ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes

Artigo Acesso aberto Revisado por pares

ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes

2008; Elsevier BV; Volume: 283; Issue: 15 Linguagem: Inglês

10.1074/jbc.m708669200

ISSN

1083-351X

Autores

Javier A. Alfaro, Ruixiang Blake Zheng, Mattias Persson, James A. Letts, Robert Polakowski, Yu Bai, S.N. Borisova, Nina O.L. Seto, Todd L. Lowary, Monica M. Palcic, Stephen V. Evans,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and an α-(1→3)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four “critical” residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/G235S bound to a panel of donor and acceptor analog substrates reveal “open,” “semi-closed,” and “closed” conformations as the enzymes go from the unliganded to the liganded states. In the open form the internal polypeptide loop (amino acid residues 177-195) adjacent to the active site in the unliganded or H antigen-bound enzymes is composed of two α-helices spanning Arg180-Met186 and Arg188-Asp194, respectively. The semi-closed and closed forms of the enzymes are generated by binding of UDP or of UDP and H antigen analogs, respectively, and show that these helices merge to form a single distorted helical structure with alternating α-310-α character that partially occludes the active site. The closed form is distinguished from the semi-closed form by the ordering of the final nine C-terminal residues through the formation of hydrogen bonds to both UDP and H antigen analogs. The semi-closed forms for various mutants generally show significantly more disorder than the open forms, whereas the closed forms display little or no disorder depending strongly on the identity of residue 176. Finally, the use of synthetic analogs reveals how H antigen acceptor binding can be critical in stabilizing the closed conformation. These structures demonstrate a delicately balanced substrate recognition mechanism and give insight on critical aspects of donor and acceptor specificity, on the order of substrate binding, and on the requirements for catalysis. The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and an α-(1→3)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four “critical” residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/G235S bound to a panel of donor and acceptor analog substrates reveal “open,” “semi-closed,” and “closed” conformations as the enzymes go from the unliganded to the liganded states. In the open form the internal polypeptide loop (amino acid residues 177-195) adjacent to the active site in the unliganded or H antigen-bound enzymes is composed of two α-helices spanning Arg180-Met186 and Arg188-Asp194, respectively. The semi-closed and closed forms of the enzymes are generated by binding of UDP or of UDP and H antigen analogs, respectively, and show that these helices merge to form a single distorted helical structure with alternating α-310-α character that partially occludes the active site. The closed form is distinguished from the semi-closed form by the ordering of the final nine C-terminal residues through the formation of hydrogen bonds to both UDP and H antigen analogs. The semi-closed forms for various mutants generally show significantly more disorder than the open forms, whereas the closed forms display little or no disorder depending strongly on the identity of residue 176. Finally, the use of synthetic analogs reveals how H antigen acceptor binding can be critical in stabilizing the closed conformation. These structures demonstrate a delicately balanced substrate recognition mechanism and give insight on critical aspects