Structural basis of pharmacological chaperoning for human β-galactosidase

S uc u al Basis o Pha macological Chape oning o Human ␤ -Galac osidase * □ S Recei ed o publica ion, Oc obe 23, 2013, and in e ised o m, Ma ch 12, 2014 Published, JBC Pape s in P ess, Ap il 15, 2014, DOI 10.1074/jbc.M113.529529 Hi ono i Suzuki ‡ , Umeha u Oh o ‡ , Ka sumi Higaki § , Te esa Mena-Ba agán ¶ , Ma ilde Aguila -Moncayo ¶ , Ca men O iz Melle ¶ , Eiji Nanba § , Jose M. Ga cia Fe nandez 储 , Yoshiyuki Suzuki** 1 , and Toshiyuki Shimizu ‡ ‡‡2 F om he ‡ G adua e School o Pha maceu ical Sciences, The Uni e si y o Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, he § Di ision o Func ional Genomics, Resea ch Cen e o Bioscience and Technology, To o i Uni e si y, 86 Nishi-cho, Yonago, To o i 683-8503, Japan, he ¶ Depa men o O ganic Chemis y, Facul y o Chemis y, Uni e si y o Se ille, P o eso Ga cía González 1, E-41012 Se ille, Spain, he 储 Ins i u e o Chemical Resea ch (IIQ), CSIC, Uni e si y o Se illa, Ame ico Vespucio 49, Isla de la Ca uja, E-41092 Se illa, Spain, he **In e na ional Uni e si y o Heal h and Wel a e G adua e School, Ki a Kanema u, O awa a, Tochigi 324-8501, Japan, and ‡‡ CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Sai ama 332-0012, Japan Backg ound: Pha macological chape one (PC) he apy has been p oposed o lysosomal s o age diseases. Resul s: Wild ype and mu an ␤ -galac osidases exhibi simila enzymological p ope ies. The ecogni ion mechanism o glycomime ic PC candida es in ol es bo h suga -like and subs i uen moie ies. Conclusion: C ys al s uc u es e eal he molecula basis o high binding po ency o PC compounds. Signi icance: Enzymological p ope ies, binding a ini ies, and ecogni ion modes a e biophysically and s uc u ally cha ac e ized. G M1 gangliosidosis and Mo quio B disease a e au osomal ecessi e diseases caused by he de ec in he lysosomal ␤ -galac- osidase ( ␤ -Gal), equen ly ela ed o mis olding and subse- quen endoplasmic e iculum-associa ed deg ada ion. Pha - macological chape one (PC) he apy is a newly de eloped molecula he apeu ic app oach by using small molecule ligands o he mu an enzyme ha a e able o p omo e he co ec old- ing and p e en endoplasmic e iculum-associa ed deg ada ion and p omo e a icking o he lysosome. In his epo , we desc ibe he enzymological p ope ies o pu i ied ecombinan human ␤ -Gal WT and wo ep esen a i e mu a ions in G M1 gan- gliosidosis Japanese pa ien s, ␤ -Gal R201C and ␤ -Gal I51T .We ha e also e alua ed he PC e ec o wo compe i i e inhibi o s o ␤ -Gal. Mo eo e , we p o ide a de ailed a omic iew o he ecogni ion mechanism o hese compounds in compa ison wi h wo s uc u ally ela ed analogues. All compounds bind o he ac i e si e o ␤ -Gal wi h he suga -mimicking moie y making hyd ogen bonds o ac i e si e esidues. Mo eo e , he binding a ini y, he enzyme selec i i y, and he PC po en ial a e s ongly a ec ed by he mono- o bicyclic s uc u e o he co e as well as he o ien a ion, na u e, and leng h o he exocyclic subs i uen . These esul s p o ide unde s anding on he mechanism o ac ion o ␤ -Gal selec i e chape oning by newly de eloped PC compounds. Human ␤ -D-galac osidase (EC 3.2.1.23, ␤ -Gal) 3 is a lyso- somal hyd olase ha ca alyzes emo al o e minal ␤ -linked galac ose in G M1 ganglioside and ke a an sul a e (1–3). In humans, de iciency o ␤ -Gal enzyme causes G M1 gangliosidosis and Mo quio B disease, wo lysosomal s o age diseases (LSDs) cha ac e ized by he p og essi e accumula ion o me aboli es in he cell (4–6). G M1 gangliosidosis is a se e e neu odegene a- i e disease ha is classi ied in o h ee ypes, in an ile, ju enile, and adul , depending on he onse and se e i y (7). Mo quio B disease is a a e bone disease wi hou cen al ne ous sys em in ol emen (7). Cu en ly, mo e han 160 mu a ions in he human ␤ -Gal gene ha e been iden i ied as ac o s causa i e o i s de iciency (8, 9). Two p incipal ea men s a egies a e cu en ly app o ed o in clinical ials o LSDs. The i s is enzyme eplacemen he - apy, whe e he de icien enzyme is supplied by egula injec ion o pu i ied ecombinan human enzyme (10–12). Howe e , li - leo noimp o emen hasbeenobse edin hecen alne ous sys em a ec a ions in LSD pa ien s because he enzyme canno *This wo k was suppo ed by G an s-in-Aid om he Minis y o Educa ion, Cul u e, Science, Spo s, and Technology o Japan ( o U. O. and T. S.) and G an s 13680918 and 14207106 ( o Y. S.), Minis y o Heal h, Labo and Wel a e o Japan H20-Koko o-022 and H22-Nanji-Ippan-002, and Japan Science and Technology Agency G an AS232Z00009G ( o Y. S.), g an s om he Takeda Science Founda ion and Mochida Memo ial Founda ion o Medical and Pha maceu ical Resea ch ( o U. O. and T. S.), and Spanish Minis e io de Economía y Compe i i idad con ac s SAF2010-15670 and CTQ2010-15848, he Fundación Ramón A eces, he Jun a de Andalucía, he Eu opean Regional De elopmen Funds (FEDER), he Eu pean Social Funds (FSE), and he Cen e o Resea ch, Technology and Inno a ion o he Uni e si y o Se ille (CITIUS). □ S This a icle con ains supplemen al da a and Figs. S1–S5. The a omic coo dina es and s uc u e ac o s (codes 3WEZ, 3WF0, 3WEZ, 3WF0, 3WF1, 3WF2, 3WF3, and 3WF4) ha e been deposi ed in he P o ein Da a Bank (h p://wwpdb.o g/). 