Correction Notice: Structural insight into mammalian sialyltransferases

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1 CORRECTION NOTICE Correction Notice: Structural insight into mammalian sialyltransferases Francesco V Rao, Jamie R Rich, Bojana Rakić, Sai Buddai, Marc F Schwartz, Karl Johnson, Caryn Bowe, Warren W Wakarchuk, Shawn DeFrees, Stephen G Withers & Natalie C J Strynadka Nat. Struct. Mol. Biol. 16, (2009) In the version of the supplementary file originally posted online, the acknowledgements were incomplete. The error has been corrected in this file as of 18 November 2009.

2 Supporting Online Material for Structural Insight into Mammalian Sialyltransferases Francesco V. Rao, Jamie R. Rich, Bojana Rakić, Sai Buddai, Marc F. Schwartz, Karl Johnson, Caryn Bowe, Warren W. Wakarchuk, Shawn DeFrees, Stephen G. Withers, Natalie C. J. Strynadka * * To whom correspondence should be addressed. natalie@byron.biochem.ubc.ca; Tel.: ; Fax: This PDF file includes: Supplementary Figure 1-4 Supplementary Table 1-2 Materials and Methods References for Supporting Online Material Acknowledgments Author Contributions S1

3 Supplementary Figure 1 Supplementary Fig. 1 Comparison of pst3gal-i (GT-29) with CstII (GT-42). The structures are shown in a ribbon representation, with red helices and blue strands. The lid domain of CstII (PDB entry 1RO7 1 ), is represented in magenta (b) and is modeled as a dashed line on pst3gal-i (a). The ligands are shown in a stick representation with yellow carbon atoms, and the catalytic base in a stick representation with cyan carbon atoms. The disulphide-linked cysteines in pst3gal-i are represented in green and labeled C1 to C3. Residues predicted to be glycosylated are shown in stick representation with black carbon atoms. (c) depicts a structure-based sequence alignment of pst3gal-i (GT- 29) and CstII (GT-42). Sequences were aligned using the program LSQMAN using the Needleman-Wunsch alignment algorithm and applying a 5 Å cut-off 2. Secondary structure and location of the lid domain for pst3gal-i and CstII are shown above and below each sequence respectively following the color scheme in (a). Identical residues and those that align structurally are depicted in black and grey respectively. The catalytic base is represented with a cyan colored box. Residues from the lid domain of pst3gal-i have not been included in the alignment as they are disordered in the crystal structure. (d) shows topology diagrams of GT-2, GT-42 and GT-29. The color scheme for secondary structure elements follows that of (a). Figures were created using TopDraw 3. S2

4 Supplementary Figure 2 Supplementary Fig. 2 Active site comparisons and reaction scheme. The pst3gal-1- disaccharide-cmp complex (a) is compared to the CstII-CMP3FNeuAc complex (GT- 42) (PDB entry 1RO7 1 ) (b), Δ24PmST1-lactose-CMP3FNeuAc (GT-80) (c) (PDB entry 2IHZ 4 ) and C2GnT-I-Gal β1,3galnac (GT-14) (d) (PDB entry 2GAM 5 ). The first domain of Δ24PmST1 (GT-B fold) is shown with pink carbon atoms (b). The proposed catalytic base for each structure is shown with cyan colored carbon atoms. Hydrogen bond interactions are shown with dashed lines. Fig. 2e. Reaction scheme of pst3gal-i. His319 deprotonates the acceptor sugar at the O3 position, resulting in nucleophilic attack on the anomeric (C2) carbon of Neu5Ac. This leads to breakage of the C2-O bond linking Neu5Ac with CMP. The oxocarbenium-ion like transition state is defined within the large brackets. S3

5 Supplementary Figure 3 Supplementary Fig. 3. Sequence alignment of pst3gal-i with other ST3 and ST6Gal-I. The pst3gal-i secondary structure and location of the lid domain are shown using the color scheme in Fig. 1a. Cysteines involved in a disulphide bond are indicated with a green triangle. Residues interacting with CMP or the disaccharide acceptor are indicated by purple and yellow diamonds respectively. Three of the predicted glycosylation sites are indicated with yellow squares. The first glycosylation site is predicted to be located on the stem region (not shown). The proposed catalytic base is represented with a cyan colored star. Sialyl motifs are represented in labeled colored boxes following the color scheme in Fig. 1b. Numbering of the above sequences is according to the UniProt accession numbers; Q02745, Q11201, Q16842, Q11203, Q11206, Q9UNP4, Q9Y274, P15907, respectively. Amino acids completely conserved or greater than 50% conserved are depicted in black and grey respectively. The sequence alignment was performed using the program ALINE 6. S4

