. juvenile/subacute and adult/chronic Tay-Sachs (ATSD),results from mutations that do not completelyprevent the formation of catalytically active HexA; with residual activities ranging from w1–8% ofnormal levels. The rare variant AB form of GM2

gangliodosis is due to mutations in the GM2A geneand produces normal levels of both Hex A and HexB when assayed with simple artificial substrates,

but no activity when assayed using GM2 gangliosideas a substrate. In the Ashkenazi Jewish population,the rate of TSD is an astounding 1 in 30. For thegeneral population, the rate is 1 in 300.1

Here, we report the crystallographic structure ofthe mature lysosomal form of Hex A from humanplacenta as a native structure to 2.8 A resolutionand co-crystallized with NAG-thiazoline (NGT) to3.25 A resolution. NGT is a mechanism-basedinhibitor,12 shown to decrease endoplasmic reticu-lum (ER) retention and hence increase residual HexA activity w3-fold in ATSD cells homozygous forthe aG269S mutation.13 The native structure revealsthe mature heterodimeric, glycosylated a-subunitand b-subunit of Hex A. Two distinct active sites arepresent in Hex A, one on the a-subunit and one onthe b-subunit. In both active sites, a glutamateresidue acts as a general acid-base that assists incleaving the terminal b-linked GalNAc or GlcNAcresidues from substrates; whereas an adjacentaspartate residue stabilizes the positively chargedoxazolinium intermediate that develops during thesubstrate-assisted catalytic mechanism carried outby human Hex18,21. In the a-active site, aAsn423and aArg424 residues promote GM2 binding byinteracting favorably with the negatively chargedsialic acid residue present on the GM2 oligosacchar-ide structure. The corresponding residues in theb-subunit active site are bAsp452 and bLeu453,which would be expected to repel the negativelycharged sialic acid moiety of GM2. The complexstructure of Hex Awith NGTreveals the mechanismby which NGT acts as a chaperone, stabilizing thenative conformation of the a-subunit and therebypromoting dimerization and allowing Hex A to exitthe ER and to be targeted to the lysosome. Thesedata provide an excellent starting point for thera-peutic advancement toward the treatment of lateon-set forms of GM2 gangliosidosis through struc-ture-based drug design.

Results and Discussion

Crystallization and overall structure of Hex A

A functionally mature glycosylated form oflysosomal Hex A isolated from human placenta(Mr 112,500) was crystallized in the absence and inthe presence of NGT (Figure 1(a)). The glycosylatedform was maintained in an attempt to view thecarbohydrate moieties involved in mannose-6-phosphate (M6P) receptor interaction(s). Numerouscrystals of both native and inhibitor-bound Hex Awere subjected to X-ray diffraction, the majority ofwhich diffracted X-rays to approximately 4 Aresolution. In the end, only one native Hex Acrystal could be found that diffracted X-rays beyond3 A resolution. Native Hex A crystallized in spacegroup C2 with four Hex A heterodimers in theasymmetric unit; a total of 4044 residues are in theasymmetric unit. Well-defined electron density(Figure 1(b)) was obtained from phasing by

Figure 1. (a) Hex A structure. Chemical structure of NGT. (b) Stereo view of the 2FoKFc map contoured at 1s onresidues aN114 to aY116, including glycosylation at aN115. (c) Stereo view of a ribbon representation for Hex A. Thea-subunit N terminus color begins with dark blue and continues to light blue, and then ends with light green at its Cterminus. The b-subunit N terminus begins with a greenish yellow color, changing to orange and ending in red at the Cterminus. NGT, located at the face of the TIM barrel, is shown in orange. (d) The individual a-subunit and (e) b-subunitare represented as viewed from the dimer interface.

Crystallographic Structure of Human Hex A 915

molecular replacement using the biological dimerof Hex B as the search model. Hex Awas built intothe experimental electron density at 2.8 A resol-ution and refined to an Rwork of 0.26 and an Rfree of0.28 (Table 1). The biological dimer of Hex A isdepicted in Figure 1(c) with the individual a andb-subunits represented in Figure 1(d) and (e),

respectively. Hex A co-crystallized with NGT wasrefined to 3.25 A resolution (Table 1). As in thenative structure, NGT-bound Hex A also crystal-lized in space group C2 with four molecules in theasymmetric unit. NGT was present in all foura-subunits and four b-subunits in the asymmetricunit Hex A (Figure 1(d) and (e)).