1 P esen add ess: Tokyo Me opoli an Ins i u e o Medical Science, 2-1-6 Kami-Ki azawa, Se agaya-ku, Tokyo 156-8506, Japan. 2 To whom co espondence should be add essed: 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-03-5841-4840; Fax: 81- 03-5841-4891; E-mail: [email p o ec ed]. 3 The abb e ia ions used a e: ␤ -Gal, ␤ -galac osidase; G M1 , monosialo e a- hexosylganglioside; LSD, lysosomal s o age disease; DGJ, 1-deoxygalac- onoji imycin; PC, pha macological chape one; NOEV, N-oc yl-4-epi- ␤ - alienamine; 6S-NBI-DGJ, 5N,6S-(N⬘-bu yliminome hylidene)-6- hio-1-de- oxygalac onoji imycin; 6S-NBI-GJ, 5N,6S-(N⬘-bu yliminome hylidene)-6- hiogalac onoji imycin; NBT-DGJ, N-(N⬘-bu yl hioca bamoyl)-1-deoxy- galac onoji imycin. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 21, pp. 14560–14568, May 23, 2014 © 2014 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. Published in he U.S.A. 14560 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014 This is an open access a icle unde he CC BY license. c oss heblood-b ainba ie .Thesecondissubs a e educ ion he apy, which uses an o ally a ailable small molecule ha inhibi s he biosyn hesis o he glycosphingolipid (13). How- e e , his ea men is nonspeci ic, leading o se ious side e ec s. An al e na i e ea men , pha macological chape one (PC) he apy, has been p oposed o G M1 gangliosidosis, Mo quio B disease, and o he LSDs (14). This he apy uses a small molecule ligand ha can bind o he mu an p o ein and s abilize he co ec con o ma ion o he p o ein a neu al pH in he endoplasmic e iculum, allowing i o be anspo ed o he lysosome whe e he ligand dissocia es a acidic pH and in he p esence o excess subs a e. Galac ose, he ca aly ic p oduc o ␣ -o ␤ -Gal, he imino- suga 1-deoxygalac onoji imycin (DGJ; Fig. 1), a mimic o galac ose, and some de i a i es ha e been well s udied as PCs o hese LSDs (15, 16). Howe e , DGJ has p omiscui y o a numbe o galac opy anoside-p ocessing isoenzymes, which may hampe clinical de elopmen . We had p e iously epo ed he alienamine de i a i e N-oc yl-4-epi- ␤ - alienamine (NOEV) and he sp 2 -iminosuga de i a i e 5N,6S-(N⬘-bu yliminome hylidene)-6- hio-1-deoxy- galac onoji imycin (6S-NBI-DGJ; Fig. 1), as e ec i e PC com- pounds ha imp o e human mu an ␤ -Gal ac i i y in cul u ed cells(8, 17–20).The e ec o NOEV and6S-NBI-DGJ on educ- ing he accumula ion o G M1 ganglioside was mu a ion speci ic. In ac , NOEV educed he accumula ion in ␤ -Gal R201C bu no in ␤ -Gal I51T cells, whe eas 6S-NBI-DGJ was e ec i e in bo h cell lines (8, 20). On he o he hand, he inhibi o y ac i i y o NOEV o e came he chape one ac i i y a mic omola concen- a ions, whe eas he chape one e ec o 6S-NBI-DGJ s eadily inc easedin adose-dependen manne up omilimola concen- a ions (20). The p esence o an ex a pseudoanome ic hyd oxyl in 5N,6S-(N⬘-bu yliminome hylidene)-6- hiogalac- onoji imycin (6S-NBI-GJ; Fig. 1) esul s in a selec i i y shi owa d ␤ -glucosidase ( ␤ -glucoce eb osidase), which has been exploi ed o he de elopmen o e icien pha macological chape ones o Gauche disease (21–23). The monocyclic N-(N⬘-bu yl hioca bamoyl)-1-deoxygalac onoji imycin (NBT- DGJ;Fig. 1) de i a i e u ned ou o be ins ead a selec i e inhib- i o o lysosomal ␣ -Gal wi h po en ial as a pha macological chape one o Fab y disease (17). We epo ed he c ys al s uc u e o human ␤ -Gal (24). Ge - ing mo e s uc u al in o ma ion on how his se o compounds in e ac wi h human ␤ -Gal and es ablishing PC䡠enzyme com- plex s uc u e-ac i i y ela ionships p o ide use ul in o ma ion abou he ea u esgo e ningchape one-enzymein e ac ionsa he molecula le el and how hey could be implemen ed in he design o new gene a ions o PC d ug candida es o G M1 gangliosidosis. In his s udy, we examined he enzymological p ope ies o ecombinan human ␤ -Gal WT and wo ep esen a i e mu a- ions in Japanese pa ien s, ␤ -Gal R201C and ␤ -Gal I51T . To da e, he chape one abili y o candida e compounds was e alua ed agains lysa es om cul u ed human ib oblas s o ansien ly ans ec ed cells. In