6 Supplementary Figure 4 Supplementary Fig. 4. Surface sequence conservation. The protein surface of pst3gal-i-disaccharide-cmp complex is shown in three different colors to reflect sequence conservation with hst6gal-i, based on the sequence alignment in Fig. 3. Dark blue denotes identical surface residues, with light blue and grey indicating similar and no sequence conservation, respectively. The location of the catalytic residue is depicted in orange. The disaccharide acceptor and CMP are shown in stick representations with yellow carbon atoms. A polar cleft (depicted in green) of suitable dimensions to bind a potential peptide or lipid (found in natural acceptors of GT-29) is shown extending appropriately from the terminal sugar of the disaccharide acceptor towards solvent. S5

7 Supplementary table 1. Kinetic parameters Substrate varied (reaction monitored) protein k cat (min -1 ) K m (mm) k cat / K m (mm -1 min -1 ) CMPNeu5Ac (hydrolase) a pst3gal-i n.d n.d n.d CMPNeu5Ac (transferase) b pst3gal-i Gal-β-1,3GalNAc-α-OBn c pst3gal-i CMPNeu5Ac (hydrolase) a Cst II CMPNeu5Ac (transferase) d Cst II Lactose f Cst II '-Sialyl Lactose f Cst II *- error range in data is between 5-15% n.d no significant value was detected. a- determined in the absence of acceptor substrate b- determined at a constant Gal-β-1,3GalNac-α-OBn concentration of 0.5 mm c- determined at a constant CMPNeu5Ac concentration of 0.3 mm d- determined at a constant lactose concentration of 160 mm f- determined at a constant CMPNeu5Ac concentration of 1 mm Comparison with the bacterial CstII sialyltransferase reveals that pst3gal-i is a much more efficient enzyme with K m values for CMP-Neu5Ac five times lower and k cat values thirty-five times higher. Interestingly, whereas CstII has been shown to additionally catalyze the hydrolysis of CMP-Neu5Ac at a significant rate in the absence of acceptor substrate, pst3gal-i has no such activity. S6

8 Supplementary table 2. Data collection, phasing and refinement statistics pst3gal-i-semet pst3gal-i-disaccharide pst3gal-i-cmp-disaccharide Data collection Space group I222 I222 I222 Cell dimensions a, b, c (Å) 73.38, 78.01, , 78.91, , 78.43, α, β, γ ( ) 90.00, 90.00, , 90.00, , 90.00, Peak Inflection Remote Wavelength Resolution (Å) ( ) ( ( ) ( ) ( ) * 1.8) R merge (0.333) (0.332) (0.504) (0.399) (0.252) I / σi 18.2 (3.4) 22.6 (3.8) 16.6 (3.3) 23.4 (2.8) 19.3 (3.5) Completeness (%) 98.3 (92.2) 98.8 (89.5) 99.3 (99.0) 99.8 (98.3) 85.0 (83.0) Redundancy 6.2 (4.5) 6.2 (4.4) 6.4 (4.5) 6.9 (4.9) 1.9 (1.7) Refinement Resolution (Å) No. reflections R work / R free 0.18/ / /0.20 No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles ( ) *Values in parentheses are for highest-resolution shell. S7