of donor and acceptor specificity, on the order of substrate binding, and on the requirements for catalysis. Glycosyltransferases synthesize carbohydrate moieties of glycoconjugates by catalyzing the sequential addition of monosaccharides from specific donors to specific acceptors. The ubiquitous presence of glycolipids and glycoproteins in all living systems underlines the importance of the glycosyltransferases superfamily, and the DNA of all domains of life encode for a large number of these enzymes (1Davies G.J. Gloster T.M. Henrissat B. Curr. Opin. Struct. Biol. 2005; 6: 637-645Crossref Scopus (238) Google Scholar). To date, crystal structures of glycosyltransferases have displayed a high degree of structural similarity even when there is low sequence homology (2Hu Y. Walker S. Chem. Biol. 2002; 9: 1287-1296Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 3Qasba P.K. Ramakrishnan B. Boeggeman E. Trends Biochem. Sci. 2005; 30: 53-62Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 4Breton C. Snajdrová L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: R29-R37Crossref PubMed Scopus (489) Google Scholar). As such, glycosyltransferases provide an excellent example of the preferential conservation of structural phenotype over the conservation of sequence identity (2Hu Y. Walker S. Chem. Biol. 2002; 9: 1287-1296Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), which indicates that the mechanism of glycosylation, although not yet fully understood, has been conserved. Elucidation of the details of substrate recognition would allow the development of new inhibitors for the treatment of microbial diseases (5Hu Y. Helm J.S. Chen L. Ginsberg C. Gross B. Kraybill B. Tiyanont K. Fang X. Wu T. Walker S. Chem. Biol. 2004; 5: 703-711Abstract Full Text Full Text PDF Scopus (88) Google Scholar), genetic ailments such as diabetes (6Somsák L. Nagya V. Hadady Z. Docsa T. Gergely P. Curr. Pharm. Des. 2003; 9: 1177-1189Crossref PubMed Scopus (208) Google Scholar), and cancer (7Greenwell P. Glycoconj. J. 1997; 14: 159-173Crossref PubMed Scopus (130) Google Scholar). The generation of inhibitors of the blood group A and B synthesizing glycosyltransferases GTA 2The abbreviations used are:GTAhuman ABO(H) blood group A α-(1→3)-N-acetylgalactosaminyltransferaseGTBhuman ABO(H) blood group B α-(1→3)-galactosaminyltransferaseHAH antigen disaccharideDAdeoxy-acceptor, 3-deoxy-Gal-H antigen disaccharideADAamino-deoxyacceptor, 2-deoxy-Fuc-3-amino-Gal-H antigen disaccharideMOPS3-(N-morpholino)propanesulfonic acidPEGpolyethylene glycolAdaN-(2-acetamido)-2-iminodiacetic acidMPDmethyl-pentanediol.2The abbreviations used are:GTAhuman ABO(H) blood group A α-(1→3)-N-acetylgalactosaminyltransferaseGTBhuman ABO(H) blood group B α-(1→3)-galactosaminyltransferaseHAH antigen disaccharideDAdeoxy-acceptor, 3-deoxy-Gal-H antigen disaccharideADAamino-deoxyacceptor, 2-deoxy-Fuc-3-amino-Gal-H antigen disaccharideMOPS3-(N-morpholino)propanesulfonic acidPEGpolyethylene glycolAdaN-(2-acetamido)-2-iminodiacetic acidMPDmethyl-pentanediol. and GTB have been reported (8Lowary T.L. Hindsgaul O. Carbohydr. Res. 1994; 251: 33-67Crossref PubMed Scopus (91) Google Scholar, 9Laferté S. Chan N.W. Sujino K. Lowary T.L. Palcic M.M. Eur. J. Biochem. 