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• W2040931402 abstract "Three subfamilies of mammalian Class 1 processing α1,2-mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation. In an effort to define the structural basis for substrate recognition among Class 1 mannosidases, we have crystallized murine Golgi mannosidase IA (space group P212121), and the structure was solved to 1.5-Å resolution by molecular replacement. The enzyme assumes an (αα)7 barrel structure with a Ca2+ ion coordinated at the base of the barrel similar to other Class 1 mannosidases. Critical residues within the barrel structure that coordinate the Ca2+ ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved. A Man6GlcNAc2 oligosaccharide attached to Asn515 in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex. In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket. A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes. Three subfamilies of mammalian Class 1 processing α1,2-mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation. In an effort to define the structural basis for substrate recognition among Class 1 mannosidases, we have crystallized murine Golgi mannosidase IA (space group P212121), and the structure was solved to 1.5-Å resolution by molecular replacement. The enzyme assumes an (αα)7 barrel structure with a Ca2+ ion coordinated at the base of the barrel similar to other Class 1 mannosidases. Critical residues within the barrel structure that coordinate the Ca2+ ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved. A Man6GlcNAc2 oligosaccharide attached to Asn515 in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex. In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket. A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes. The processing of Asn-linked oligosaccharides in mammalian organisms is initiated in the endoplasmic reticulum by the action of α-glucosidases and α-mannosidases that cleave the Glc3Man9GlcNAc2 structure primarily to a specific Man8GlcNAc2 isomer (Man8B) 1The abbreviations used are: Man8B, an isomer of Man8GlcNAc2 where the central branch mannose residue had been removed from the standard Man9GlcNAc2 structure (see Fig. 1); dMNJ, 1-deoxymannojirimycin; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; EDEM, ER degradation-enhancing α-mannosidase-like protein; NAG, N-acetylglucosamine; MES, 4-morpholine-ethanesulfonic acid; r.m.s., root mean square. (Fig. 1). Following transport to the Golgi complex, further trimming of α1,2-mannose residues results in the production of a Man5GlcNAc2 structure (1Moremen K. Ernst B. Hart G. Sinay P. Oligosaccharides in Chemistry and Biology: A Comprehensive Handbook. II. John Wiley and Sons, Inc., New York2000: 81-117Google Scholar). These trimming reactions are followed by additional modifications, catalyzed by glycosyltransferases and hydrolases, to yield the array of complex structures on cellular and secreted glycoproteins (2Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar). The Class 1 mannosidases (family 47 glycosidases) play several roles in the oligosaccharide-trimming reactions in the ER and Golgi (for reviews, see Refs. 1Moremen K. Ernst B. Hart G. Sinay P. Oligosaccharides in Chemistry and Biology: A Comprehensive Handbook. II. John Wiley and Sons, Inc., New York2000: 81-117Google Scholar and 3Herscovics A. Biochim. Biophys. Acta. 1999; 1426: 275-285Crossref PubMed Scopus (123) Google Scholar, 4Herscovics A. Pinto B.M. Comprehensive Natural Products Chemistry. 3. Elsevier, New York1999: 13-35Google Scholar, 5Herscovics A. Biochim. Biophys. Acta. 1999; 1473: 96-107Crossref PubMed Scopus (240) Google Scholar, 6Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (325) Google Scholar, 7Daniel P.F. Winchester B. Warren C.D. Glycobiology. 1994; 4: 551-566Crossref PubMed Scopus (169) Google Scholar). Three subfamilies of mammalian Class 1 mannosidases have been identified; the ER mannosidase I subfamily cleaves a single residue from Man9GlcNAc2 to generate the Man8B structure (Fig. 1, A and B) (1Moremen K. Ernst B. Hart G. Sinay P. Oligosaccharides in Chemistry and Biology: A Comprehensive Handbook. II. John Wiley and Sons, Inc., New York2000: 81-117Google Scholar), the Golgi mannosidase I subfamily cleaves Man9-8GlcNAc2 structures to Man5GlcNAc2 (Fig. 1, A and C), and the EDEM subfamily of mannosidase-related proteins does not appear to have an intrinsic hydrolase activity but appears to be required for disposal of terminally misfolded glycoproteins by ER-associated degradation (ERAD) (8Oda Y. Hosokawa N. Wada I. Nagata K. Science. 