9 Materials & Methods Cloning, expression and crystallization DNA encoding porcine ST3Gal-I (Genbank accession NP ) starting at Glu46 was synthesized with codon selection optimized for bacterial expression (Blue Heron Biotechnology). The pst3gal-i open reading frame was cloned by PCR, restriction digest, and ligation into the BamHI and XhoI sites of pcwin2-mbp 11. For protein expression, pcwin2-mbp-pst3gal-i was transformed into Origami- 2 cells (Novagen). Cells were grown in rich media (typically LB or 2BN (2% martone B- 1 (Marcor), 2% yeast extract, trace metals 12, 1% NaCl, and 1 mm MgSO 4 ), with kanamycin at 50 µg ml -1 ) to mid-logarithmic phase, shifted to 20 C for at least 30 minutes, and induced overnight with 0.2 mm IPTG. Induced cells were harvested by centrifugation and stored at -80 C. Induced cells were thawed and resuspended in lysis buffer (10 mm Tris ph 8.2, 5 mm NaCl) at 6-10 ml g -1 wet cells, and lysed with two to four passes through a microfluidizer. The lysate was clarified by centrifugation for one hour at 27,000 x g, 4 C, concentrated approximately five-fold, and diafiltered against lysis buffer to 1.4 ms cm -1 using two Pellicon-2 mini ultrafiltration cassettes (Millipore). The lysate was applied to a Source 30Q column (GE Healthcare) pre-equilibrated with 20 mm Tris ph 8.2, 10 mm NaCl, and eluted with a gradient to 100 mm NaCl. Fractions containing active, full length MBP-tagged ST3Gal-I were pooled and diluted three-fold with 20 mm Tris ph 8.2. The diluted material was then applied to a Source 15Q column (GE Healthcare) preequilibrated with Q column buffer, and eluted with a gradient to 80 mm NaCl. Fractions containing active, full length MBP-tagged pst3gal-i were pooled and buffer exchanged to 10 mm sodium phosphate, ph 8.0, 10 mm NaCl by centrifugal ultrafiltration (Centricon Plus 70, Millipore). The material was then applied to a ceramic hydroxyapatite column (20 µm, Type-I, Bio-Rad) pre-equilibrated with 10 mm sodium phosphate, ph 8.0, 10 mm NaCl, and eluted with a gradient to 130 mm sodium phosphate. Fractions containing active, full length MBP-tagged pst3gal-i were pooled, concentrated, buffer exchanged to 20 mm Tris ph 8.2, 10 mm NaCl by centrifugal ultrafiltration, and mixed with 1 volume of glycerol prior to storage at -20 C. For the expression of selenomethionine (Se-Met)-labeled MBP-pST3Gal-I, cells were grown and induced under similar conditions using synthetic complete media (50 mm KH 2 PO 4 and Na 2 HPO 4, 25 mm (NH 4 ) 2 SO 4, 0.5% glycerol, 0.05% glucose, 1 mm MgSO 4, trace metals, 500 mg l -1 adenine, guanosine, thymine, and uracil, 15 mg l -1 p- aminobenzoic acid, 1 mg l -1 thiamine, 1 mg l -1 biotin, 304 mg l -1 leucine, 150 mg l -1 myoinositol, alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, lysine, phenylalanine, proline, serine, tryptophan, tyrosine, valine, and selenomethionine (media recipe modified from 12,13. Se-Met-labeled MBP-tagged pst3gal-i was purified in smaller lots using methods similar to those described above. Purified MBP-tagged pst3gal-i was digested with 25 Units of Factor Xa (Novagen) and dialyzed overnight into 25 mm Tris ph 8.2, 150 mm NaCl, at 4 C. Dialyzed protein sample was applied to a gravity column (Bio-Rad) containing 10 ml of pre-equilibrated (25 mm Tris ph 8.2, 150 mm NaCl) amylose resin (NEB) and washed with 3 column volumes of buffer. The flow-through was applied to a gravity column (Bio-Rad) containing 2 ml of pre-equilibrated Xarrest agarose (Novagen) and washed S8

10 with 3 column volumes of buffer. The flow-through containing pure pst3gal-i was spin concentrated to approximately 5 mg ml -1. Microbatch crystallization experiments were set-up by mixing 0.5 µl of protein and 0.5 µl of mother liquor (30% PEG 4000 and 0.1 M MES, ph 6.8), which was overlaid with Al s oil. Thin plate-shaped crystals would appear after 6 days, growing to a maximum size of approximately 0.2 x 0.2 x 0.1 mm. Data Collection, structure solution and refinement The crystals were cryoprotected by a five second immersion in a solution containing 27% PEG 4000, 0.09 M MES, ph 6.8 and 10% glycerol, and frozen in liquid nitrogen. A complex with a synthetic acceptor was obtained by adding 10 mm Galβ1,3GalNAc α-onitrophenyl to the protein and mother liquor drop. A complex with CMP was obtained by adding 0.2 µl of 10 mm CMP to the crystals for 3 minutes, followed by cryoprotection. Co-crystallization and soaking experiments with the inert donor substrate analog CMP- 3FNeu5Ac were unsuccessful. A six-fold redundant 1.8 Å MAD data set was collected from Se-Met labeled protein crystals, at beamline at the Advanced Light Source (Berkeley, CA). Data were processed using MOSFLM and scaled with SCALA 14. Initial phases were calculated with SOLVE 15 (figure of merit = 0.51), which located 3 Se sites (from a possible 4). Solvent flattening and initial model building with RESOLVE 16 yielded an interpretable 1.8 Å experimental map. This was used as input for ArpWarp 17, which was able to build 281 out of 298 possible residues. Iterative model building with COOT 18 and refinement with REFMAC 19 and PHENIX 20, yielded the final model with the statistics shown in Table S1. Topologies for the ligands were generated with PRODRG 21. Enzymology A continuous coupled assay 22, in which the release of CMP is coupled to the oxidation of NADH ( λ = 340nm, ε = 6.22 mm -1 cm -1 ), was used to monitor the activity of pst3gal-i. Absorbance measurements were obtained using a Cary 300 UV-Vis spectrophotometer equipped with a circulating water bath. GraFit version (Erithacus) was used to calculate kinetic parameters by direct fit of initial rates to the Michaelis-Menten equation. The assay conditions in the 200 µl quartz cuvette are as follows: 20 mm HEPES (ph 7.5), 50 mm KCl, 10 mm MgCl 2, 10 mm MnCl 2, 0.7 mm PEP, 0.25 mm or 0.3 mm NADH, 2 mm ATP, 1.0 mg ml -1 BSA, 5.9U of PK, 8.4U of LDH, 7.5U of NMPK, and varying concentrations of donor and acceptor substrates. The cuvettes were left at 37 C until a stable rate of decreasing absorbance at 340 nm was established. This period is required to deplete the CMP present in the donor solution and any NDPs present in the ATP or NMPK solutions. The stable rate of change in absorbance is the result of the spontaneous hydrolysis of the relatively labile CMP Neu5Ac substrate. Once a stable background rate is established (20-30 min), 4 µl of transferase enzyme solution was added to each cuvette and the solutions were mixed thoroughly to initiate the assay. The concentration of enzyme was chosen such that the rate of chance in absorbance resulting from transferase activity is constant for a period of more than 10 minutes. The initial rate of transferase activity (µm min. -1 ) is calculated as follows (((Slope Background) /2) / 6220 M -1 cm -1 ) x A simple TLC assay was used to monitor the sialylation of bodipy-labeled acceptor substrates. Direct comparison of reactions carried out in the presence of EDTA S9