Table 1. X-ray diffraction data collection and atomicrefinement

A. Crystal informationData set Native NGTSpace group C2 C2Solvent content (%, v/v) 50.7 50.7Matthew’s coefficienta 2.5 2.5Molecules/asymmetricunitb

4 (4044) 4 (3933)

Residues/asymmetric unit

B. Data collectionUnit cell dimensionsa (A) 321.1 322.2b (A) 110.5 109.8c (A) 129.7 132.8b (deg.) 90.9 91.5Wavelength (A) 1.1158 1.1271Resolution range (A) 40.00 – 2.80 35.0 – 3.25High-resolution (A) 2.90 – 2.80 3.37 – 3.25Total observations 406,584 144,007Unique reflections 111,512 71,535hI/sIic,d 9.7 (2.0) 8.7 (1.9)Completeness (%)e 99.4 (99.7) 97.8 (97.4)B-value, Wilson plot (A2) 75 92Multiplicity 3.6 2.0Rmerge

e 0.089 (0.750) 0.065 (0.475)

C. RefinementRwork

f 0.26 0.27Rfree

g 0.28 0.32Number of atoms 32,139 32,007Water 151 11r.m.s.d from idealBond lengths (A) 0.006 0.009Bond angles (deg.) 0.818 1.09Ramachandran plotMost favored (%)h 2903 (86.0) 2884 (85.5)Allowed (%) 461 (13.7) 481 (14.3)Generously allowed (%) 13 (0.4) 10 (0.3)Disallowed (%) 0 0

a VM, A3/Da.

b Z, the number of molecules in the unit cell.c Statistics for the highest resolution shell are in parentheses.d hI/sIi is the ratio between the mean intensity and the mean

error of the intensity.e RmergeZ

Phkl

PjIjðhklÞKhIðhklÞij

Phkl

Pj jhIðhklÞi, with Ij(hkl)

representing the intensity of measurement j and hI(hkl)i is themean of measurements for the reflection hkl. Although the Rmerge

in the outer shell is high, the appropriate resolution limits werededuced from the Wilson plot.

f RworkZP

hkl jjFobsðhklÞjKFcalcðhklÞjj=P

hkl jFobsðhklÞj, whereFobs and Fcalc are the observed and calculated structure factors,respectively.

g Rfree is calculated in the same manner on 5% of structurefactors that were not used in the model refinement.

h Numbers in parentheses represent the percentage ofresidues in each area of the Ramachandran plot.


The four heterodimers in the asymmetric unit ofthe Hex A crystals are structurally comparable,having an average r.m.s.d. of 0.40 A for 920G23matching Ca atoms. (See Supplementary Data forindividual r.m.s.d. values for all structural super-impositions.) The NGT-bound Hex A has slightlybetter structural agreement, with an averager.m.s.d. of 0.31 A for 961G4 matching Ca atoms.When the four heterodimers from the Hex Astructure are superimposed with the four NGT-bound Hex A heterodimers, the average r.m.s.d. is0.36 A with 938G20 matching Ca atoms.

The overall structure of the Hex A heterodimeris similar to the structure of the Hex B homodimer,

having an average r.m.s.d. of 0.65 A for 915G8matching Ca atoms. The NGT-bound Hex A iscomparable when superimposed with Hex B,giving an average r.m.s.d. of 0.66 A for 920G5matching Ca atoms.

Subunit structure of Hex A

The a-subunit of Hex A is post-translationallycleaved to give the mature form,14 consisting oftwo polypeptide chains: aLys23 to aGly74, andaThr89 to aGln528 (Figure 1(d)).15 The b-subunit ofHex A is also cleaved post-translationally,16 to givethe mature form consisting of three polypeptides:bAla50 to bGly107, bThr122 to bSer311, andbLeu316 to bMet556 (Figure 1(e)).17 With only60% sequence identity, the structures of thea-subunits and b-subunits are comparable withan r.m.s.d. of 0.71 A for 460G10 matching Ca atomswhen structurally aligned. When the individuala-subunits and b-subunits from the NGT-boundHex A structure are aligned, the average r.m.s.d. is0.67 A for 466G4 matching Ca atoms.

In our X-ray structure of Hex A, both thea-subunit and b-subunit reveal similar topologies.Each subunit consists of two domains. Domain I,residues Leu23 to Pro168 in the a-subunit andbAla50 to bPro201 in the b-subunit, is an N-terminaldomain having two parallel a-helices sandwichedbetween a six-stranded anti-parallel b-sheet anddomain II. The function of domain I in Hex A isunknown. Domain II, residues 165 to 529 in thea-subunit and 202 to 556 in the b-subunit, consistsof a core TIM barrel fold ((b,a)8-barrel) with a helicalinsertion, aThr327 to aAsp347 in the a-subunit andbGlu362 to bThr378 in the b-subunit, as well as anextension at the C terminus (Figure 1(d) and (e)).