2000; 267: 4840-4849Crossref PubMed Scopus (25) Google Scholar), including an inhibitor-bound structure (10Nguyen H.P. Seto N.O.L. Cai Y. Leinala E.K. Borisova S.N. Palcic M.M. Evans S.V. J. Biol. Chem. 2003; 278: 49191-49195Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). human ABO(H) blood group A α-(1→3)-N-acetylgalactosaminyltransferase human ABO(H) blood group B α-(1→3)-galactosaminyltransferase H antigen disaccharide deoxy-acceptor, 3-deoxy-Gal-H antigen disaccharide amino-deoxyacceptor, 2-deoxy-Fuc-3-amino-Gal-H antigen disaccharide 3-(N-morpholino)propanesulfonic acid polyethylene glycol N-(2-acetamido)-2-iminodiacetic acid methyl-pentanediol. human ABO(H) blood group A α-(1→3)-N-acetylgalactosaminyltransferase human ABO(H) blood group B α-(1→3)-galactosaminyltransferase H antigen disaccharide deoxy-acceptor, 3-deoxy-Gal-H antigen disaccharide amino-deoxyacceptor, 2-deoxy-Fuc-3-amino-Gal-H antigen disaccharide 3-(N-morpholino)propanesulfonic acid polyethylene glycol N-(2-acetamido)-2-iminodiacetic acid methyl-pentanediol. Most glycosyltransferases are observed to lie in one of two major fold families, GT-A and GT-B (not to be confused with the GTA and GTB enzymes discussed here) (2Hu Y. Walker S. Chem. Biol. 2002; 9: 1287-1296Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 3Qasba P.K. Ramakrishnan B. Boeggeman E. Trends Biochem. Sci. 2005; 30: 53-62Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 11Bourne Y. Henrissat B. Curr. Opin. Struct. Biol. 2001; 11: 593-600Crossref PubMed Scopus (359) Google Scholar). Structural studies have revealed that specific internal sections of polypeptide adjacent to the active site are often observed to be flexible or completely disordered. These internal loops have been suggested to restrict water access to the active site, as well as act in donor recognition and catalysis (3Qasba P.K. Ramakrishnan B. Boeggeman E. Trends Biochem. Sci. 2005; 30: 53-62Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), including the inverting enzymes β4Gal-T1 (12Ramakrishnan B. Balaji P.V. Qasba P.K. J. Mol. Biol. 2002; 318: 491-502Crossref PubMed Scopus (115) Google Scholar), GnT-I (13Unligil U.M. Zhou S. Yuwaraj S. Sarkar M. Schachter H. Rini J.M. EMBO J. 2000; 19: 5269-5280Crossref PubMed Scopus (238) Google Scholar), GlcAT-I (14Pedersen L.C. Tsuchida K. Kitagawa H. Sugahara K. Darden T.A. Negishi M. J. Biol. Chem. 2000; 275: 34580-34585Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and GlcAT-P (15Kakuda S. Shiba T. Ishiguro M. Tagawa H. Oka S. Kajihara Y. Kawasaki T. Wakatsuki S. Kato R. J. Biol. Chem. 2004; 279: 22693-22703Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar); the retaining enzymes EXTL2 (16Pedersen L.C. Dong J. Taniguchi F. Kitagawa H. Krahn J.M. Pedersen L.G. Sugahara K. Negishi M. J. Biol. Chem. 2003; 278: 14420-14428Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) α-(1→3)-GalT (17Boix E. Zhang Y. Swaminathan G.J. Brew K. Acharya K.R. J. Biol. Chem. 2002; 277: 28310-28318Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Jamaluddin H. Tumbale P. Withers S.G. Acharya K.R. Brew K. J. Mol. Biol. 2007; 369: 1270-1281Crossref PubMed Scopus (43) Google Scholar), GTA, and GTB (19Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (201) Google Scholar, 20Letts J.A. Persson M. Schuman B. Borisova S.N. Palcic M.M. Evans S.V. Acta Crystallogr. Sect. D Biol. Crystallogr. 2007; 63: 860-865Crossref PubMed Scopus (10) Google Scholar); the microbial inverting SpsA (21Charnock S.J. Davies G.J. Biochemistry. 1999; 38: 6380-6385Crossref PubMed Scopus (309) Google Scholar) and CstII (22Chiu C.P. Watts A.G. Lairson L.L. Gilbert M. Lim D. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Mol. Biol. 2004; 11: 163-170Crossref PubMed Scopus (177) Google Scholar); and the retaining microbial enzyme LgtC (23Persson K. Ly H.D. Dieckelmann M. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Biol. 2001; 8: 166-175Crossref PubMed Scopus (312) Google Scholar). The retaining α-(1→3)-galactosyltransferase (α-(1→3)-GalT) is the enzyme most homologous to GTA/GTB in sequence and structure, and it has been reported to display substrate-induced conformational changes (18Jamaluddin H. Tumbale P. Withers S.G. Acharya K.R. Brew K. J. Mol. Biol. 2007; 369: 1270-1281Crossref PubMed Scopus (43) Google Scholar). This enzyme transfers Gal from UDP-Gal to oligosaccharides terminating in lactose or LacNAc (β-d-Gal-(1→4)-β-d-GlcNAc) (24Blanken W.M. van den Eijnden D.H. J. Biol. Chem. 1985; 260: 12927-12934Abstract Full Text PDF PubMed Google Scholar). Like GTA and GTB, α-(1→3)-GalT is a retaining enzyme with a GTA fold. Unlike GTA and GTB, the structure of α-(1→3)-GalT displays a completely ordered internal loop in the unliganded state, which has been reported to lie in different conformations for different mutants and in substrate-bound and unbound complexes (17Boix E. Zhang Y. Swaminathan G.J. Brew K. Acharya K.R. J. Biol. Chem. 2002; 277: 28310-28318Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Jamaluddin H. Tumbale P. Withers S.G. Acharya K.R. Brew K. J. Mol. Biol. 2007; 369: 1270-1281Crossref PubMed Scopus (43) Google Scholar). GTA and GTB are responsible for the generation of the human ABO(H) blood group A and B antigens (25Hearn V.M. Smith Z.G. Watkins W.M. Biochem. J. 1968; 109: 315-317Crossref PubMed Scopus (67) Google Scholar, 26Kobata A. Grollman E.F. Ginsburg V. Biochem. Biophys. Res. Commun. 1968; 32: 272-277Crossref PubMed Scopus (70) Google Scholar). GTA catalyzes the transfer of GalNAc from UDP-GalNAc to the H antigen acceptor (α-l-Fuc-(1→2)-β-d-Gal-O-R, where R is glycolipid or glycoprotein) to form the A antigen, whereas GTB catalyzes the transfer of Gal from UDP-Gal to the H antigen acceptor to form the B antigen (27Watkins W.M. Morgan W.T. Vox Sang. 1959; 4: 97-119Crossref PubMed Scopus (108) Google Scholar, 28Kabat E.A. Blood Group Substances: Their Chemistry and Immunochemistry. Academic Press, New York1956: 135-139Google Scholar). Initial high resolution structural studies of both GTA and GTB revealed two regions of disordered polypeptide (19Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (201) Google Scholar). One region consisted of the last 10 residues of the C terminus, whereas the other was an internal polypeptide loop composed of residues 177-195. Subsequent studies have shown that part of the disorder of the internal loop was because of the presence of a heavy atom, and that crystals of the mutant enzyme GTB/C209A grown in the absence of heavy atoms display a smaller disordered segment of the internal loop consisting of residues 177-187 (20Letts J.A. Persson M. Schuman B. Borisova S.N. Palcic M.M. Evans S.V. Acta Crystallogr. Sect. D Biol. Crystallogr. 2007; 63: 860-865Crossref PubMed Scopus (10) Google Scholar). GTA and GTB are the two most homologous glycosyltransferases known that utilize different nucleotide donors and differ by only 4 of 354 amino acids as follows: Arg/Gly176, Gly/Ser235, Leu/Met266, and Gly/Ala268 in GTA and GTB, respectively (29Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (883) Google Scholar). The role of each critical residue in donor and acceptor recognition has been studied through the generation of chimeric GTA/GTB enzymes. A nomenclature based on these four critical amino acid residues has been developed to describe GTA and GTB chimera, where GTA can be referred to as AAAA and GTB as BBBB with each letter corresponding to one critical residue in increasing order, such that the ABBB chimera would correspond to the GTB/G176R mutant enzyme and AABB would correspond to the GTB/G176R/S235G mutant enzyme. Critical residues Leu/Met266 and Gly/Ala268 have been shown to be responsible for discrimination between the two donor molecules (30Yamamoto F. Hakomori S. J. Biol. Chem. 1990; 265: 19257-19262Abstract Full Text PDF PubMed Google Scholar, 31Seto N.O.L. Compston C.A. Evans S.V. Bundle D.R. Narang S.A. Palcic M.M. Eur. J. Biochem. 