11 with those carried out in the presence of Mn 2+ or Mg 2+ revealed no significant rate differences. Synthesis 2-Nitrophenyl 2-acetamido-2-deoxy-(b-D-galactopyranosyl)-(1 3)-a-Dgalactopyranoside. 2-Nitrophenyl 2-acetamido-2-deoxy-(β-D-galactopyranosyl)-(1 3)-α-Dgalactopyranoside 2-nitrophenyl 2-acetamido-2-deoxy-α-D-galactopyranoside (5.8 mg; 17 µmol; 5.6 mm in reaction) and UDP-galactopyranose, disodium salt (11.8 mg; 19 µmol; 6.3 mm in reaction) were combined as solids and dissolved in 2290 µl of deionized water. To this solution were added sequentially NaOAc, ph 6.0 (300 µl of 500 mm; 50 mm in reaction), MnCl 2 (300 µl of 100 mm; 10 mm in reaction), DTT (9 µl of 325 mm; 1 mm in reaction), recombinant β1,3-galactosyltransferase (CgtB OH4384 ΔC30- C-term MalE; 100 µl of 5 mg ml -1 in 50 mm NaOAc, ph 6.0), 23 and alkaline phosphatase (Sigma, 0.5 µl; 65 units). After 24 hours, the reaction mixture was applied to a plus tc18 SepPak cartridge (1g; Waters), prewashed with MeOH (8 ml) and equilibrated with water (15 ml). Elution was performed with water (20 ml), followed by a gradient of methanol in water. The title compound (6.9 mg; 14 µmol; 81%) eluted at 25% MeOH. 1 H NMR (300MHz, D 2 O, δ) 7.84 (dd, 1H, Ar, J = 1.6, 8.2 Hz), 7.54 (ddd, 1H, Ar, J = 1.5, 7.6, 8.5 Hz), 7.35 (d, 1H, Ar, J = 8.3 Hz), 7.13 (dt, 1H, Ar, J = 0.7, 7.6 Hz), 5.75 (d, 1H, H-1, J 1,2 = 3.7 Hz), 4.46 (d, 1H, H-1, J 1,2 = 7.5 Hz), 4.43 (dd, 1H, H-2, J 2,3 = 11.3 Hz), 4.23 (d, 1H, H-4, J 4,3 = 2.9 Hz), 4.12 (dd, 1H, H-3), 3.98 (dd, 1H, H-5 or H-5, J = 4.6, 6.1 Hz), 3.82 (d, 1H, H-4, J 4,3 = 3.2 Hz), (m, 5H, H-6a, H-6b, H-6a, H-6b, H-5 or H-5 ), 3.54 (dd, 1H, H-3, J 3,2 = 9.9 Hz), 3.43 (dd, 1H, H-2 ), 1.91 (s, 3H, NHAc). 13 C NMR (75 MHz, D 2 O, δ) (C=O), 148.9, 140.2, 135.1, 125.8, 123.1, 118.3, 104.8, 97.0, 77.2, 75.1, 72.7, 72.4, 70.8, 68.7, 68.5, 61.08, 61.06, 48.5, HRMS calc d for C 20 H 28 N 2 O 13 Na: Found: References for supporting online material 1. Chiu, C.P. et al. Nat Struct Mol Biol 11, (2004). 2. Kleywegt, G.J. Acta Crystallogr D Biol Crystallogr 55, (1999). 3. Bond, C.S. Bioinformatics 19, (2003). 4. Ni, L. et al. Biochemistry 46, (2007). 5. Pak, J.E. et al. J Biol Chem 281, (2006). 6. Bond, C.S. & Schuttelkopf, A.W. Acta Crystallogr D Biol Crystallogr 65, (2009). 7. Jeanneau, C. et al. J Biol Chem 279, (2004). 8. Vallejo-Ruiz, V. et al. Biochim Biophys Acta 1549, (2001). 9. Kono, M. et al. Glycobiology 7, (1997). 10. Rearick, J.I., Sadler, J.E., Paulson, J.C. & Hill, R.L. J Biol Chem 254, (1979). 11. Johnson, K.F., Bezila, D., Ngo, W., and Hakes, D PCT patent application publication WO2005/ Studier, F.W. Protein Expr Purif 41, (2005). 13. Doublie, S. Methods Mol Biol 363, (2007). 14. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, (1994). 15. Terwilliger, T.C.. Acta Crystallogr D Biol Crystallogr 55, (1999). 16. Terwilliger, T.C. Acta Crystallogr D Biol Crystallogr 59, (2003). 17. Perrakis, A., Morris, R. & Lamzin, V.S. Nat Struct Biol 6, (1999). 18. Emsley, P. & Cowtan, K. Acta Crystallogr D Biol Crystallogr 60, (2004). 19. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Acta Crystallogr D Biol Crystallogr 53, (1997). 20. Adams, P.D. et al. Acta Crystallogr D Biol Crystallogr 58, (2002). 21. Schuttelkopf, A.W. & van Aalten, D.M. Acta Crystallogr D Biol Crystallogr 60, (2004). 22. Gosselin, S., Alhussaini, M., Streiff, M.B., Takabayashi, K. & Palcic, M.M. Anal Biochem 220, 92-7 (1994). 23. Bernatchez, S. et al. Glycobiology 17, (2007). S10

12 Acknowledgments We thank the staff at the Advanced Light Source beamline and the Canadian Light Source Inc. ID08-1 for data collection time and assistance. We thank the Canadian Institutes for Health Research (CIHR) for financial support. N.C.J.S. also thanks the Howard Hughes Medical Institute (HHMI) International Scholar program and the Michael Smith Foundation for Health Research (MSFHR) for operational and salary support, as well as the Canada Foundation for Innovation (CFI), the MSFHR and the British Columbia Knowledge Development Fund (BCKDF) for infrastructure funding. S.G.W. thanks the Canada Research Chairs Program for salary support. F.V.R. is supported by long-term European Molecular Biology Organization (EMBO) and MSHFR fellowships. J.R.R is supported by a MSHFR fellowship. Author contributions F.V.R. performed purification, crystallization and structure determination; J.R.R. synthesized acceptor sugars; B.R. performed the enzymology; S.B., M.F.S., K.J., C.B. and S.D performed construct design and purification; F.V.R., J.R.R., S.G.W. and N.C.J.S. analyzed the data and wrote the manuscript; F.V.R. made the figures; W.W.W. provided feedback on the manuscript. S11