Important differences exist between the a-subunitand the b-subunit. The a280GSEP283 loop in thea-subunit is post-translationally cleaved in theb-subunit after bSer311 and before bAsp316(Figure 1(c)). In addition, the a396IPV398 loopfound in the a-subunit is not encoded by theHEXB mRNA for the b-subunit. From the structureof Hex B, a model of Hex A was generated, ontowhich the structure of the GM2A protein wasdocked.18 The model suggested the necessity for aflexible a280GSEP283 loop in order for the GM2Aprotein to interact with Hex A. It was demonstratedsubsequently through biochemical studies withmutant forms of Hex A in which these loops hadbeen deleted that the flexible a280GSEP283 loopplays the most important role in this interaction.19

Our current Hex A structure is consistent with thebiochemical data and confirms the validity of theprevious model derived from Hex B.

The active site of Hex A and the proposedmechanism of action

Two active sites are present in the Hex A dimer;one comprising residues from the a-subunit(Figure 2(a)) and a second one from residues of

Figure 2. NGT bound in the active site of Hex A. (a) NGT (shown in blue) bound in the active site of the a-subunit(green) showing a minor contribution of bY456 from the b-subunit (pink). (b) NGT bound in the active site of theb-subunit (pink) showing a minor contribution of aY427 from the a-subunit (green). Unrefined FoKFc density shown forNGT is contoured at 2.5s.


the b-subunit (Figure 2(b)). These active sites arelocated at the opening of the TIM barrels at theinterface between the a and b-subunits. In thea-subunit, NGT is stabilized via hydrogen bondingwith aArg178, aGlu462, aAsn423 aTyr421 andaAsp322 (Figure 2(a)). In the b-subunit, NGTforms hydrogen bonds with bArg211, bGlu491,bAsp452, bTyr450, and bAsp354 (Figure 2(b)).There is residue sharing in both active sites:bTyr456 is found in the a-subunit active site,whereas aTyr427 is found in the b-subunit activesite. Although not observed in our structure ofNGT-bound Hex A, previous analyses of the NGT-bound structure of Hex B solved at 2.5 A demon-strated that a water molecule along with, bTyr456,stabilizes active site residues aGlu462 and aAsn423in the a-subunit. These residues participate inhydrogen bonding with NGT bound within thea-subunit. A complementary stabilization takesplace within the b-subunit, where aTyr427 hydro-gen bonds with water to coordinate bGlu491 and

bAsp452 in the active site of the b-subunit. Theintimate interactions shared between the two activesites of both Hex A and Hex B suggest thatdimerization is essential for activity in each subunitof these isoenzymes. These data are consistentwith the lack of any biochemical evidence forthe existence of an active a or b-monomeric formof Hex.2

GM2 is presented to Hex A by the GM2 activatorprotein (GM2AP). Hex A removes the terminalb-linked GalNAc from the GM2 ganglioside toproduce the GM3 ganglioside (Figure 3(a)).18 Thishydrolysis is catalyzed only by the a-subunit of HexA. Residues emanating from the C termini of theb-strands comprising the (ba)8 barrel participate inGM2 hydrolysis. From the structures of other Family20 glycoside hydrolases, it has been demonstratedthat Family 20 members use substrate-assistedcatalysis with retention of configuration(Figure 3(b)) to remove the terminal b-linkedGalNAc and/ or GlcNAc residues from their

(a)

(b)

Figure 3. Proposed catalytic mechanism for Hex A. (a) Hydrolysis of the GM2 ganglioside by Hex A results in the lossof GalNAc to produce a GM3 ganglioside. (b) Proposed catalytic mechanism for Hex A showing substrate-assistedcatalysis. aGlu323 in the a-subunit and bGlu355 in the b-subunit act as the general base, while aAsp322 in the a-subunitand bAsp354 in the b-subunit act to orient the C2-acetamido group into position for nucleophilic attack andsubsequently stabilizes the oxazolinium ion intermediate. The hydroxyl residues and C6 have been removed from thepyranose ring of the substrate for clarity. The exact positions for these groups have not been determined.


oligosaccharide substrates.13,18,20–22 In Hex A,aGlu323 (a-subunit) and bGlu355 (b-subunit) arethe general acid-base residues for protonation of theglycosidic oxygen atom; aAsp322 (a-subunit) andbAsp354 (b-subunit) provide the negativelycharged carboxylate groups that stabilize thedeveloping positive charge on the nitrogen atomof the oxazolinium ion during the nucleophilicattack of theN-acetamido oxygen atom on the C1 0 ofthe substrate. In addition, there are strong sub-strate-orienting effects from the aromatic rings ofaTrp373, aTrp392, and aTrp460 in the a-subunit,and bTrp405, bTrp424 and bTrp489 in the b-subunit.Hydrogen bonding from aTyr421 in the a-subunitand bTyr450 in the b-subunit helps to orient thenucleophilic carbonyl oxygen atom as well as tostabilize the oxazolinium ion intermediate(Figure 2). This environment protects the acyl centerof the oxazolinium ion from attack and guides anincoming water molecule for the correct attack atthe anomeric center of the intermediate to produce aproduct with net retention of the b-configuration.