1999; 259: 770-775Crossref PubMed Scopus (88) Google Scholar, 32Kamath V.P. Seto N.O.L. Compston C.A. Hindsgaul O. Palcic M.M. Glycoconj. J. 1999; 16: 599-606Crossref PubMed Scopus (18) Google Scholar), whereas Gly/Ser235 and Leu/Met266 significantly impact acceptor recognition (33Letts J.A. Rose N.L. Fang Y.R. Barry C.H. Borisova S.N. Seto N.O.L. Palcic M.M. Evans S.V. J. Biol. Chem. 2006; 281: 3625-3632Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar); however, the function of the conserved mutation Arg/Gly176 has been elusive. Structural studies in the past have been hampered by the fact that Arg/Gly176 lies at the edge of the internal disordered loop from residues 176-195; however, the development of crystallization conditions for BBBB (GTB), ABBB, and AABB in the absence of heavy atoms permits a structural investigation of the influence of residue 176 on loop ordering and substrate binding. We now report the kinetic characterization of several chimeric enzymes along with high resolution structures of GTB (BBBB) and the chimeric enzyme ABBB in their unliganded states, BBBB and ABBB in the presence of UDP, BBBB and AABB in the presence of synthetic H antigen disaccharide α-l-Fucp-(1→2)-β-d-Galp-O(CH2)7CH3, BBBB and ABBB in the presence of UDP, and the H antigen acceptor analog α-l-2-deoxy-Fucp-(1→2)-β-d-3-amino-Galp-O(CH2)7CH3, BBBB, and ABBB and in the presence of both UDP and H antigen disaccharide, and AABB in complex with UDP-Gal and the H antigen acceptor analog α-l-Fucp-(1→2)-β-d-3-deoxy-Galp-O(CH2)7CH3. Construction of the Synthetic Glycosyltransferase Chimeric Genes ABBB and AABB—The synthetic wild-type GTA (designated AAAA, amino acids 53-354) gene was constructed from synthetic oligonucleotides as described previously (34Seto N.O.L. Palcic M.M. Compston C.A. Li H. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The synthetic gene was designed with unique restriction sites to facilitate mutagenesis. Glycosyltransferase chimeric mutants ABBB and AABB were synthesized by digesting the AAAA gene with KpnI/SphI and ligating in the appropriate oligonucleotides to form the desired gene sequence. The -10/ABBB and -10/AABB genes (amino acids 63-354) were made by PCR amplification using the wild-type ABBB and AABB genes as templates. The forward primer 5′-ATA TGA ATT CAT GGT TTC CCT GCC GCG TAT GGT TTA CCC GCA GCC GAA-3′ (MIN2) introduced an EcoRI site in the 5′ end, and the reverse primer 5′-ATA ATT AAG CTT CTA TCA CGG GTT ACG AAC AGC CTG GTG GTT TTT-3′ (PCR-3B) introduced a HindIII site in the 3′ end. The PCR profile used was 94 °C/3 min (94 °C, 30 s, 55 °C, 30 s, and 72 °C, 1 min) for 30 cycles. After gel purification, the PCR products were digested with EcoRI and HindIII for 2 h at 37 °C and were ligated into pCWΔlac, which had been opened with EcoRI/HindIII. Each ligation was transformed into BL21-competent cells. The DNA sequences were confirmed on both strands. All insert and plasmid purifications were made by Qiagen plasmid purification system (Qiagen, Chatsworth, CA). All ligations were made by the use of T4 DNA ligase (Invitrogen) at room temperature for 1 h. All restriction enzymes were purchased from New England Biolabs. For GTB R188S, R188K, and R188H (amino acids 63-354), site-directed mutagenesis was carried out using a QuikChange kit (Stratagene). The primers used for