2003; 299: 1394-1397Crossref PubMed Scopus (392) Google Scholar, 9Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (389) Google Scholar, 10Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 11Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (304) Google Scholar, 12Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (219) Google Scholar, 13Wang T. Hebert D.N. Nat. Struct. Biol. 2003; 10: 319-321Crossref PubMed Scopus (26) Google Scholar). ER mannosidase I has also been shown to play a critical role in ERAD by acting as a timing step to create a key Man8GlcNAc2 isomer required for initiating the disposal process (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 15Wu Y. Swulius M.T. Moremen K.W. Sifers R.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8229-8234Crossref PubMed Scopus (151) Google Scholar, 16Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Recent data indicate that further processing to structures smaller than Man8GlcNAc2 may also be required for ERAD, indicating that the Golgi family of mannosidases may play a role in ERAD (16Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 17Frenkel Z. Gregory W. Kornfeld S. Lederkremer G.Z. J. Biol. Chem. 2003; 278: 34119-34124Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Ermonval M. Kitzmuller C. Mir A.M. Cacan R. Ivessa N.E. Glycobiology. 2001; 11: 565-576Crossref PubMed Scopus (60) Google Scholar, 19Kitzmuller C. Caprini A. Moore S.E. Frenoy J.P. Schwaiger E. Kellermann O. Ivessa N.E. Ermonval M. Biochem. J. 2003; 376: 687-696Crossref PubMed Scopus (52) Google Scholar). In comparing the substrate specificities of ER and Golgi mannosidase I, these enzymes were found to have differences in both degree of mannose trimming as well as branch specificity for substrate recognition. ER mannosidase I preferentially cleaves the α1,2-mannose residue on the central branch of the Man9GlcNAc2 structure (residue M10; Fig. 1B) to produce the Man8B isomer (3Herscovics A. Biochim. Biophys. Acta. 1999; 1426: 275-285Crossref PubMed Scopus (123) Google Scholar, 20Lipari F. Herscovics A. Glycobiology. 1994; 4: 697-702Crossref PubMed Scopus (27) Google Scholar, 21Puccia R. Grondin B. Herscovics A. Biochem. J. 1993; 290: 21-26Crossref PubMed Scopus (30) Google Scholar, 22Ziegler F.D. Trimble R.B. Glycobiology. 1991; 1: 605-614Crossref PubMed Scopus (45) Google Scholar). In contrast, the Golgi mannosidase I family members (designated IA, IB, and IC) preferentially cleave residue M11 or M9, followed by residue M8 (Fig. 1C (23Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar, 24Tremblay L.O. Campbell Dyke N. Herscovics A. Glycobiology. 1998; 8: 585-595Crossref PubMed Scopus (54) Google Scholar)). The order of initial mannose removal (M11 versus M9) varies among the Golgi mannosidase I family members, but all of the mammalian family members tested thus far have a relatively poor efficiency for cleavage of the α1,2-mannose designated M10 (the target of ER mannosidase I action). Thus, the ER and Golgi mannosidase I families of enzymes have complementary and largely nonoverlapping substrate specificities, despite their similarities in protein sequence and presumed protein fold (1Moremen K. Ernst B. Hart G. Sinay P. Oligosaccharides in Chemistry and Biology: A Comprehensive Handbook. II. John Wiley and Sons, Inc., New York2000: 81-117Google Scholar). The structural basis for glycone recognition by Class 1 mannosidases was revealed through the co-crystallization of human ER mannosidase I with the mannose mimics, 1-deoxymannojirimycin (dMNJ) and kifunensine (Fig. 1D) (25Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The inhibitors were found to occupy the -1 mannose site (based on established glycosidase subsite nomenclature (26Davies G.J. Wilson K.S. Henrissat B. Biochem. J. 1997; 321: 557-559Crossref PubMed Scopus (852) Google Scholar)) through a direct coordination of the O-2′ and O-3′ hydroxyls to an enzyme-bound calcium ion and a collection of hydrogen bonds and hydrophobic interactions that stabilized the binding of the inhibitors in the equivalent of an unusual 1C4 sugar conformation. The structural basis for the branch specificity in substrate recognition by ER mannosidase I was previously described for the enzyme from Saccharomyces cerevisiae (27Vallee F. Lipari F. Yip P. Sleno B. Herscovics A. Howell P.L. EMBO J. 2000; 19: 581-588Crossref PubMed Scopus (86) Google Scholar). The x-ray structure of this enzyme revealed an oligosaccharide attached to one protein unit of the crystal lattice extending into the active site of an adjoining protein molecule in a proposed enzyme-product complex. Sugar residues of the oligosaccharide extended from the proposed +1 enzyme subsite, with the -1 mannose subsite unoccupied. A key Arg residue (Arg273), conserved among the ER mannosidase I subfamily of enzymes, was found to interact with several residues in the substrate and contribute to branch specificity. These interactions with Arg273, along with other hydrogen bonding interactions with the glycan across the oligosaccharide binding cleft, resulted in the insertion of residue M7 into the +1 binding site. This enzyme-product complex presumably mimics the insertion of residues M7 and M10 of the nascent Man9GlcNAc2 substrate into the respective +1 and -1 binding sites during a catalytic cycle that would result in glycoside bond cleavage and the production of the Man8GlcNAc2 isomer B (28Romero P.A. Vallee F. Howell P.L. Herscovics A. J. Biol. Chem. 2000; 275: 11071-11074Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Mutation of Arg273 to Leu resulted in a low efficiency enzyme with a hybrid activity between ER mannosidase I and Golgi mannosidase I; it cleaved Man9GlcNAc2 to Man5GlcNAc2 by the removal of M10, M11, M8, and finally M9 (28Romero P.A. Vallee F. Howell P.L. Herscovics A. J. Biol. Chem. 2000; 275: 11071-11074Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Recent structural studies on Class 1 α-mannosidases from Tricoderma reesei (29Van Petegem F. Contreras H. Contreras R. Van Beeumen J. J. Mol. Biol. 2001; 312: 157-165Crossref PubMed Scopus (34) Google Scholar) and Penicillium citrinum (30Lobsanov Y.D. Vallee F. Imberty A. Yoshida T. Yip P. Herscovics A. Howell P.L. J. Biol. Chem. 2002; 277: 5620-5630Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), enzymes that have a similar substrate specificity to the Golgi mannosidase I subclass of enzymes, suggest that the sequence alteration of the key Arg to a smaller amino acid side chain and a more spacious substrate binding site may provide the basis for broader substrate specificity of these enzymes. In an effort to define the structural basis for substrate recognition among the Golgi mannosidase I subclass of enzymes, we chose to study murine Golgi mannosidase IA (23Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar, 31Lal A. Schutzbach J.S. Forsee W.T. Neame P.J. Moremen K.W. J. Biol. Chem. 1994; 269: 9872-9881Abstract Full Text PDF PubMed Google Scholar, 32Vallee F. Lal A. Moremen K.W. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 571-573Crossref PubMed Scopus (5) Google Scholar). The structure of this enzyme revealed a protein fold and catalytic site that was similar to other Class 1 mannosidases. Surprisingly, the single N-glycan on the murine glycoprotein was found to extend into the +1 mannose position of the enzyme binding site in an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex analogous to the structure of S. cerevisiae ER mannosidase I. In contrast to the yeast enzyme structure, an alternative oligosaccharide conformation was bound to the active site pocket, and a distinct monosaccharide was found in the presumed +1 binding site. A comparison of the geometry and topology of the oligosaccharide binding cleft between the various Class 1 mannosidases indicates that ER and Golgi mannosidases have a high potential for selective substrate binding resulting from substrate interactions in a constricted binding cleft. Comparisons of the glycan structures bound in the active sites of the yeast and murine enzymes provide insights into the structural basis for substrate recognition among these Class 1 mannosidases. Golgi ManIA Purification and Crystallization—The cDNA encoding the soluble catalytic domain of mouse Golgi mannosidase IA (31Lal A. Schutzbach J.S. Forsee W.T. Neame P.J. Moremen K.W. J. Biol. Chem. 1994; 269: 9872-9881Abstract Full Text PDF PubMed Google Scholar) was subcloned into the Pichia pastoris expression vector, pHIL-S1, as a fusion protein with the yeast α-factor signal sequence (23Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar). The final construct was transformed into the GS115 Pichia host strain by selection for reversion in His auxotrophy as previously described (23Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar). The strain was grown in a 100-ml shake flask culture in BMGY medium overnight and used to inoculate a 7-liter culture in minimal medium (33Cregg J.M. Vedvick T.S. Raschke W.C. Bio/Technology. 1993; 11: 905-910Crossref PubMed Scopus (850) Google Scholar) in a New Brunswick BioFlow 3000 fermentor. After growth to a cell density of 300 g/liter using glycerol as a carbon source, the culture was starved for ∼10 h and grown on 0.5% MeOH as an inducing agent and carbon source for 3 days (maintained using a MeOH probe and monitor (Raven Biotech) to control feed rate). The medium was harvested and clarified by centrifugation at 6000 × g for 20 min. The enzyme was purified in batches of 2 liters by dialysis against 8 liters of 10 mm sodium succinate (pH 6.5) using 24,000 molecular weight cut-off dialysis tubing with two changes of buffer. Prior to column chromatography, the dialysate was adjusted to pH 5.5 with HCl. The dialyzed medium was loaded at 10 ml/min onto an SP-Sepharose column (1.5 × 20 cm) pre-equilibrated with Buffer A (10 mm sodium succinate, pH 5.5), washed with >100 ml of Buffer A, and eluted with a gradient of 0-0.5 m NaCl in Buffer A. Fractions containing mannosidase activity were pooled, adjusted to 1.0 m (NH4)2SO4 and pH 6.5 with 0.1 m Na-HEPES buffer. The pooled enzyme sample was then loaded onto a phenyl-Sepharose column (1.5 × 20 cm); washed with 1.0 m (NH4)2SO4, 10 mm Na-HEPES (pH 6.5); and eluted using a linear gradient of 1.0 to 0.5 m (NH4)2SO4 in 10 mm Na-HEPES (pH 6.5). Fractions containing enzyme activity were pooled and concentrated using an Amicon YM10 membrane following the addition of NDSB-201 (Calbiochem) to 0.75 m. Aliquots of 5 ml of concentrated enzyme sample were applied to Super-dex-75 column (1 × 100 cm) pre-equilibrated with 20 mm Na-MES (pH 6.5), 150 mm NaCl, 5 mm CaCl2, 0.75 m NDSB-201. The final purified enzyme was concentrated to 30 mg/ml in the same buffer and stored at 4 °C. The enzyme was screened for crystallization conditions as previously described (32Vallee F. Lal A. Moremen K.W. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 571-573Crossref PubMed Scopus (5) Google Scholar) using a microbatch technique. The best crystals were obtained using precipitant conditions of 15-20% polyethylene glycol 4000 and pH 4.5-6.5. Co-crystallization of the enzyme in the presence of dMNJ was attempted using the hanging drop vapor diffusion method. For crystallization, the above enzyme solution (19 μl) was incubated with 1 μl of a 200 mm stock solution of dMNJ (Oxford Glycosystems) at 37 °C for 2 h. The hanging drop was then prepared by mixing an equal volume of protein/dMNJ solution (1 μl) with a crystallization solution containing 100 mm MES, 100 mm Tris-HCl, 25-35% polyethylene glycol 4000, pH 6.0. Single crystals were formed in 2 days at 18 °C and used for structure determination. Data Collection—Data were collected with a Smart 6000 CCD detector (Bruker AXS) on an FRD rotating copper anode source equipped with HiRes2 optics (Rigaku/MSC). Data collection was carried out in three passes with 600 images collected in each pass (Δω = 0.3°) at a crystal to detector distance of 9 cm. For pass 1, 2θ = 25° and ϕ = 0°; for pass 2, 2θ = 25° and ϕ = 90°; for pass 3, 2θ = 0° and ϕ = 0°. Integration and scaling were performed with the Proteum software suite (Bruker AXS). The structure was solved by molecular replacement using the CNS software suite (34Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) using the coordinates of human ER mannosidase I as a probe (Protein Data Bank code 1FMI) (25Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Simulated annealing refinement (CNS (34Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar)) was followed by automated model rebuilding with ARP/wARP (35Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar). Manual rebuilding (Xfit (36McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar)) was repeatedly iterated with refinement of coordinates and isotropic temperature factors (Refmac5 (37Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1010) Google Scholar) and CCP4 (38Winn M.D. Ashton A.W. Briggs P.J. Ballard C.C. Patel P. Acta. Crystallogr. D Biol. Crystallogr. 2002; 58: 1929-1936Crossref PubMed Scopus (42) Google Scholar)). Anisotropic refinement of temperature factors was introduced once the crystallographic residual was reduced to 17.1% (19.0% for free reflections) and resulted in only a slight drop of the residuals and improved conformance with geometric parameters (39Engh R.A. Huber R. Acta Crystallogr. A. 1991; 47: 392-400Crossref Scopus (2548) Google Scholar). Protein structural alignments and r.m.s. deviation measurements were generated using Swiss-PdbViewer (version 3.7) (40Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar). All protein and glycan structure figures were prepared using MacPymol (version 0.95; Delano, W. L. Pymol Molecular Graphics System (2002) available on the World Wide Web at www.pymol.org) to generate rasterized images. Expression, Purification, and Crystallization of Golgi Mannosidase IA—The soluble catalytic domain (amino acid residues 66-655 (31Lal A. Schutzbach J.S. Forsee W.T. Neame P.J. Moremen K.W. J. Biol. Chem. 1994; 269: 9872-9881Abstract Full Text PDF PubMed Google Scholar)) of murine Golgi mannosidase IA was expressed as a secreted form in the recombinant fungal host, P. pastoris (23Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar). Expression was accomplished by fermentation in minimal medium with initial growth in glycerol as a carbon source followed by growth and induction using methanol as a carbon source once high cell density was achieved (32Vallee F. Lal A. Moremen K.W. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 571-573Crossref PubMed Scopus (5) Google Scholar). Optimized fermentation strategies using this recombinant host have routinely yielded as much as 100 mg/liter crude recombinant enzyme in the culture medium. Chromatographic purification using a combination of ion exchange, phenyl-Sepharose, and gel filtration columns resulted in the isolation of the homogeneous enzyme. An important element in the purification strategy was the inclusion of NDSB-201 in all of the buffers following the phenyl-Sepharose step through to protein crystallization. This compound has been found to aid in blocking protein aggregation during in vitro protein folding and crystallization studies (42Vuillard L. Rabilloud T. Goldberg M.E. Eur. J. Biochem. 1998; 256: 128-135Crossref PubMed Scopus (60) Google Scholar, 43Goldberg M.E. Expert-Bezancon N. Vuillard L. Rabilloud T. Fold. Des. 1995; 1: 21-27Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 44Vuillard L. Madern D. Franzetti B. Rabilloud T. Anal. Biochem. 1995; 230: 290-294Crossref PubMed Scopus (25) Google Scholar, 45Vuillard L. Marret N. Rabilloud T. Electrophoresis. 1995; 16: 295-297Crossref PubMed Scopus (27) Google Scholar, 47Vuillard L. Braun-Breton C. Rabilloud T. Biochem. J. 1995; 305: 337-343Crossref PubMed Scopus (75) Google Scholar, 48Vuillard L. Rabilloud T. Leberman R. Berthet-Colominas C. Cusack S. FEBS Lett. 1994; 353: 294-296Crossref PubMed Scopus (33) Google Scholar). Significant losses in the recovery of recombinant Golgi mannosidase IA by aggregation and precipitation were avoided by inclusion of NDSB-201 in all of the buffers. Previous purification and crystallization of recombinant mouse Golgi mannosidase IA under different conditions (32Vallee F. Lal A. Moremen K.W. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 571-573Crossref PubMed Scopus (5) Google Scholar) resulted in crystals that diffracted to 2.8 Å (space group P2221, unit cell dimensions a = 54.9 Å, b = 135.1 Å, c = 69.9 Å). The structure of this prior crystal form was not solved. In the present study, the crystals were obtained with a P212121 space group (unit cell dimensions of a = 55.3 Å, b = 72.2 Å, c = 129.6 Å) and diffracted to 1.51 Å (Table I). A single monomer was found in the asymmetric unit. The structure was determined by molecular replacement using the human ER mannosidase I structure (25Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) as a probe, followed by simulated annealing, automated and manual model building, and anisotropic refinement of temperature factors to an Rcryst of 17.1 and an Rfree of 19.0. The calcium ion and oligosaccharide structure were modeled into the remaining densities following the construction of the protein model. The structure model traces a continuous amino acid sequence from residues 178-644 indicating that 112 NH2-terminal and 11 COOH-terminal amino acids were cleaved from the recombinant protein during expression and purification. NH2-terminal protein sequence data, SDS-PAGE mobility, and matrix-assisted laser desorption ionization time-of-flight analysis of the protein all indicated that proteolysis had occurred, resulting in a protein of similar size to the sequence resolved in the crystal structure (not shown). In addition, the side chains of 18 surface-exposed residues (predominately Lys, Arg, and Glu residues) were not resolved in the final structure, presumably due to flexibility of the side chains in the solvent.Table IData collection and refinement statisticsParameterValueUnique reflections73,142Mean redundancy6.38Resolution (high resolution shell, Å)62.50-1.51 (1.63-1.51)Completeness (%)89.2 (56.8)Rsym (%)6.0 (14.7)Space groupP212121Cell dimensions (Å)a = 55.3 b = 72.2 c = 129.6Resolution range in refinement (Å)62.50-1.51Unique reflections in refinement (free set)65,697 (3,689)Mean B value (from Wilson plot, Å2)12.2 (9.7)R (Rfree, %)17.1 (19.0)r.m.s. bond lengths (Å)0.012r.m.s. bond angles (degrees)1.4 Open table in a new tab Overall Structure of the Molecule—The overall structure of the molecule consists of an (αα)7 barrel (Fig. 2), similar to other family 47 glycosylhydrolases (25Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Vallee F. Lipari F. Yip P. Sleno B. Herscovics A. Howell P.L. EMBO J. 2000; 19: 581-588Crossref PubMed Scopus (86) Google Scholar, 29Van Petegem F. Contreras H. Contreras R. Van Beeumen J. J. Mol. Biol. 2001; 312: 157-165Crossref PubMed Scopus (34) Google Scholar, 30Lobsanov Y.D. Vallee F. Imberty A. Yoshida T. Yip P. Herscovics A. Howell P.L. J. Biol. Chem. 2002; 277: 5620-5630Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), with consecutively alternating antiparallel pairs of helices forming a concentric set of inner and outer helical layers in the barrel structure. One end of the barrel has hairpin loop crossovers between the alternating inner and outer helices (short connection side (SC side), Fig. 2), and this side of the barrel also contains a COOH-terminal β-hairpin that extends into the mouth of the cylinder to form a plug at the bottom of the cavity. Residue Thr635, at the apex of the β-hairpin in the interior of the barrel cavity, is directly involved in coordination of a calcium ion in the core of the barrel (through the carbonyl oxygen and Oγ). Other conserved acidic residues in the interior of the barrel cavity are involved in forming a hydrogen-bonding network through six water molecules to result in a calcium ion containing an 8-coordinate pentagonal bipyramid geometry common to other family 47 glycosidases (25Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Vallee F. Lipari F. Yip P. Sleno B. Herscovics A. Howell P.L. EMBO J. 2000; 19: 581-588Crossref PubMed Scopus (86) Google Scholar, 29Van Petegem F. Contreras H. Contreras R. Van Beeumen J. J. Mol. Biol. 2001; 312: 157-165Crossref PubMed Scopus (34) Google Scholar, 30Lobsanov Y.D. Vallee F. Imberty A. Yoshida T. Yip P. Herscovics A. Howell P.L. J. Biol. Chem. 2002; 277: 5620-5630Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). A single disulfide bond is found between Cys478 and Cys510 to bridge helices α10 and α11. An equivalent conserved and essential disulfide is found in several other family 47 glycosidases (25Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Vallee F. Lipari F. Yip P. Sleno B. Herscovics A. Howell P.L. EMBO J. 2000; 19: 581-588Crossref PubMed Scopus (86) Google Scholar, 30Lobsanov Y.D. Vallee F. Imbe" @default.
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• W2040931402 title "Structure of Mouse Golgi α-Mannosidase IA Reveals the Molecular Basis for Substrate Specificity among Class 1 (Family 47 Glycosylhydrolase) α1,2-Mannosidases" @default.
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