A model of GM2 ganglioside, based on thepreviously published model,18 was docked ontothe a-subunit of Hex A (Figure 4). Only residuesinteracting with the sugar residues are shown. Theremaining HexA residues and GM2AP, whichinteracts with the acyl chains of the GM2 ganglio-side, have been removed for clarity. The onlyresidue that required adjustment in order toaccommodate GM2 in the a-subunit active site wasaArg424, which was rotated about the Cd–Cb

torsion angle. The model of GM2 docked into HexA demonstrates that aArg424 would stabilize thenegatively charged carboxylate group of theN-acet-ylneuraminic acid (NANA) via hydrogen bonding.In addition, it appears that aArg424, which in theunbound structure stacks against aTyr456, moves tostack against aTyr421 in the presence of GM2. Manyof the residues in the a and b-active sites areconserved with the exception that aAsn423 andaArg424 in the a-subunit are replaced withbAsp452 and bLeu453 in the b-subunit. Thenegatively charged carboxylate group of the

Figure 4. Model of GM2 docked onto the a-subunit active site of Hex A. A model of the GM2 ganglioside (yellow) wasdocked into the active site of the a-subunit of Hex A based on the model of GM2 bound to the a-subunit active site of HexB.18 For clarity, only residues interacting with the sugar residues of GM2 are shown. GM2AP, which interacts with the acylchains of the GM2 ganglioside, has also been removed. aArg424, a positively charged residue unique to the a-subunit ofHex A, is found within hydrogen bonding distance from the negatively charged carboxylate of the NANA group of GM2.


NANAwould be repelled by the carboxylate groupof bAsp452 in the b-subunit, making the formationof a productive complex unlikely. Mutagenesis dataalso support this rationale for Hex A substratespecificity. A double mutant was prepared wherebyin Hex B, bAsp452 and bLeu453 were replaced withan Asn and Arg, respectively, in order to mimic theenvironment of the a-subunit active site.23 Kineticstudies with this Hex B double mutant resulted in a30-fold increase in the rate of the GM2AP-indepen-dent hydrolysis of the negatively charged artificial6-sulfated substrate 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy)-b-D-glucopyranoside(MUGS) compared with the wild-type Hex B.

Structural flexibility of Hex A

The fact that four Hex Amolecules were found inthe asymmetric unit gives us an opportunity toassess the structural variability among the differentHex A molecules and the effect of the NGT binding,a chemical chaperone, on the Hex A structure.There is close structural agreement between all fourHex A dimers in the asymmetric unit for both thenative and NGT-bound Hex A. (See SupplementaryData, Table 1.) The structural alignment of theheterodimers of native Hex A gives r.m.s.d. valuesranging from 0.36 A to 0.44 A, with an averager.m.s.d. of 0.39 A. When NGT is associated with HexA, the structural alignment of the heterodimersresults in r.m.s.d. values that are somewhat lower,ranging from 0.26 A to 0.35 A with the average

r.m.s.d. of 0.31 A. The small deviations fromstructural superimpositions can be attributed todifferences in crystal packing between unboundand NGT-bound Hex A that, interestingly, occur inthe a280GSEP283 loop. Flexibility in this loop regionis expected for GM2AP docking.

Hex A glycosylation

The a and b-subunits of Hex A display manyN-linked glycosyl residues and there are oligo-saccharides bound at most of the known glycosyla-tion sites on Hex A (Figure 1(b), (c), (d) and (e)).There are three glycosylation sites on the a-subunitof Hex A: aAsn115, aAsn157 and aAsn295.24 Fourglycosylation sites on the b-subunit have beenidentified: bAsn84, bAsn142, bAsn190, andbAsn327.24,25 The mannose residues of the glycosy-lated aAsn115, aAsn295 and bAsn84 are preferen-tially phosphorylated in order to be recognized bythe M6P receptor. In our structure, electron densityfor glycosylation at aAsn115 and aAsn157 wasobserved in all four a-subunits. In only twoa-subunits was electron density for glycosylationseen at aAsn295. In the four b-subunits, all bAsn190had electron density for glycosylation, whereaselectron density for glycosylation was observedin only one bAsn327. No or weak electron densitywas observed for the remaining glycosylationsites in the b-subunit. In several instances,two N-acetylglucosamine residues followed bymannose are visible.