mutagenesis were as follows: GTB R188S (sense) 5′-ATG CGT TCC ATG GAA ATG ATC AGC GAC TTC TGC-3′ and antisense 5′-CAT TTC CAT GGA ACG CAT GGA AAC GTC CTG CC-3′. Cloning of R188K was as follows: (sense) 5′-TGG CAG GAC GTT TCC TGC GTAAAA TGG AAA TGA TCA GCG AC-3′ and antisense 5′-GTC GCT GAT CAT TTC CATTTT ACG CAT GGA AAC GTC CTG CC-3′; and cloning of R188H was as follows: (sense) 5′-CAG GAC GTT TCC ATG CGT CAT ATG GAA ATG ATC AGC-3′ and antisense 5′-GCT GAT CAT TTC CAT ATG ACG CAT GGA AAC GTC CTG-3′. The altered nucleotides are shown in boldface. Protein Purification—Mutant enzymes were purified from Escherichia coli by methods described previously (36Seto N.O.L. Compston C.A. Szpacenko A. Palcic M.M. Carbohydr. Res. 2000; 324: 161-169Crossref PubMed Scopus (45) Google Scholar), with the exception of R188H and R188K where cells were disrupted at 1.35 kbar with a constant system cell disrupter. Expression levels for mutants were good, and the yields of final purified proteins were ABBB 36 mg/liter, AABB 50 mg/liter, R188S 8 mg/liter, R188K 66 mg/liter, and R188H 15 mg/liter. Kinetic Characterization—Kinetics using α-l-Fucp-(1→2)-β-d-Galp-O-R as an acceptor were carried out with a radiochemical assay, where a Sep-Pak reverse-phase cartridge is used to isolate radiolabeled reaction products created when the label is transferred from a radioactive donor to the hydrophobic acceptor (37Palcic M.M. Heerze L.D. Pierce M. Hindsgaul O. Glycoconj. J. 1988; 5: 49-63Crossref Scopus (279) Google Scholar). Assays were performed at 37 °C in a total volume of 12 μl containing substrates and enzyme in 50 mm MOPS buffer, pH 7.0, 20 mm MnCl2, and bovine serum albumin (1 mg/ml). Seven different concentrations of donor and acceptor were employed, and initial rate conditions were linear with no more than 10% of the substrate consumed in the reaction. For the donors, the Km values were determined at 1.0 mm acceptor, and the Km for the acceptor was determined at 1.0 mm donor. The kinetic parameters kcat and Km were obtained by nonlinear regression analysis of the Michaelis-Menten equation with the Graph Pad PRISM 3.0 program (GraphPad Software, San Diego). Two-substrate kinetic analysis was performed for the AABB and ABBB mutants to obtain KA (acceptor Km), KB (donor Km), Kib, and Kia, as described previously (34Seto N.O.L. Palcic M.M. Compston C.A. Li H. Bundle D.R. Narang S.A. J. Biol. Chem. 1997; 272: 14133-14138Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Kib is the apparent Michaelis constant for donor that is independent of the concentration of acceptor and thus corresponds to the dissociation constant of the enzyme·UDP-Gal or enzyme·UDP-GalNAc complexes. Kia is the dissociation constant of the enzyme·acceptor complex. Crystallization—All proteins were crystallized using conditions different from those reported previously (10Nguyen H.P. Seto N.O.L. Cai Y. Leinala E.K. Borisova S.N. Palcic M.M. Evans S.V. J. Biol. Chem. 2003; 278: 49191-49195Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Patenaude S.I. Seto N.O.L. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (201) Google Scholar, 33Letts J.A. Rose N.L. Fang Y.R. Barry C.H. Borisova S.N. Seto N.O.L. Palcic M.M. Evans S.V. J. Biol. Chem. 2006; 281: 3625-3632Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 35Lee H.J. Barry C.H. Borisova S.N. Seto N.O.L. Zheng R.B. Blancher A. Evans S.V. Palcic M.M. J. Biol. Chem. 2005; 280: 525-529Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 38Marcus S.L. Polakowski R. Seto N.O.L. Leinala E. Borisova S.N. Blancher A. Roubinet F. Evans S.V. Palcic M.M. J. Biol. Chem. 2003; 278: 