his epo , all assays we e pe o med using pu i ied ecombinan human ␤ -Gal, so ha he chape one e ec canbe es ima edwi hou anyin e e ence. Wealsode e - mined c ys al s uc u es o he complexes o he s ong ␤ -Gal inhibi o s, NOEV and 6S-NBI-DGJ, and o he much weake ligands 6S-NBI-GJ and NBT-DGJ bound o human ␤ -Gal WT . ␤ -Gal I51T mu an s uc u es complexed wi h galac ose o 6S-NBI-DGJ we e also de e mined. EXPERIMENTAL PROCEDURES ␤ -Gal Inhibi o s/Chape ones—The alienamine de i a i e NOEV, and he h ee sp 2 -iminosuga de i a i es 6S-NBI-DGJ, 6S-NBI-GJ, and NBT-DGJ, we e syn hesized ollowing p e i- ously epo ed p ocedu es (Fig. 1) (17, 20, 25, 26). All com- pounds a e s able a oom empe a u e and soluble in wa e up o 10 mM(NOEV and 6S-NBI-DGJ) o 20 mM(6S-NBI-GJ and NBT-DGJ). The molecula weigh s a e 287.4, 260.4, 278.4, and 276.4, espec i ely. S ock solu ion was p epa ed in s e ile H 2 O and s o ed a ⫺20 °C. NOEV and 6S-NBI-DGJ inhibi human ␤ -Gal in i o wi h high selec i i y, whe eas 6S-NBI-GJ and NBT-DGJ a e e y weak inhibi o s o ␤ -Gal bu low mic omo- la inhibi o s o ␤ -glucoce eb osidase o ␣ -Gal, espec i ely. Cloning, Exp ession, and Pu i ica ion o Human ␤ -Gal—The de ails o cloning, exp ession, and pu i ica ion o ␤ -Gal we e epo ed p e iously (27). Mu a ions we e in oduced by P ime- STAR Max DNA polyme ase (Taka a) using oligonucleo ide p ime s ollowing he manu ac u e ’s p o ocol. B ie ly, ␤ -Gal WT and wo mu an p o eins, ␤ -Gal R201C and ␤ -Gal I51T ( esidues 24–677), used o he N- e minal hexahis idine and FLAG ag, we e exp essed in yeas Pichia pas o is KM71 and pu i ied o homogenei y in a nickel-Sepha ose column. In he cou se o pu i ica ion, polysaccha ide moie ies a ached o he p o ein we e immed o by endoglycosidase H ea men . Fo c ys alliza ion, ␤ -Gals we e subjec ed o limi ed p o eolysis wi h bo ine ypsin and u he pu i ied by ca ion-exchange column ch oma og aphy, and Supe dex 200 size exclusion col- umn (GE Heal hca e) equilib a ed wi h bu e A (0.01 MMES, pH 6.0, 0.1 MNaCl). Finally, pu i ied ␤ -Gals we e concen a ed o 10 mg/ml in bu e A wi h o wi hou 10 mMgalac ose. FIGURE 1. S uc u es o DGJ de i a i es and PC compounds discussed in his s udy. S uc u al Insigh s in o Pha macological Chape ones MAY 23, 2014•VOLUME 289•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14561 Kine ics o Pu i ied ␤ -Gals and E alua ion o Inhibi o s— ␤ -Gal ac i i y was measu ed by using 4-me hylumbelli e yll- ␤ - D-galac opy anoside in bu e B (0.15 Msodium ci a e, pH 4.5, and 0.2 MNaCl) as a subs a e, and incuba ing a 37 °C o 6 min. The eac ion was e mina ed by adding 0.2 Mglycine- NaOH bu e (pH 10.7). The libe a ed 4-me hylumbelli e yll was measu ed wi h a luo escence pla e eade (exci a ion 355 nm; emission 460 nm; luo oskan ascen , The mo elec on Co p.). A kine ic expe imen on pu i ied ␤ -Gal was pe o med using a ious concen a ions (75–375 mM) o 4-me hylumbelli e yll- ␤ -Gal as a subs a e o de e mine he maximum eloci y (V max ) and Michaelis cons an (K m ) alues. Fo inhibi ion assays, he ligand compounds (galac ose, 25 mM; DGJ, 250 ␮ M; NOEV, 2.5 ␮ M; 6S-NBI-DGJ, 250 ␮ M; 6S-NBI-GJ, 2.5 mM; and NBT-DGJ, 2.5 mM) we e added o he pu i ied ␤ -Gal, and hen he ac i i y o ␤ -Gal was measu ed as desc ibed abo e. The inhibi ion con- s an (K i ) alues (Table 1) we e calcula ed om he plo s o slope e sus inhibi o concen a ion. E ec s o Pha macological Chape ones on he S abili y o he ␤ -Gals—To compa e he s abili y o he ␤ -Gal WT and ha o mu an p o eins unde a ious pH condi ions in a bu e , 10 ␮ g/ml o ␤ -Gal was incuba ed in ei he 0.1 Msodium ci a e bu e (pH 3.5–7.0) o 0.1 MHepes bu e , pH 7.5–8.0, wi h 0.1 MNaCl a 48 °C o 40 min. A e incuba ion, ␤ -Gal ac i i y was assayed immedia ely unde pH 4.5. To assess he e ec s o PC compounds on he s abili y o WT and mu an ␤ -Gal, a ious concen a ions(galac ose,0.2,2,and20mM;DGJ,2, 20,and 200 ␮ M; NOEV, 0.02, 0.2, and 2 ␮ M;6S-NBI-DGJ 2, 20, and 200 ␮ M) o PC compounds we e added o 10 ␮ g/ml o ␤ -Gal in bu e C (0.1 Msodium ci a e, pH 7.0, and 0.1 MNaCl), ollowed by incuba ion a 48 °C o a ious imes (0, 10, 20, and 40 min). As a con ol, he same expe imen was ca ied ou wi h o wi hou PCs (galac ose, 20 mM; DGJ, 200 ␮ M; NOEV, 2 ␮ M;6S-NBI- DGJ, 200 ␮ M) unde he acidic condi ion (0.1 Msodium ci a e, pH 4.8, and 0.1 MNaCl). Values a e a e ages o h ee indi idual expe imen s. CD Spec um—Ci cula dich oism (CD) spec a we e eco ded a 20 °C on a Jasco J-720W spec opola ime e equipped wi h a Julabo F25-ED empe a u e con olle . The wild ype and wo mu an p o eins o ␤ -Gal we e dilu ed o 0.1 mg/ml in 20 mMsodium ace a e, pH 5.0, wi h o wi hou galac- ose (50 mM). CD spec a we e collec ed o e he 200–280 nm wa eleng h ange and wi h a esolu ion o 0.1 nm, a bandwid h o 1 nm, and a esponse ime o 1 s. Final spec a we e he sum o 16 scans accumula ed a a speed o 50 nm/min. Iso he mal Ti a ion Calo ime y—Iso he mal i a ion cal- o ime e Mic oCal iTC200 was employed o de e mine he a ini ies be ween ␤ -Gal and he wo PCs, NOEV o 6S-NBI- DGJ, a 25 °C. The calo ime ic cell was illed wi h 50 ( o NOEV) o 100 ␮ M( o 6S-NBI-DGJ) ␤ -Gal, and PCs (0.5 mM NOEV and 1 mM6S-NBI-DGJ) we e injec ed in o he cell wi h a 60- ␮ l sy inge. The eleased hea was measu ed by in eg a ing he calo ime ic ou pu cu es. Da a we e i wi h he model o one se o binding si es in O igin (O iginLab). C ys alliza ion and Da a Collec ion—C ys alliza ion expe i- men s we e pe o med by si ing-d op apo di usion me hods a 4 °C. C ys als o ␤ -Gal WT 䡠6S-NBI-DGJ complex we e g own om he equi olume o 10 mg/ml o ␤ -Gal WT in bu e A con- aining1mM6S-NBI-DGJ and he ese oi solu ioncon aining 20–26% PEG3350, 0.2 Mammonium sul a e, and 0.1 MHepes, pH 7.5. The o he s we e i s p epa ed as ␤ -Gal WT o ␤ -Gal I51T 䡠galac ose complex c ys als, and hen he c ys als we e soaked in o he solu ion con aining each PC compound. C ys als o ␤ -Gal WT o ␤ -Gal I51T 䡠galac ose complex we e g own om he equi olume o 10 mg/ml o ␤ -Gal WT o ␤ -Gal I51T in bu e A con aining 10 mMgalac ose and he es- e oi solu ion con aining 20–26% PEG3350, 0.2 Mammonium sul a e, and 0.1 MHepes, pH 7.5. Then, ␤ -Gal WT o ␤ -Gal I51T - galac ose c ys als we e ans e ed in o mo he liquo (25% PEG3350, 0.2 Mammonium sul a e, and 0.1 MHepes, pH 7.5) supplemen ed wi h PC compounds (NOEV o 6S-NBI-DGJ, 1 mM;6S-NBI-GJ o NBT-DGJ, 10 mM) and incuba ed o 3–55 min (NOEV, 55 min; 6S-NBI-DGJ, 40 min; 6S-NBI-GJ, 15 min; NBT-DGJ, 3 min) a 4 °C. Di ac ion da ase s we e collec ed a beamline BL-17A and AR-NE3Aa he Pho on Fac o y (Tsukuba,Japan).P io o da a collec ion, he c ys als o he each PC compound䡠 ␤ -Gal com- plex we e soaked o a ew seconds in he ese oi solu ion supplemen ed wi h he co esponding PC compound (galac- ose 10 mM; NOEV and 6S-NBI-DGJ, 1 mM;6S-NBI-GJ and NBT-DGJ, 2 mM) and 15% e hylene glycol, and lash-cooled o 95 K. The da ase s we e p ocessed wi h he HKL2000 package (Table 2) (28). S uc u e De e mina ion and C ys allog aphic Re inemen — S uc u es o ␤ -Gal WT complexed wi h PC compounds (NOEV, 6S-NBI-DGJ, 6S-NBI-GJ, o NBT-DGJ) and ␤ -Gal I51T complexed wi h galac ose o 6S-NBI-DGJ we e de e mined by he molecula eplacemen me hod using he ␤ -Gal WT -galac- ose complex s uc u e (PDB code 3THC) as a sea ch model and he p og am Mol ep implemen ed in he CCP4 sui e (29). Modelbuilding andadjus men we eca iedou usingp og am COOT (30). C ys allog aphic e inemen was pe o med using he p og am REFMAC (31) implemen ed in he CCP4 package un il he R ac o was con e ged (Table 2). The a omic coo di- na es and s uc u e ac o s ha e been deposi ed in he P o ein TABLE 1 Enzymological pa ame e s o he wild- ype and mu an ␤ -Gals, and inhibi o y cons an alues o PC compounds The enzyme ac i i y o ␤ -Gal was luo ome ically de e mined wi h 4-me hylumbelli e yll- ␤ -D-galac opy anoside as a subs a e. K i Speci ic ac i i y V max K i Galac ose DGJ NOEV 6S-NBI-DGJ 6S-NBI-GJ NBT-DGJ ␮ mol/min/mg ␮ M/min mMmM ␮ M WT 15.6 1.78 0.49 4.14 61.8 1.1 68.3 253.1 372.7 R201C 13.1 1.74 0.44 5.61 49.0 0.94 53.3 320.4 388.3 I51T 16.3 1.82 0.55 4.91 44.7 1.2 68.8 319.6 329.0 S uc u al Insigh s in o Pha macological Chape ones 14562 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014 Da a Bank (3WEZ ( ␤ -Gal WT -NOEV), 3WF0 ( ␤ -Gal WT -6S- NBI-DGJ), 3WF1 ( ␤ -Gal WT -6S-NBI-GJ), 3WF2 ( ␤ -Gal WT - NBT-DGJ), 3WF3, ( ␤ -Gal I51T -galac ose) and 3WF4 ( ␤ -Gal I51T - 6S-NBI-DGJ)) (supplemen al da a). RESULTS Enzymological P ope ies o ␤ -Gal—The Michaelis-Men en pa ame e so hepu i ied ecombinan ␤ -Galwe ede e mined (Table 1). The V max alues o ␤ -Gal WT , ␤ -Gal R201C , and ␤ -Gal I51T we e1.8, 1.7, and 1.8 ␮ M/min,and he K m alueswe e 0.5, 0.4, and 0.6 mM, espec i ely. The esul s sugges ha he wild ypeand mu an p o eins showsimila enzymeac i i yand subs a e a ini y. These obse a ions u he suppo he p omise o PCs capable o escuing he mu an p o ein om endoplasmic e iculum-associa ed deg ada ion and p omo ing a icking o he lysosome o he ea men o G M1 gangli- osidosis. The co esponding inhibi ion cons an (K i ) alues a e p esen ed in Table 1. Da a o D-galac ose and DGJ ha e been collec ed o compa a i e pu poses. Galac ose is a e y weak inhibi o , al hough DGJ shows 100- old s onge inhibi ion han galac ose. NOEV exhibi ed he mos po en inhibi ion o ␤ -Gal among he se ies, wi h K i close o 1 ␮ M.6S-NBI-DGJ inhibi s ␤ -Gal o he same deg ee as DGJ, whe eas he s uc u - ally ela ed de i a i es 6S-NBI-GJ and NBT-DGJ showed much weake inhibi o y p ope ies. Consis en wi h he deg ee o he inhibi ion, a iso he mal i a ion calo ime y expe imen dem- ons a ed ha NOEV exhibi ed a s ong binding a ini y wi h he dissocia ion cons an o submic omola , whe eas 6S-NBI- DGJ exhibi ed a ⬃100- old educed a ini y (supplemen al Fig. S1). I should be no ed ha all he compounds inhibi ed WT and mu an ␤ -Gal wi h i ually iden ical po encies. The S abili y o WT and Mu an ␤ -Gal unde Va ious pH Condi ions—The ac i i y o WT and mu an ␤ -Gal a e hea ea men (48 °C, 40 min) was examined unde a ious pH con- di ions (Fig. 2). As p e iously epo ed (32), each ␤ -Gal was mo eac i e a acidic pH (4.5–5.5), whe eas he ca aly ic ac i i y was g ea ly educed unde s ong acidic (pH ⬍3.5) and basic (pH ⬎8.0) condi ions. When compa ing wild ype and mu an enzymes, i was ound ha ␤ -Gal I51T was only sligh ly mo e sensi i e o hea -induced inac i a ion in he whole ange o pH alues om 3.5 o 8.0, whe eas he ac i i y o ␤ -Gal R201C was signi ican ly educed unde a ious pH condi ions compa ed wi h ␤ -Gal WT . As a conclusion om hese esul s, ␤ -Gal mu a- ions associa ed wi h G M1 gangliosidosis lead o he educ ion o enzyme s abili ies bu do no lose he ca aly ic ac i i y a all, sugges ing ha bo h ␤ -Gal R201C and ␤ -Gal I51T could p ocess G M1 ganglioside and p e en / educe i s accumula ion p o ided hey a e p ope ly ans e ed o he lysosome wi h he help o PCs. TABLE 2 Da a collec ion and c ys allog aphic e inemen s a is ics ␤ -Gal WT WT WT WT I51T I51T PC compound NOEV 6S-NBI-DGJ 6S-NBI-GJ NBT-DGJ Galac ose 6S-NBI-DGJ Da a collec ion X- ay sou ce PF-BL17A PF-BL17A PF-AR NE3A PF-AR NE3A PF-AR NE3A PF-AR NE3A Wa eleng h (Å) 0.98000 0.98000 1.00000 1.00000 1.00000 1.00000 Resolu ion ange (Å) 50–2.1 (2.14–2.10) 50–2.2 (2.24–2.20) 50–2.0 (2.03–2.00) 50–2.3 (2.34–2.30) 50–2.2 (2.19–2.15) 50–2.3 (2.34–2.30) Space g oup P2 1 P2 1 P2 1 P2 1 P2 1 P2 1 Uni cell pa ame e s a(Å) 94.8 95.0 95.0 95.1 95.0 95.0 b(Å) 115.6 116.5 116.0 116.3 115.9 116.2 c(Å) 140.3 140.4 140.6 141.8 140.3 140.7 ␤ (°) 92.3 92.2 92.3 92.4 92.2 92.3 No. o obse ed e lec ions 709,280 367,282 743,452 597,215 515,585 505,251 No. o unique e lec ions 172,353 139,532 199,457 133,461 160,267 134,623 Redundancy 4.1 (3.8) 2.6 (2.5) 3.7 (3.6) 4.5 (4.5) 3.2 (3.3) 3.8 (3.7) Comple eness (%) 98.6 (96.4) 90.4 (93.7) 97.5 (96.4) 98.3 (98.1) 96.4 (98.6) 99.1 (100.0) A e age I/ ␴ (I) 16.7 (3.2) 9.0 (2.0) 15.8 (2.2) 13.5 (2.5) 15.5 (3.5) 11.5 (2.3) R me gea 0.136 (0.602) 0.148 (0.571) 0.128 (0.605) 0.171 (0.763) 0.154 (0.478) 0.173 (0.677) Re inemen Resolu ion ange (Å) 47.4–2.1 43.4–2.2 25.9–2.0 26.0–2.3 27.3–2.2 25.3–2.3 P o ein esidues 2,415 2,416 2,416 2,416 2,416 2,416 No. o a oms 19,291 19,302 19,305 19,243 19,280 19,263 Ligand 80 68 72 72 48 68 N-Ace ylglucosamine 224 224 224 224 224 224 Wa e molecules 1,513 1,248 1,596 1,093 1,429 1,241 SO 4 2⫺ /Cl ⫺ /EG b 40/4/32 40/4/32 40/4/32 40/4/32 40/4/32 40/4/32 A e age B ac o (Å 2 ) 22.9 21.9 28.8 32.0 28.9 29.0 R wo kc /R eed (%) 17.9/22.4 17.7/23.0 18.6/23.3 17.4/24.0 19.6/24.2 17.9/24.0 Roo mean squa e de ia ion Bond leng h (Å) 0.012 0.013 0.014 0.016 0.014 0.016 Bond angles (°) 1.54 1.59 1.69 1.84 1.70 1.86 Ramachand an plo (%) Fa o ed 96.6 96.6 97.3 96.0 96.5 96.1 Allowed 3.4 3.3 2.6 3.8 3.4 3.8 Disallowed 0.0 0.1 0.1 0.2 0.1 0.1 Values in pa en heses a e o he shell wi h he highes esolu ion. a R me ge (I)⫽⌺兩I⫺具I典兩/⌺I,whe e Iis he di ac ion in ensi y. b EG indica es e hylene glycole. c R⫽⌺兩F o ⫺F c 兩/⌺F o , whe e F o and F c a e he obse ed and calcula ed s uc u e ampli udes, espec i ely. d R ee is an R alue o a 5% subse o all e lec ions, bu was no used in he e inemen . S uc u al Insigh s in o Pha macological Chape ones MAY 23, 2014•VOLUME 289•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14563 E ec s o he Pha macological Chape ones on he S abili y o he ␤ -Gal—Theabili yo aligand o p e en hea -induced inac- i a ion o a gi en glycosidase has been p e iously used as an indica ion o i s pha macological chape one po en ial (33). In ou case, galac ose, DGJ, NOEV, and 6S-NBI-DGJ we e added o ␤ -Gal a neu al pH in bu e s, and hen he emaining enzyme ac i i ies we e examined a e hea ea men (Fig. 3). All compounds we e able o inc ease he esidual ac i i ies o he ␤ -Gal in a dose-dependen manne . ␤ -Gal WT ac i i y was dec eased o ⬃20% a e 40 min a 48 °C incuba ion, bu galac- ose- ea ed ␤ -Gal e ained ⬃70% ac i i y. In acco dance wi h he inhibi o y ac i i y, NOEV- ea ed ␤ -Gal, and DGJ and 6S-NBI-DGJ- ea ed ␤ -Gal eached a simila ac i i y imp o e- men a ⬃10,000- and ⬃100- old lowe concen a ions, espec- i ely, compa ed wi h galac ose, sugges ing ha hese com- pounds bea po en ial as PCs. Li le chape one e ec s we e obse ed unde he acidic condi ion (supplemen al Fig. S2). S uc u al Basis o Pha macological Chape one In e ac ion wi h Human ␤ -Gal—To gain a omic insigh in o he PC com- pounds binding o ␤ -Gal, we de e mined c ys al s uc u es o ␤ -Gal WT complexed wi h NOEV, 6S-NBI-DGJ, 6S-NBI-GJ, and NBT-DGJ. The o e all s uc u es o ␤ -Gal emained la gely unchanged among hese ou complexes and all s uc- u es showed a s ong o e all ag eemen wi h galac ose-bound ␤ -Gal WT (PDB code 3THC) (24) wi h oo mean squa e de ia- ions anging om 0.17 o 0.26 Å. ␤ -Galwas oldedin o h eedomains: he TIMba el domain, ␤ -domain 1, and ␤ -domain 2 (supplemen al Fig. S3). The galac- ose mime ics we e embedded in he same ligand binding pocke o he TIM ba el domain in ␤ -Gal, wi h he alkyl chain a he exocyclic ni ogen a om o ien ed owa d he en ance o he ac i e si e pocke (Fig. 4, A–D). In he case o NOEV, all hyd oxyl g oups made di ec hyd ogen bonds wi h ␤ -Gal in a simila manne o galac ose. In addi ion, a hyd ogen bond was o med be ween he exocyclic N a om o NOEV and Glu-188 o ␤ -Gal, s abilizing he o ien a ion o he oc yl chain. The hyd o- phobic ail ex ended along he p o ein su ace consis ing o Ty -485, T p-273, Leu-274, His-276, and Asn-321 (Fig. 4A). The e minal me hyl g oup (C16 ca bon a om) was in con ac wi h he CE1 a om o His-276, and CB and CG a oms o Asn- 321 (Fig. 5A). Simila o NOEV, hyd oxyls OH2, OH3, and OH4 o he sp 2 -iminosuga 6S-NBI-DGJ made di ec hyd ogen bonds wi h ␤ -Gal (Fig. 4B). The exocyclic N a om o 6S-NBI-DGJ also made a hyd ogen bond wi h Glu-188 o ␤ -Gal, which o ien s he bu yl chain owa d he hyd ophobic pocke lanked by Ty - 485 and T p-273 (Fig. 5B). Con a y o NOEV, o which he endocyclic double-bond imposes a hal -chai con o ma ion a he alienamine ing, he 6-membe ed ing o 6S-NBI-DGJ exhibi s an almos ideal chai con o ma ion close o ha encoun e ed o galac ose in he galac ose䡠 ␤ -Gal complex. The 5-membe ed ing is used o he 6-membe ed ing a an angle o 110° (supplemen al Fig. S4). Because 6S-NBI-DGJ lacks he p i- ma y hyd oxyl equi alen o he OH6 g oup in galac ose, he side chain o ien a ion o Ty -333 is shi ed so as o ill he co - esponding space. This ea u e is unique o he 6S-NBI-DGJ䡠 ␤ - Gal complex s uc u e. The p esence o a pseudoanome ic hyd oxyl g oup, OH1, in he galac onoji imycin analogue 6S-NBI-GJ led o a e y signi - ican dec easein hebindinga ini y owa d ␤ -Galascompa ed wi h he DGJ congene 6S-NBI-DGJ. Ac ually his compound is a a he selec i e inhibi o o ␤ -glucosidase (21). Whe eas 6S-NBI-GJ has been shown o exis exclusi ely in he 4 C 1 chai con o ma ion in wa e solu ion, wi h OH1 axially o ien ed in he ␣ -con igu a ion (34, 35), in he co esponding complex wi h ␤ -Gal he opposi e ␤ -con igu a ionwas encoun e ed, wi h he 6-membe ed ing and he 5-membe ed ing in he same plane(supplemen alFig.S4).The ␤ -o ien edOH1g oupisnow in ol ed in hyd ogen bonding wi h Glu-188 (Fig. 4C), wi h he bu yl chain ex ended o Ty -485 and T p-273 (Fig. 5B). The monocyclic de i a i e NBT-DGJ eco e ed he hyd o- gen bonding in e ac ion be ween he p ima y hyd oxyl OH6 and Ty -333 (Fig. 4D). Ac ually, all hyd oxyl g oups o NBT- DGJ made di ec hyd ogen bonds wi h ␤ -Gal as in he NOEV䡠 ␤ -Gal complex. Howe e , he exocyclic N and S a oms had no in e ac ions wi h ␤ -Gal, which is in ag eemen wi h he weak binding a ini y obse ed in he kine ic inhibi ion s udies. We also de e mined c ys al s uc u es o ␤ -Gal I51T com- plexed wi h galac ose o 6S-NBI-DGJ (Fig. 6). The quali y o he elec on densi y maps is su icien ly high o allow modeling o he SNP esidue (Fig. 6B). These o e all s uc u es a e essen- ially iden ical o hose o ␤ -Gal WT bound o he co esponding ligand. In ac , he main chain a oms in each complex could be supe imposed on hose o he galac ose o 6S-NBI-DGJ com- plex wi h oo mean squa e de ia ions o 0.16 and 0.17 Å, espec i ely. CD spec um demons a ed ha s uc u es o he wild ype and mu an p o eins a e essen ially he same ega d- less o he p esence o absence o he PC compounds (supple- men al Fig. S5). DISCUSSION In his s udy, we de e mined he enzymological p ope ies o pu i ied ecombinan human ␤ -Gal ( ␤ -Gal WT ) and wo ep e- sen a i e mu an p o eins, ␤ -Gal R201C and ␤ -Gal I51T . Mo e- o e , we de e mined he c ys al s uc u es o ␤ -Gal WT com- plexed wi h ou ligand compounds, wo o which, NOEV and 6S-NBI-DGJ, ha e shown pha macological chape one ac i i y FIGURE 2. Enzyma ic ac i i y o he WT and wo mu an p o eins o ␤ -Gal unde a ious pH condi ions. The pu i ied WT and wo mu an p o eins o ␤ -Gal we e incuba ed unde he indica ed pH condi ions in a bu e a 48 °C o 40 min. Then, enzyme assay was pe o med unde pH 4.5 condi ions. The esidual enzyme ac i i y is shown as a pe cen age o he alues a he ime 0. Values a e a e ages o h ee indi idual expe imen s and exp essed as he pe cen a io o he ac i i y a a pa icula pH condi ion o he alue a he ime 0. S uc u al Insigh s in o Pha macological Chape ones 14564 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014 o se e al G M1 gangliosidosis-associa ed mu a ions, whe eas he o he wo, 6S-NBI-GJ and NBT-DGJ, showed only weak a ini y o he enzyme despi eha ing a s uc u e closely ela ed o ha o 6S-NBI-DGJ. C ys al s uc u es o ␤ -Gal I51T com- plexed wi h galac ose o 6S-NBI-DGJ ha e also been de e - mined. Al hough NOEV and 6S-NBI-DGJ exhibi mic omola and ens o mic omola K i alues, hese compounds we e shown o signi ican ly enhance he ␤ -Gal ac i i ies (up o 6- old) in G M1 ib oblas s, demons a ing hei e icacy in i o. I was also demons a ed ha he PC compounds amelio a ed accumula ion o G M1 ganglioside in a mouse model (human ␤ -Gal R201C ), including he b ain, a e o al adminis a ion (8, 18, 20). The Ile-51 mu a ion is loca ed in he inne egion o ␤ -Gal, whe eas he A g-201 mu a ion is loca ed ins ead on he la e al ace o he TIM ba el domain, being exposed o he sol en . In bo h he A g-201 and Ile-51 mu an p o eins, he mu a ed amino acid is a om he ac i e si e, indica ing ha he mu a- ions a e unlikely o a ec he ac i e si e di ec ly. Acco dingly, ␤ -Gal I51T and ␤ -Gal R201C showed simila enzymological pa ame e s as ␤ -Gal WT (Table 1). This coincides wi h he ac ha no signi ican con o ma ional change in ␤ -Gal I51T was obse ed in he c ys al s uc u e o he mu an p o ein as com- pa ed wi h he wild ype enzyme (Fig. 6A). By con as , ␤ -Gal R201C was mo e uns able han ␤ -Gal WT and ␤ -Gal I51T unde a ious pH condi ions (Fig. 2). A g-201 o ms a sal b idge o Asp-198 (24) and he loss o his sal b idge p obably a ec s he s abili y o he p o ein, inc easing dena u a ion p o- pensi y. These enzymological p ope ies, ob ained using ecombinan ␤ -Gal, we e consis en wi h p e iously epo ed da a (32). The s uc u al s udy p o ides an a omic basis o he binding mechanism o ac i e si e-di ec ed pha macological chape ones o ␤ -Gal, by highligh ing he impo ance o he suga -like agly- con moie y, he na u e o he exocyclic subs i uen and he con- o ma ional p ope ies o he ligand. NOEV and 6S-NBI-DGJ we e ecognized in a simila manne ; howe e , NOEV was a igh binding inhibi o , whe eas 6S-NBI-DGJ binds ⬎60- old weake (Table 1). This high a ini y o NOEV esul s om he exocyclic N a om ecogni ion, he leng h and he o ien a ion o he ex ended hyd ophobic ail and hal -chai con o ma ion imposedby hedouble bond.The sp 2 -iminosuga ype inhibi o 6S-NBI-DGJ is cha ac e ized by a igid bicyclic co e de i ed om DGJ. 6S-NBI-DGJ lacks OH6, exhibi s a di e en leng h and o ien a ion o he hyd ophobic ail, and adop s he chai con o ma ion, esul ing in he weake a ini y. As a conse- FIGURE 3. E ec s o PC compounds on he s abili y o he WT and wo mu an p o eins o ␤ -Gals a pH 7.0. The pu i ied WT and wo mu an p o eins o ␤ -Gal we e incuba ed in 0.1 Msodium ci a e, pH 7.0, wi h o wi hou galac ose, DGJ, NOEV, and 6S-NBI-DGJ a 48 °C o he incuba ed imes. Then, he enzyme assay was pe o med a pH 4.5. The esidual enzyme ac i i y is shown as a pe cen age o he alues a he ime 0. Values a e a e ages o h ee indi idual expe imen s and a e exp essed as he pe cen a io o he ac i i y a a pa icula pH condi ion o he alue a he ime 0. S uc u al Insigh s in o Pha macological Chape ones MAY 23, 2014•VOLUME 289•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14565 quence, he co esponding hyd ogen bond in e ac ion wi h Ty -333 is missing, which is expec ed o ha e a de imen al impac in complex s abili y. No ewo hy, his scena io leads o a shi o Ty -333 in he 6S-NBI-DGJ䡠 ␤ -Gal complex, occupy- ing he space whe e OH6 is loca ed in he co esponding com- plex wi h NOEV, e ealing a ce ain deg ee o lexibili y a his egion o ␤ -Gal. The sho ened alkyl subs i uen in 6S-NBI- DGJ as compa ed wi h NOEV, bu yl ins ead o oc yl, is also expec ed o a ec he binding a ini y. In ac , N-alkyl-4-epi- ␤ - alienamine de i a i es wi h longe alkyl chains han NOEV ha e been shown o exhibi highe a ini y o bo ine ␤ -Gal (36), whe eas N-hexyl-4-epi- ␤ - alienamine had 3- old weake a in- i y han NOEV (14). Consis en ly, he N⬘-oc yl analogue o 6S-NBI-DGJ was also ound o be a 4- old s onge inhibi o o bo ine ␤ -Gal han 6S-NBI-DGJ, bu i was disca ded o PC chape one s udies wi h he human lysosomal enzyme due o oxici y issues (17). P obably he longe alkyl chain in e ac s wi h he neu al g oo e, o med by His-276, Asn-321, P o-323, and Ala-325, in a wide su ace a ea han he bu yl chain in 6S-NBI-DGJ, he eby inc easing he binding a ini y (Fig. 5). Unlike he oc yl chain in NOEV, he bu yl chain does no ex end o e he neu al g oo e a he en ance o he ac i e si e FIGURE 4. Binding o PC compounds o he ac i e si e o human ␤ -Gal. A–D,le , PC compounds bound o ␤ -Gal iewed om he en ance o ac i e si e pocke : A, NOEV; B, 6S-NBI-DGJ; C, 6S-NBI-GJ; D, NBT-DGJ. Hyd ogen bonds de ined by ccp4 mg (37) a e shown by dashed lines.InB, a unique side chain a angemen o Ty -333 in he 6S-NBI-DGJ complex is shown in c imson. The F o ⫺F c elec on densi y maps o PC compounds a e shown in g ay.Aand Ba e con ou ed a 2.0 ␴ ,Ca 1.0 ␴ , and Da 1.5 ␴ .Righ , schema ic ep esen a ion o he in e ac ion be ween ␤ -Gal and each PC compound: A, NOEV; B, 6S-NBI-DGJ; C, 6S-NBI-GJ; and D, NBT-DGJ. Hyd ogen bonds a e shown as do ed lines, and an de Waals in e ac ions a e shown by a cs. FIGURE 5. Compa ison o he binding o he PC compounds. Elec os a ic po en ial su ace o he su ounding o he ac i e si e pocke . A, NOEV (o ange) complex s uc u e is supe imposed on NBT-DGJ (g ay) complex. B, 6S-NBI-DGJ (g een) and 6S-NBI-GJ (cyan) a e d awn in a simila manne as in A. S uc u al Insigh s in o Pha macological Chape ones 14566 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014 in ␤ -Gal (Fig. 5B), which in pa is esponsible o i s mode a e binding a ini y o ␤ -Gal. The p esen s udy e ealed ha he s abili y o he mu an p o eins (R201C, I51T) coincides wi h he se e i y o he dis- ease and he PC e ec owa d ␤ -Gal WT and ␤ -Gal R201C a e well consis en wi h he p e ious da a (8, 20). Howe e , NOEV has been shown o exhibi di e ences in hei PC e ec s owa d some ␤ -Gal mu an p o eins in i o (20). This beha io does no co ela e, howe e , wi h he p esen da a on he s abiliza- ion e ec owa d hea -induced inac i a ion o pu i ied ecom- binan ␤ -Gal,whe e NOEV p o ed mo ee icien han 6S-NBI- DGJ i espec i e o he mu a ion. Mu an enzyme escued by PCs is a complex p ocess ha also in ol es s abiliza ion o he al eady olded enzyme o p omo e a icking and ma u a ion. How close a e he olded s a es is p obably s ongly mu a ion- dependen , which may explain he abo e disc epancy be ween in i o and cellulo esul s. Fu he in es iga ion will be equi ed. The hyd ogen bond in e ac ions in ol ing OH2, OH3, and OH4 o he suga -like moie y and wo glu amic acid esidues (Glu-129 and Glu-268) a he ac i e si e o ␤ -Gal a e c i ical o binding in bo h he NOEV and 6S-NBI-DGJ complexes and likely de ine he D-galac o con igu a ional selec i i y o he enzyme. A hi d glu amic acid, Glu-188, in e ac s wi h he exo- cyclic basic ni ogen a om. This hyd ogen bond con ibu es subs an ially o ␤ -Galbinding a ini y and i s absence is likely o be a he o igin o he much lowe inhibi o y po encies o he s uc u ally ela ed sp 2 -iminosuga s 6S-NBI-GJ and NBT-DGJ as well as o he monosaccha ide galac ose. The weakening o he ␤ -Gal binding a ini y o NOEV and 6S-NBI-DGJ obse ed a acidic pH (8, 17) can be a ionalized in e ms o he expec ed dec ease in he s eng h o he key hyd ogen bonds a e p o o- na ion o he glu amic acid esidues in he p o ein and he basic ni ogen unc ionali y in he chape one. The sha p dec ease in ␤ -Gal binding a ini y o 6S-NBI-GJ and NBT-DGJ as compa ed wi h 6S-NBI-DGJ u he illus- a es he s ong dependenceo he inhibi o y/chape oneac i - i y o sp 2 -iminosuga glycomime ics on sub le chemical modi- ica ions. The indings p esen ed he e may be pa icula ly use ul o he a ional design o second gene a ion PCs o he ea men o he mu an ␤ -Gal-associa ed LSDs G M1 gangli- osidosis and Mo quio B disease. Thus, acco ding o he x- ay da a, s uc u al changes a he exocyclic subs i uen in he 6S-NBI-GJ sca old a e expec ed o be ole a ed by he enzyme, o e ing po en ial o d ug op imiza ion. In addi ion, he inco - po a ion o an app op ia e subs i uen a he i e-membe ed ing me hylene ca bon migh lead o a o able in e ac ions wi h Ty -333, which could be exploi ed in he design o highe a ini y ligands. Resea ch in ha di ec ion is cu en ly sough in ou labo a o ies. Acknowledgmen —We hank he beamline s a s a he Pho on Fac- o y o assis ance wi h da a collec ion. REFERENCES 1. Alpe s, D. 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