Characterization of Hex A mutations on thebasis of structure

The reduction in the rate of GM2 gangliosidehydrolysis below a surprisingly low criticalthreshold, estimated to be w10% of normal,11

leads to its accumulation in the neural tissues andconcomitant neurodegeneration. Reduced Hex Aactivity can occur via numerous types of mutationsthroughout the HEXA and HEXB genes. A largenumber of these defects have been identified†.2 Inmany cases, genotype can easily be used to predictthe ITSD phenotype, e.g. partial gene deletions,mRNA splicing, and nonsense mutations. Missensemutations also play a major role in dysfunctionalHex A and can lead to phenotypes ranging fromacute to chronic, making predictions based only ongenotype difficult. Interestingly, more disease-associated missense mutations in the a-subunithave been identified than in the b-subunit. Thismay reflect the lower inherent stability, i.e. greaterflexibility of the a-subunit as compared to theb-subunit, which would make the a-subunit moresusceptible to destabilizing missense mutations. Invitro mutagenesis and expression experiments thatduplicated a-point mutation in the aligned site inthe b-subunit support this hypothesis.26

We have mapped all known missense mutationsonto the Hex A molecule according to the severityof disease (Figure 5). In the a and b-subunits of HexA, these include the residues listed in Table 2. Eachmissense mutation has been colored according tothe severity of the GM2 gangliosidosis phenotype,red for acute to subacute, green for chronic and bluefor asymptomatic (mutations that lower Hex Aactivity, but not below the critical threshold neededto prevent storage). The majority of residuesinvolved in acute and chronic TSD are locatedthroughout domain II of the a-subunit, distributedamongst the b-strands and helices comprising theTIM barrel. Notably, only a fewmutations are foundamong residues of the active site.

As noted in Table 2, we have attempted to predictthe effect of these missense mutations on thestructure of Hex A in addition to listing the cellularphenotype and the severity of disease associatedwith that mutation. The majority of mutationscharacterized for Hex A tend to shift the equili-brium from fully folded toward misfolded proteinproduction, leading to the retention of the defectivea or b-subunits in the ER and degradation by theER-associated degradation pathway, ERAD.27 InERAD, misfolded proteins in the ER are detected byresident ER proteins and undergo retrogradetransport back into the cytosol, where they areubiquitinated and targeted for degradation by theproteasome.28 Indeed, it has been shown foranother lysosomal storage disease, GM1 gangliosi-dosis, that the neuronal cell death associated withlysosomal b-galactosidase deficiency is attributed

† http://www.hexdb.mcgill.ca/

not to the accumulation of GM1, but to the unfoldedprotein response that results in the up-regulation ofchaperones and apoptotic factors.29 Therefore,characterization of Hex A misfolding is essentialto correlate the mutation with the severity ofdisease.

Because the extent of protein folding andmisfolding in the ER for the subunits of Hex Acan vary, the classification of misfolded mutants isdifficult. Nevertheless, there are two main bio-chemical phenotypes associated with the destabiliz-ing missense mutations for Hex A. Firstly, themutations that result in subunits that are comp-letely unable to fold correctly and, because theycannot form heterodimers and exit the ER, noneobtains the lysosomal targeting label. These mutantsubunits are either not easily detectable because ofrapid degradation by the ERAD pathway, or can beextracted from cells only in the presence ofdetergent, because they form ERAD-resistantaggregates. These aggregates may exacerbate theclinical phenotype.30 The second type of missensemutations are presumably less destabilizing andallow a small proportion of newly synthesizedmutant subunits to fold. These properly foldedsubunits can form heterodimers, can exit the ER andtherefore obtain the lysosomal targeting label. Inthese cases, the levels of residual activity generallycorrelate with the levels of mature lysosomalprotein, indicating that little if any change hasoccurred in the catalytic capacity of the mutantenzymes. These latter mutations include aTyr180-His, aGly269Ser, bPro504Ser, bArg505Gln andbAla543Ser, resulting in the less severe phenotypesof GM2 gangliosidosis. The reduction in Hex Aactivity caused by these mutations is a result of thevarious biochemical consequences of both theposition of the mutated residue in the subunit andthe degree of conservation of the amino-acidsubstitution. We see from the overall distributionof the currently characterized Hex A mutations andtheir biochemical phenotypes that prediction of theseverity of a mutation, with the exception of thosearising in the active site, would be difficult. Tay-Sachs mutations arising in Hex A that have beenstudied in more detail are described below,including examples of acute, chronic and asympto-matic phenotypes.

Active site mutation aArg178: acute, B1-variant

Analysis of naturally occurring mutants revealeda rare phenotype of “normally folded but reducedactivity” class of active site mutants termed the B1variant. The B1 variant mutation occurs predomi-nantly at aArg178,31 and results in the formation ofa normal Hex A heterodimer that can hydrolyze thecommon neutral artificial substrate MUG, but isnearly catalytically inactive towards the a-specificsubstrates MUGS and the GM2 ganglioside.32 WithGalNAc from the GM2 substrate docked into theactive site of Hex A (Figure 6(a)), we can see thataArg178 is involved in substrate binding by

http://www.hexdb.mcgill.ca/

Figure 5. Known mutations of Hex A contributing to Tay-Sachs and Sandhoff disease. (a) Stereo view of a ribbonrepresentation of Hex A (wheat), with residues known to disrupt Hex A activity: acute to sub-acute, red; chronic, green;asymptomatic, cyan. (b) A stereo view of the a-subunit of Hex A and residues associated with Tay-Sachs disease. (c) Astereo view of the b-subunit and residues associated with Sandhoff disease.


interacting with the 3 0 hydroxyl group of the non-reducing bGalNAc. By modeling the aArg178H,aArg178C and aArg178L mutations, we see adisruption in the hydrogen bonding network in

the active site that would reduce GM2 binding andseverely affect the activity of the mutant a-activesite of Hex A. Patients homozygous for aArg178Hishave a sub-acute phenotype, whereas heterozygotes

Table 2. Missense mutations identified in the a-subunits and b-subunits of Hex A


Table 2 (continued)

Missensemutations identified in the a-subunit and b-subunits of Hex A are listed according to their severity for Tay-Sachs and Sandhoffdisease. The color of the text corresponds to the mutations mapped on the structure shown in Figure 6. na: not assessed. References foreach individual mutation can be found at http://www.hexdb.mcgill.ca/.2


with a second null allele present with the moresevere acute phenotype.33 Other substitutions of thesame residue, aArg178Cys or aArg178Leu, result ina more severe phenotype, because of these lessconservative substitutions that may also destabilizethe a-subunit, as well as decrease its catalyticcapacity severely.

aAsp258His mutation: severe subacute,B1-variant like

The aAsp258His mutation34 was identified in aTSD patient with a phenotype termed severesubacute. Samples from this patient displayed ahigher than expected residual Hex A activityutilizing MUG as a substrate (w15% of normal),but were nearly inactive when the MUGS substratewas used. Thus, biochemically this appeared to beB1-like.35,36 aAsp258 is located adjacent to the activesite in the a-subunit. It participates in stronghydrogen bonding with residues aThr259 andaAsp322 (Figure 6(b)), the latter hydrogen bondswith GalNAc, providing a negatively chargedcarboxylate that stabilizes the developing positivecharge on the nitrogen atom of the oxazolinium ionduring the nucleophilic attack of the N-acetamidooxygen atom on the C1 0 of the substrate. Thesubstitution of aAsp258 for a more bulky Hisresidue would disrupt the coordination ofaAsp322 and may displace aGlu323, which acts asthe general acid in the GM2 hydrolysis. Conse-quently, this mutation would be expected to inhibitsubstrate hydrolysis indirectly by the a-subunitactive site of Hex A, as well as destabilize the initialfolding of the a-subunit.

aArg504His mutation: subacute

An Arg504His substitution37–39 results insynthesis and proper folding of the a-subunitprecursor, such that it was unable to form dimersand thus be transported to the lysosome. Thisconclusion was based on the observations that nomature (lysosomal) forms of the a-subunit could bedetected but treatment of the cells of this patientwith NH4Cl could induce the secretion of someinactive, phosphorylated a-monomers. aArg504 islocated at the interface between the a and b-sub-units and hydrogen bonds with bAsp494 of theb-subunit (Figure 6(c)). This interaction, along withother hydrogen bonding interactions such asaGln515 with bAsn497 and aAsn518 with bHis212at the center of the subunit interface, plays a role inHex A dimerization. (See Supplementary Data,Figure 1.) Substitution of aArg504 for a His residuewould weaken this interaction at the core of thesubunit interface.

aGlu482Lys mutation: acute

This mutation accounts for 2% of cases of TSDfound in Moroccan Jews.40 When aGlu482 issubstituted for Lys, the protein cannot exit the ERand results in expression of insoluble aggregates.As a result, this single amino acid substitutionresults in ITSD. aGlu482 is a buried residue thatparticipates in hydrogen bonding with aArg499(Figure 6(d)). In addition, aGlu482 hydrogen bondswith aTrp26 of domain I. This salt-bridge issurrounded by hydrophobic residues that comprisethe interface between domain I and domain II of

http://www.hexdb.mcgill.ca/


Hex A. (See Supplementary Data, Figure 2 forinterface interactions between domain I anddomain II.) Substitution of Lys for aGlu482 woulddisrupt this hydrogen bonding and may disrupt theinteractions between domain I and domain II.Therefore, it is possible that the interaction betweendomain I and domain II plays a role in proteinfolding and/or facilitates dimer formation. Curren-tly, the role of domain I in Hex A is unknown.

The most common (chronic) ATSD mutation,aGly269Ser

The aGly269Ser is the predominant mutationfound in adult TSD (ATSD).41 Although this

Figure 6 (legen

mutation is rare in all populations, its expressionin Ashkenazi Jews is greatest, as it can pair with oneof the two high-frequency ITSD alleles. The overallTSD carrier rate in this population is 1 in 30, withthe ATSD allele accounting for w3% of the totalmutant TSD alleles.1 Whether homozygous orheterozygous for this allele, patients present withthe ATSD phenotype. This mutation was predictedto disrupt the stability of Hex A, resulting inretention of the majority of the mutant a-subunitsin the ER. As a result, these ATSD patients haveresidual activity between 4% and 8%.13 Modelingthe aGly269Ser mutation in Hex A shows thatthe Cb of Ser269 would clash with the Cb ofGlu220 (Figure 6(e)). Examination of the region

d next page)

Figure 6. Model of Hex A mutants. (a) Stereo view representation of the Ca trace for wt-Hex A (a-subunit, green; b-subunit, pink) superimposed with the aArg178H,C,L Hex A substitutions (yellow). GalNAc (cyan) from a GM2 substratehas been docked into the active site. (b) Stereo view representation of the Ca trace for wt-Hex A (a-subunit, green)superimposed with the aAsp258His Hex A mutant (yellow). (c) A stereo view representation of the Ca trace for wt-HexA (a-subunit, green; b-subunit, pink) superimposed with the aAsp258His substitution (yellow). (d) A stereo viewrepresentation of the Ca trace for wt-Hex A (a-subunit, green) superimposed with the aGlu482Lys Hex A substitutions(yellow). (e) A stereo view representation of the Ca trace for wt-Hex A (a-subunit, green) superimposed with theaGly269Ser Hex A substitutions (yellow). (f) A stereo view representation of the Ca trace for wt-Hex A (a-subunit, green)superimposed with the aArg247W Hex A mutant (yellow). Stick representations are shown for residues surroundingsubstitutions as well as other key residues described in the text.


surrounding aGly269 reveals a random coil, whilethe aGlu220 is found on a 310 coil that appears to berigidified via hydrogen bonding with neighboringresidues. A mutation at aGly269 with a more bulkyand polar residue such as Ser would result in adisplacement of its randomly coiled backbone. ThisaGly269Ser mutation is proximal to aHis262, a keyresidue found in the active site that plays anessential role as a proton donor to aGlu323 andaAsp207 in the active site. Thus, disruption of the

backbone from aGly269Ser may result in a move-ment of aHis262 and may disrupt the coordinationof residues in the active site. The fact that NGT canrescue aGly269Ser Hex A from misfolding to someextent,13 indicates that upon NGT binding a slightconformational change may occur in a sufficientmanner to enable proper folding and targeting ofHex A from the ER to the Golgi, and ultimately tothe lysosomal compartment via the mannose-6-phosphate receptor. Binding of NGT may shift the


equilibrium for protein folding toward a morestable conformation that is able to evade theERAD pathway.

The asymptomatic aArg247Trp and aArg249Trpsubstitutions in Hex A

Missense aArg247Trp and aArg249Trp substi-tutions in Hex A have been identified in patientshaving low levels of Hex A similar to those havingTSD, but with no symptom of disease.42 The lowlevels of Hex A have been attributed to instabilityof the a-subunit. Using pulse-chase studies, also ithas been shown that the rate of conversion fromthe precursor a-subunit to its mature form isdelayed.43 This may reflect an effect on folding ordimerization. Interestingly, once formed, theseHex A mutants are not heat-labile, nor do theyhave trouble being processed or targeted to thelysosomal compartment. They appear to havenormal catalytic capacities. Both residues arelocated on the surface of Hex A at the interfacebetween domain I and domain II. The aArg247Trpsubstitution (Figure 6(f)), located in domain II,interacts via hydrogen bonding with aSer59 andaCys104 found in domain I and aGlu244 ofdomain II of Hex A. (See Supplementary Data,Figure 2 for interface interactions between domainI and domain II.) The aArg249Trp substitution(model not shown), also located in domain II,participates in hydrogen bonding with aArg67 ofdomain I, as well as aAsp191 and aTyr245 foundin domain II. Substitutions of aArg247Trp andaArg249Trp would disrupt these hydrogen bondsand influence the interaction between domains Iand II in the a-subunit. As stated above, it ispossible that the interaction between domain I anddomain II plays a role in protein folding and/orfacilitates dimer formation. In addition, it has beenshown that single amino acid mutations can resultin an increased association of a protein withchaperones leading to their retention in the ER: aD18G transthyretin mutation increases its associ-ation with BiP,44 and the human ether-a-go-go(hERG) N470D mutation increases its associationwith calnexin.45 It is possible that the asympto-matic aArg247Trp and aArg249Trp mutationsfound on the surface of Hex A may increase itsassociation with ER chaperones leading to reten-tion in the ER.

The determination of the three-dimensionalstructure of Hex A provides the first glimpse ofthe molecule responsible for TSD, and provides anopportunity to develop structure-based inhibitorsand novel chemical chaperones (CC). Substratedeprivation therapy with N-butyl-DNJ is currentlybeing used to treat Gaucher diseases,46,47 and isbeing tested for the treatment of ATSD. This druginhibits the synthesis of glucosylceramide (stored inGaucher), which is the precursor to neutral andacidic (ganglioside) glycolipid synthesis. N-Butyl-DNJ, however, has a number of unpleasantside-effects, which increase in a dose-dependent

manner.48 Thus, more specific drugs such as CC areneeded as alternative methods of treatment. Poten-tially, CC could be used in conjunction withsubstrate reduction therapy to lower the amountof N-butyl-DNJ needed to treat Gaucher or ATSD.Information obtained from the structure of NGTbound to Hex A is important in understanding themechanism by which this chemical acts as achaperone for Hex A protein folding. Primaryscreening using live cell assays is being developedin order to identify novel CC. Promising candidatescan then be docked and hopefully co-crystallizedwith Hex A. In addition, the three-dimensional-structure of Hex A can be used for in silico dockingin order to identify new potential inhibitors or CCs.Moreover, a structure of Hex A will enablestructure-based design of new pharmacologicalchaperones for the treatment of those afflictedwith ATSD.

Materials and Methods

Purification and crystallization

Hex A was purified from human placenta asdescribed,49 and was crystallized using the vapor-diffusion method in 13% (w/v) PEG 8000, 0.1 Msodium acetate, 0.2 M thiocyanate (pH 5.5). The proteinwas used in its mature glycosylated state for allexperiments. Initially, small multilayered crystals grewwithin one week. Macroseeding was used to obtainwell-ordered crystals with dimensions of 100 mm!100 mm!50 mm. Crystals were then soaked briefly inmother liquor containing 20% (v/v) ethylene glycolfollowed by flash-cooling in liquid nitrogen. NGT-bound HexA crystals were obtained by soaking 5 mMNGT for one to two days into drops containingcrystals.

Structure determination and model building

All diffraction data were collected at the AdvancedLight Source (ALS) at Lawrence Berkeley National Lab,BL8.3.1 equipped with a Quantum 210 ADSC CCDdetector. Intensity data were processed using DENZOand SCALEPACK.50 Phases were calculated usingMOLREP for Hex A,51 and PHASER for Hex A-NGT,52

with physiological Hex B (1NOU.pdb) as the searchmodel.Four Hex A molecules, each consisting of an a-subunit

and a b-subunit, were located in the asymmetric unit. TheHex B structure served as a backbone model to facilitateHex A tracing. The structure was subjected to rigid bodyrefinement followed by successive rounds of restrainedrefinement using REFMAC5,53 followed by modelbuilding using Xfit.54 Both NCS and TLS were used inthe refinement to facilitate tracing. Water molecules wereplaced manually and checked with WATERTIDY in theCCP4 package.55 NCS was omitted for the final round ofrefinement. The final model has good geometry with noamino acid residue in the disallowed region of theRamachandran plot, as determined by PROCHECK.56

GM2 and the GM2AP complex structure were dockedmanually onto the Hex A structure on the basis of the HexA-NGT structure, and models generated by the Hex B


structure.18 Figures were prepared with PYMOL†. Thesuperimposition of similar and related molecules werecarried out with ALIGN.57

Protein Data Bank accession number

The atomic coordinates and structure factors have beendeposited with RCSB Protein Data Bank as entry pdb2GJX for native Hex A and 2GKI for NGT-bound Hex A.

Acknowledgements

This work has been supported by the CanadianInstitute for Health Research (CIHR) and theCanadian Protein Engineering Network (PENCE).We thank Ernst Bergman, Jonathan Parrish (AlbertaSynchrotron Institute, University of Alberta) andJames Holden (BL8.3.1, Advanced Light Source,Berkeley National Laboratory) for help with thedata collection. X-ray diffraction data were collectedat beamline 8.3.1 of the Advanced Light Source(ALS) at Lawrence Berkeley Lab, under an agree-ment with the Alberta Synchrotron Institute (ASI).The ALS is operated by the Department of Energyand supported by the National Institutes of Health.Beamline 8.3.1 is funded by the National ScienceFoundation, the University of California and HenryWheeler. The ASI synchrotron access program issupported by grants from the Alberta Science andResearch Authority (ASRA) and the Alberta Heri-tage Foundation for Medical Research (AHFMR)and Western Economic Diversification (WED),Canada. We thank Amy Leung (Hospital for SickChildren) for purifying the Hex A used in thisproject. We thank all members of the Jameslaboratory, Dr M. Wang and Dr B. Biswal for theirassistance during structure determination andmanuscript preparation, Dr J. Parrish for datacollection, and Shiraz Khan for his assistance withcrystal shipping. B.L.M. was supported by scholar-ships from the CIHR and AHFMR. M.J.L. issupported by fellowship scholarships with CIHRand AHFMR. M.N.G.J. acknowledges support fromthe Canada Research Chairs Program.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2006.04.004

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Edited by M. Guss

(Received 27 February 2006; received in revised form 30 March 2006; accepted 1 April 2006)Available online 27 April 2006

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