12403-12405Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 39Persson M. Letts J.A. Hosseini-Maaf B. Borisova S.N. Palcic M.M. Evans S.V. Olsson M.L. J. Biol. Chem. 2007; 282: 9564-9570Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Whereas the first crystals of GTB were grown from relatively low protein concentrations (∼8-15 mg/ml) and as a mercury derivative, the crystals in this paper were initially generated from higher protein concentrations (∼60-75 mg/ml). The first crystals of the ABBB and AABB mutants grew in stock solutions containing 20 mm MOPS, pH 7.0, 75 mm NaCl, 15 mm β-mercaptoethanol, 0.05% NaN3 and stored at 4 °C for several months. Crystals of ABBB and AABB were washed with mother liquor consisting of 7% PEG-4000, 70 mm Ada buffer (N-(2-acetamindo)iminodiacetic acid), pH 7.5, 30 mm sodium acetate buffer, pH 4.6, 40 mm ammonium sulfate, and 5 mm MnCl2. Crystals of BBBB were obtained by the hanging drop method from 30 to 40 mg/ml fresh protein solutions containing 1% PEG, 4.5% methyl-pentanediol (MPD), 0.1 m ammonium sulfate, 0.07 m NaCl, 0.05 m Ada buffer, pH 7.5, 5 mm MnCl2 against a reservoir containing 2.7% PEG-4000, 7% MPD, 0.32 m ammonium sulfate, 0.25 m NaCl, and 0.2 m Ada buffer, pH 7.5. Crystals of BBBB, ABBB, and AABB in complex with UDP, synthetic H antigen disaccharide α-l-Fucp-(1→2)-β-d-Galp-O(CH2)7CH3 (HA) (8Lowary T.L. Hindsgaul O. Carbohydr. Res. 1994; 251: 33-67Crossref PubMed Scopus (91) Google Scholar), or the analogs α-l-2-deoxy-Fucp-(1→2)-β-d-3-amino-Galp-O(CH2)7CH3 (ADA) (40Bai Y. Lin S.J. Qi G. Palcic M.M Lowary T.L. Carbohydr. Res. 2006; 341: 1702-1707Crossref PubMed Scopus (3) Google Scholar) and α-l-Fucp-(1→2)-β-d-3-deoxy-Galp-O(CH2)7CH3 (DA) (41Lowary T.L. Hindsgaul O. Carbohydr. Res. 1993; 249: 163-195Crossref PubMed Scopus (61) Google Scholar) in various combinations were obtained by soaking substrate into the unliganded crystals. Crystals were washed with mother liquor consisting of 7% PEG-4000, 70 mm Ada buffer, pH 7.5, 30 mm sodium acetate buffer, pH 4.6, 40 mm ammonium sulfate, and 5 mm MnCl2. The concentration of UDP was usually 25 mm, but as little as 10 mm was often sufficient, and 50 mm was used for BBBB+UDP. The H antigen acceptor analogs HA, DA, and ADA concentrations ranged from 10 to 20 mm. The concentration of UDP-Gal ranged from 35 to 50 mm. The concentration of MnCl2 was 5 mm. All substrates were added incrementally over a period of a few minutes to a few hours so as to prevent crystal fracture. In the case of AABB+UDP-Gal+DA, additional UDP-Gal was added to the crystal minutes before freezing to minimize the extent of UDP-Gal hydrolysis. No UDP was added to AABB+UDP, as the UDP appeared to follow the protein through the purification process. The UDP was removed to generate the AABB+H structure by washing the crystal with 10 mm EDTA to remove the manganese that bound the UDP to the protein. Data Collection and Reduction—X-ray diffraction data were collected at -160 °C for all crystals using a CryoStream 700 crystal cooler. Each crystal was incubated with a cryoprotectant solution that consisted of mother liquor with 30% (v/v) glycerol replacing a corresponding volume of water, except AABB+UDP-Gal+DA where a corresponding volume of MPD was used. Data were collected on a Rigaku R-AXIS IV2+ area detector at distances of 72 mm and exposure times between 4.0 and 7.0 min for 0.5° oscillations. X-rays were produced by an MM-002 generator (Rigaku/MSC, College Station, TX) coupled to Osmic “Blue” confocal x-ray mirrors with power levels of 30 watts (Osmic, Auburn Hills, MI). The data were scaled, averaged, and integrated using d*trek and CrystalView (42Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1415) Google Scholar). Structure Determination—Although the structures were nearly isomorphous, for completeness all structures were solved by molecular replacement using the CCP4 module MOLREP (43Vagin A. Teplyakov A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1622-1624Crossref PubMed Scopus (690) Google Scholar, 44Vagin A.A. Isupov M.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1451-1456Crossref PubMed Scopus (186) Google Scholar) with the structure of wild-type GTB as a starting model (Protein Data Bank accession code 1LZ7), and subsequently refined using the CCP4 module REFMAC5 (45Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19733) Google Scholar). All figures were produced using Setor (46Evans S.V. J. Mol. Graphics. 2003; 11: 134-138Crossref Scopus (1249) Google Scholar) and SetoRibbon. 3S. V. Evans, unpublished. The details of data collection and refinement for the enzyme complexes are provided in Table 1 for BBBB structures and Table 2 for ABBB and AABB structures. In the 12 structures determined, BBBB, ABBB, and AABB crystals were soaked with combinations of UDP, UDP-Gal, H antigen disaccharide (HA), 3-deoxy-Gal-H antigen disaccharide (deoxy-acceptor, DA), and 2-deoxy-Fuc-3-amino-Gal-H antigen disaccharide (aminodeoxy-acceptor, ADA). These 12 structures are labeled as BBBB, BBBB+UDP, BBBB+HA, BBBB+UDP+HA, BBBB+UDP+ADA, ABBB, ABBB+UDP, ABBB+UDP+HA, ABBB+UDP+ADA, AABB+UDP, AABB+HA, and AABB+ UDP-Gal+DA. The maximum resolution of the diffraction data collected varied from 1.75 to 1.41 Å with a final Rwork ranging from 19.4 to 22.3% and an Rfree ranging from 21.7 to 24.6%.TABLE 1Data collection and refinement results for crystal structures of BBBBBBBBBBBB+UDPBBBB+HABBBB+UDP+HABBBB+UDP+ADAResolution (Å)20-1.4320-1.7520-1.5520-1.6920-1.69Space groupC2221C2221C2221C2221C2221A (Å)52.5452.5452.5652.4552.43B (Å)149.91149.93150.25149.60149.93C (Å)79.2179.2879.3878.7478.92Rmerge (%)aRmerge, ∑|Iobs - Iave|/∑Iave.,bValues in parentheses represent highest resolution shell.0.034 (0.322)0.051 (0.338)0.037 (0.318)0.048 (0.324)0.044 (0.325)Completeness (%)bValues in parentheses represent highest resolution shell.98.1 (96.9)98.5 (96.2)98.2 (96.0)99.5 (99.9)97.0 (98.4)Unique reflections57,03031,52545,17134,92334,204RefinementRwork (%)cRwork, ∑||Fo| - |Fo||/∑|Fo|.21.219.621.119.820.0Rfree (%)cRwork, ∑||Fo| - |Fo||/∑|Fo|.,d10% of reflections were omitted for Rfree calculations.22.123.123.522.223.2No. of waters236213240200196r.m.s. bond (Å)er.m.s. is root mean square.0.0090.0140.0110.0140.017r.m.s. angle (°)er.m.s. is root mean square.1.281.441.281.411.61Protein Data Bank ID2RIT2RIX2RIY2RJ82RJ9a Rmerge, ∑|Iobs - Iave|/∑Iave.b Values in parentheses represent highest resolution shell.c Rwork, ∑||Fo| - |Fo||/∑|Fo|.d 10% of reflections were omitted for Rfree calculations.e r.m.s. is root mean square. Open table in a new tab TABLE 2Data collection and refinement results for crystal structures of ABBB and AABBABBBABBB+UDPABBB+UDP+HAABBB+UDP+ADAAABB+UDPAABB+HAAABB+UDP-Gal+DAResolution (Å)20-1.4520-1.5220-1.5520-1.4720-1.4520-1.4120-1.70Space groupC2221C2221C2221C2221C2221C2221C2221A (Å)52.5352.5352.4552.4852.5952.5252.36B (Å)149.58149.35149.12149.74149.02149.06148.65C (Å)79.6479.6579.6479.7979.6579.6179.52Rmerge (%)aRmerge, ∑|Iobs - Iave|/∑Iave.,bValues in parentheses represent highest resolution shell.2.5 (23.0)4.2 (36.0)4.6 (33.0)3.4 (28.9)2.8 (20.7)3.7 (31.5)5.7 (31.0)Completeness (%)bValues in parentheses represen

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ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes