Crystal Structures of Sialyltransferase from Photobacterium Damselae

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Title Crystal structures of sialyltransferase from Photobacterium damselae.

Permalink https://escholarship.org/uc/item/5gm463dj

Journal FEBS letters, 588(24)

ISSN 1873-3468

Authors Huynh, Nhung Li, Yanhong Yu, Hai et al.

Publication Date 2014-12-20

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California FEBS Letters 588 (2014) 4720–4729

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Crystal structures of sialyltransferase from Photobacterium damselae q ⇑ Nhung Huynh c,1,3, Yanhong Li a,3, Hai Yu a, Shengshu Huang a, Kam Lau a,2, Xi Chen a, , ⇑ Andrew J. Fisher a,b, a Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA b Department of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616, USA c Cell Biology Graduate Program, University of California, One Shields Avenue, Davis, CA 95616, USA article info abstract

Article history: Sialyltransferase structures fall into either GT-A or GT-B glycosyltransferase fold. Some sialyltransfe- Received 2 October 2014 rases from the Photobacterium genus have been shown to contain an additional N-terminal immu- Revised 7 November 2014 noglobulin (Ig)-like domain. Photobacterium damselae a2–6-sialyltransferase has been used Accepted 7 November 2014 efﬁciently in enzymatic and chemoenzymatic synthesis of a2–6-linked sialosides. Here we report Available online 15 November 2014 three crystal structures of this enzyme. Two structures with and without a donor substrate analog Edited by Richard Cogdell CMP-3F(a)Neu5Ac contain an immunoglobulin (Ig)-like domain and adopt the GT-B sialyltransferase fold. The binary structure reveals a non-productive pre-Michaelis complex, which are caused by crystal lattice contacts that prevent the large conformational changes. The third structure lacks Keywords: a2–6-Sialyltransferase the Ig-domain. Comparison of the three structures reveals small inherent ﬂexibility between the Protein crystal structure two Rossmann-like domains of the GT-B fold. Sialic acid Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. CMP-3F(a)Neu5Ac Photobacterium damselae

1. Introduction to function as key recognition sites that can be used for multi-pur- poses by cells such as modulating cellular interactions or masking Sialic acids are a-keto acids usually found as the terminal car- molecules for invasion of host systems [2,3]. The important roles of bohydrate moieties attached to cell surface glycoconjugates of sialic acids in numerous pathological and physiological processes, higher eukaryotes [1]. The terminal position allows the sialic acids including tumor metastasis and bacterial and viral infections, make sialyltransferases, the key enzymes involved in the biosynthesis of Abbreviations: CMP-3F(a)Neu5Ac, cytidine 50-monophosphate-3-fluoro(axial)- sialic acid-linked glycans, potential therapeutic targets [4]. N-acetylneuraminic acid; CMP-Neu5Ac, cytidine 50-monophosphate-N-acetylneu- Sialyltransferases are enzymes that catalyze the transfer of sia- raminic acid; GalNAc, Gal, N-acetylgalactosamine; EDTA, ethylenediaminetetraace- lic acid from its activated sugar nucleotide form, cytidine 50-mono- tic acid; EGTA, ethylene glycol tetraacetic acid; GlcNAc, N-acetylglucosamine; phosphate sialic acid (CMP-sialic acid or CMP-Sia), to the terminal Neu5Ac, N-acetylneuraminic acid; Gal, galactose; Ig, immunoglobulin; Sia, sialic acid; D15Pd2,6ST(N), Photobacterium damselae a2–6-sialyltransferase lacking the positions of glycans on glycoproteins and glycolipids [2,5]. Sial- N-terminal 15 residues and the C-terminal 178 residues; D112Pd2,6ST(N), Photo- yltransferases can link sialic acids either through an a2–3- or an bacterium damselae a2–6-sialyltransferase lacking the N-terminal 112 residues and a2–6-bond to galactose (Gal), an a2–6-bond to N-acetylgalactos- the C-terminal 178 residues; D16Psp26ST, Vibrionaceae Photobacterium sp. JT-ISH- amine (GalNAc) or N-acetylglucosamine (GlcNAc), or an a2–8/9- 224 a2–6-sialyltransferase lacking the N-terminal 16 residues bond to another sialic acid to form polysialic acid. Some bacterial q Protein coordinates have been deposited in the Protein Data Bank [IDs: 4R83 sialyltransferases also display multifunctionality. For example, (ligand-free D15Pd2,6ST(N)), 4R84 (D15Pd2,6ST(N) with CMP-3F(a)Neu5Ac) and 4R9V (ligand-free D112Pd2,6ST(N)]. Pasteurella multocida sialyltransferase 1 (PmST1) has optimal activ- ⇑ Corresponding authors at: Department of Chemistry, University of California, ities as an a2–3-sialyltransferase at pH 7.5–9.0, an a2–6-sialyl- One Shields Avenue, Davis, CA 95616, USA. Fax: +1 530 752 8995 (A.J. Fisher). transferase at pH 4.5–6.0, an a2–3-sialidase at pH 5.0–5.5 and an Fax: +1 530 752 8995 (X. Chen). a2–3-trans-sialidase at pH 5.5–6.5 [6]. Multifunctionalities includ- E-mail addresses: [email protected] (X. Chen), ajfi[email protected] (A.J. ing trans-sialidase and sialidase activities in addition to sialyltrans- Fisher). 1 Current address: The Scripps Research Institute, Department of Integrative ferase activities have also been found for Campylobacter jejuni Structural and Computational Biology, La Jolla, CA 92037, USA. sialyltransferase CstII [7] and Photobacterium damselae a2–6-sialyl- 2 Current address: Institute for Glycomics, Gold Coast Campus, Griffith University, transferase Pd2,6ST [8]. Investigation of the structures of sial- Queensland 4222, Australia. yltransferases will hence contribute to further understanding of 3 Co-first authors. These authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.febslet.2014.11.003 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729 4721 the detailed mechanism and various functions of this important centrifuge with a swinging bucket rotor at 4000g for 2 h. The cell class of enzymes. pellet was resuspended in 20 mL/L cell culture in lysis buffer (pH Among the sialyltransferases known to date, crystal structures 8.0, 100 mM Tris–HCl containing 0.1% Triton X-100) supplemented of representative members of Carbohydrate Activated enZyme with lysozyme (1 mg/L culture) and DNaseI (50 lg/L culture) and (CAZy, http://www.cazy.org) [9,10] sialyltransferases from families incubated at 37 °C for 50 min with shaking (125 rpm). The cell GT29, GT42, GT52, and GT80, have been reported. Based on their lysate was collected by centrifugation (Sorvall RC-5B centrifuge protein sequence similarity, all mammalian sialyltransferases are with a S5–34 rotor) at 12000g for 30 min and the lysate was grouped into CAZy GT29 family. The crystal structures of two applied to a Ni2+-NTA affinity column to purify the target protein. members of this mammalian sialyltransferase family, including After loading, the Ni2+ column was washed with 10 column vol- porcine ST3Gal I [11] and rat ST6Gal I [12], have been reported. umes of binding buffer (5 mM imidazole, 0.5 M NaCl, and 20 mM Among bacterial sialyltransferases, the structures of a multifunc- Tris–HCl, pH 7.5), 15 column volumes of washing buffer tional C. jejuni a2–3/8-sialyltransferase Cst-II [13] and C. jejuni (20–40 mM imidazole, 0.5 M NaCl, and 20 mM Tris–HCl, pH 7.5), a2–3-sialyltransferase Cst-I [14] belonging to CAZy GT42 family followed by 8 volumes of elute buffer (200 mM imidazole, 0.5 NaCl, and Neisseria meningitidis lipopolysaccharide a2–3-sialyltransfer- 20 mM Tris/HCl, pH 7.5). The fractions containing the purified ase [15] belonging to CAZy GT52 family have been solved by enzyme were collected. The combined sample was dialyzed against X-ray crystallography. In addition, the protein crystal structures Tris–HCl buffer (20 mM, pH7.5), and stored at 4 °C. of several GT80 family bacterial sialyltransferases, including PmST1 [2,16,17], Vibrionaceae Photobacterium sp. JT-ISH-224 2.2. Crystallization of D15Pd2,6ST(N) and D112Pd2,6ST(N) a2–6-sialyltransferase (Psp26ST) [18], and Photobacterium phosphoreum a/b-galactoside a2–3-sialyltransferase (Pp23ST) D15Pd2,6ST(N) and D112Pd2,6ST(N) were concentrated to [19] have been reported. However, structures have not been solved 10 mg/mL using centrifugal filter units (EMD Millipore, Billerica, for polysaccharide synthases catalyzing the formation of sialic MA, USA) and crystallized by hanging drop vapor diffusion in a acid-containing polysaccharides in mammals and bacteria, such 1:1 ratio of protein and reservoir solution at 21 °C. The reservoir as mammalian a2–8-polysialyltransferases belonging to GT29, N. solution for D15Pd2,6ST(N) contained PEG6000 (20%, w/v), NaCl meningitidis serogroups B and C and Escherichia coli K-1 and K-92 (0.2 M), and HEPES buffer (0.1 M, pH 7.0). The reservoir solution a2–8/9-polysiayltransferases belonging to GT38, and capsular for D112Pd2,6ST(N) contained PEG1000 (20%, w/v), Ca(OAc)2 polysaccharide synthases of N. meningitidis serogroups W135 and (0.2 M), and imidazole (0.1 M) at pH 8.0. D15Pd2,6ST(N) binary Y belonging to CAZy GT4 family [20]. structure with CMP-3F(a)Neu5Ac (‘a’ stands for the axial position Among solved sialyltransferase crystal structures, members of the fluorine at carbon 3) was obtained by soaking with belonging to GT29, GT42, and GT52 families all have one single 1.25 mM of CMP-3F(a)Neu5Ac (final concentration) for 30 min Rossmann domain and fall into glycosyltransferase GT-A or prior to cryocooling and data collection. The CMP-3F(a)Neu5Ac GT-A-like structures [21,22]. In contrast, crystal structures of analog was synthesized as previously described [16,28]. The crys- GT80-family sialyltransferases identified so far belong to glycosyl- tals were placed in a reservoir solution containing 20% ethylene transferase GT-B type, which contain two Rossmann-like domains. glycol (for both crystals of D15Pd2,6ST(N)) or Paratone-N oil (for Here we present the structures of a2–6-sialyltransferase from P. crystal of D112Pd2,6ST(N)) and flash cooled in liquid nitrogen damselae, D15Pd2,6ST(N) [23] in the ligand-free form and com- prior to data collection. plexed with cytidine 50-monophosphate-3-fluoro(axial)-N-acetylneuraminic acid [CMP-3F(a)Neu5Ac] as well as D112Pd2,6ST(N) 2.3. Data collection, structure determination, model building, and in the ligand-free form resolved to resolutions of 1.93 Å, 1.70 Å, refinement and 2.30 Å, respectively. Pd2,6ST is an a2–6-sialyltransferase that catalyzes the transfer of sialic acid from the activated sugar nucle- X-ray diffraction data for the crystals were collected at Stanford otide donor CMP-Sia to the Gal or the GalNAc in an acceptor [24].It Synchrotron Radiation Lightsource (SSRL) beam line 7-1 at 100 K. was the first bacterial a2–6-sialyltransferase that was cloned [25]. The SSRL data were indexed and integrated with MOSFLM [29], Pd2,6ST has weaker a2–6-trans-sialidase and a2–6-sialidase activ- and then scaled with SCALA [30]. A complete data set for the ities [8] and is grouped to the CAZy GT80 family (EC 2.4.99.1). The ligand-free D15Pd2,6ST(N) structure was collected to a resolution structures presented here of D15Pd2,6ST(N) and D112Pd2,6ST(N) of 1.93 Å (Table 1). Soaking the crystal with the non-hydrolyzable can provide valuable information for developing potential drugs analog of CMP-3F(a)Neu5Ac actually improved the resolution of and generating mutants for chemoenzymatic synthesis of oligosac- the crystal. A complete data set was collected to 1.70 Å resolution charides [26,27]. (Table 1). Finally, the D112Pd2,6ST(N) crystal only diffracted to 2.30 Å resolution for collecting a complete data set (Table 1). 2. Materials and methods The D112Pd2,6ST(N) ligand-free structure was obtained by molecular replacement using a previously solved sialyltransferase 2.1. Expression and purification of D15Pd2,6ST(N) and structure from Vibrionaceae Photobacterium sp. JT-ISH-224 D112Pd2,6ST(N) (D16Psp26ST) (PDBID: 2Z4T) [18]. The program PHASER [31] as a part of the PHENIX suite [32] was used for molecular replacement Cloning, expression, and purification were performed as previ- by splitting the GT-B domain of D16Psp26ST structure into two ously reported [23]. For expression, E. coli BL21 (DE3) cells contain- separate Rossmann-like search domains. The D15Pd2,6ST(N) ing recombinant plasmid in pET15b vector were cultured in LB-rich structure was also solved by molecular replacement using the medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) sup- two Rossmann-like domains of the D112Pd2,6ST(N) structure plemented with 100 lg/mL ampicillin. The cells were grown to and the N-terminal Ig-like domain from D16Psp26ST (PDBID:

OD600 nm = 0.8–1.0, induced with 0.1 mM of isopropyl-1-thio-b-D- 2Z4T). The atomic model building was carried out with the molec- galactopyranoside (IPTG) for over-expression of the target protein, ular graphics program COOT [33]. The structures were reﬁned and incubated at 20 °C with shaking at 250 rpm in a C25KC incuba- using the program PHENIX [34] using 95% of the measured data tor shaker (New Brunswick Scientiﬁc, Edison, NJ) for 24 h. The cells as a target function. Non-crystallographic restraints (for were harvested by centrifugation at 4 °C in a Sorvall Legend RT D15Pd2,6ST(N)) and TLS parameters [35] were included during 4722 N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729

Table 1 Data collection and reﬁnement statistics.

D15Pd2,6ST(N) ligand-free D15Pd2,6ST(N) soaked w/Neu5Ac-3F(a)-Neu5Ac D112Pd2,6ST(N) ligand-free X-ray source SSRL BL 7-1 SSRL BL 7-1 SSRL BL 7-1 Wavelength (Å) 0.97946 0.97946 0.97607 Space group P1 P1 C2 Cell parameters a = 64.7 Å a = 64.3 Å a = 95.4 Å b = 81.3 Å b = 81.4 Å b = 56.5 Å c = 111.8 Å c = 111.2 Å c = 87.4 Å a = 98.60° a = 99.02° a =90° b = 92.76° b = 91.59° b = 119.95° c = 100.85° c = 101.20° c =90° Monomers/ASU 4 4 1 3 VM (Å /Da) 2.53 2.50 2.30 Percent solvent 51.4% 50.9% 46.5% Resolution (Å) 1.93 (1.98–1.93) 1.70 (1.79–1.70) 2.30 (2.36–2.30) No of reflections 311576 (23059) 455871 (61700) 63335 (4671) No. of unique reflections 156324 (11570) 229782 (31361) 17873 (1287) Completeness (%) 94.2 (94.0) 95.7 (89.3) 99.1 (99.2) a Rmerge (%) 2.9 (39.7) 4.2 (31.5) 6.7 (32.0) I/r 18.4 (2.16) 8.8 (2.2) 16.9 (3.6) Refinement statistics Resolution (Å) 1.93 1.70 2.30 No. of reflections (F > 0) 156284 229738 17868 b Rf (%) 18.3 17.1 18.4 c Rfree (%) 22.4 20.0 24.8 RMS bond length (Å) 0.008 0.007 0.008 RMS bond angle (°) 1.123 1.091 1.075 Wilson B-factor (Å2) 31.9 23.8 38.3 Refined average B-factor (Å2) 45.0 33.1 44.3 Ramachandran plot statistics Most favorable (%) 1851 (98.1%) 1850 (98.0%) 372 (96.9%) Allowed (%) 31 (1.6%) 34 (1.8%) 12 (3.1%) Disallowed (%) 5 (0.3%) 4 (0.2%) 0 (0.0%) Asymmetric unit content Non-hydrogen protein atoms 15261 15274 3099 Calcium ions 4 4 1 CMP-3F(a)Neu5Ac atoms 0 168 (4 molecules) 0 Waters 1395 2441 126 PDB ID 4R83 4R84 4R9V P P P P a Rmerge =[ h Pi|Ih Ihi|/ h iIhiP] where Ih is the mean of Ihi observations of reflection h. Numbers in parenthesis represent highest resolution shell. b c R and Rfree = ||Fobs| |Fcalc||/ |Fobs| 100 for 95% of recorded data (R) or 5% data (Rfree).

refinement. The final R and R-free for all three structures, along 178 residues, which constitute a PhoU domain. The PhoU domain is with the quality of the model based on PROCHECK [36] are listed typically involved in phosphate regulation, specifically in regulat- in Table 1. ing phosphate uptake [37]. The role or function of this domain at the C-terminal end of the sialyltransferase in P. damselae is 2.4. Effects of ethylene glycol tetraacetic acid (EGTA) and Ca2+ on the unknown. Both constructs also include 21 additional residues at a2–6-sialyltransferase activity of D15Pd2,6ST(N) the N-terminus corresponding to a His6-Tag, which was not cleaved prior to crystallization. Because the D15Pd2,6ST(N) con-

EGTA (10 mM), CaCl2 (10 mM), or the mixture of EGTA (15 mM) struct was more complete, diffracted to higher resolution, and sub- and CaCl2 (5 mM) were used in Tris–HCl buffer (100 mM, pH 8.5) strate could be soaked into the active site, much of the discussion containing cytidine 50-monophosphate-N-acetylneuraminic acid will be focused on the structures of this construct. (CMP-Neu5Ac) (1 mM) and LacbMU (1 mM) to analyze their effects Crystals of D112Pd2,6ST(N) were grown in a couple of days at on the a2–6-sialyltransferase activity of D15Pd2,6ST(N) (16.5 ng/ room temperature. They belong to the monoclinic space group C2 lL). A reaction without EGTA or CaCl2 was used as a control. Reac- and contain one monomer in the asymmetric unit. Residues 112– tions were allowed to proceed for 15 min at 37 °C followed by 497 of this crystal structure are well ordered in the electron den- quenching with ice-cold acetonitrile (12%, v/v) and tested by high sity map (residue 112 is from the pET15b vector). Crystals of the performance liquid chromatography (HPLC) [23]. larger D15Pd2,6ST(N) construct were also grown in a few days, but were crystallized in the triclinic space group P1. The 3. Results and discussion D15Pd2,6ST(N) crystals contain four monomers per asymmetric unit, with each monomer consisting of ordered residues 24–497 3.1. Overall native structure organized into three separate domains (Fig. 1A). In each monomer, the first domain (residues 24–111, Ig-like domain) is the smallest Two soluble Pd2,6ST constructs, D15Pd2,6ST(N) and domain and adopts the Ig-like beta-sandwich fold that has been D112Pd2,6ST(N) deleting the first 15 and 112 residues respec- found in some members of the glycosyl hydrolase family tively, were expressed and crystallized. Residues 1–15 contain a (Fig. 1B) [38]. The Ig-like domain was also found in D16Psp26ST, putative membrane anchor, and residues 24–111 contain an Ig-like where it might play a regulatory role because deleting the Ig-like domain of unknown function. Both constructs lack the C-terminal domain increased the specific activity of the sialyltransferase by N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729 4723

Fig. 1B. A ribbon representation of the D15Pd2,6ST(N) monomer. The N-terminal Ig-like domain is at the bottom and is colored red, while the two-domain GT-B fold is at the top. The N-terminal GT-B domain is colored green (dark green for b-strands and light green for a-helices), while the C-terminal domain is colored blue (dark blue for b-strands and light blue for a-helices). The bound CMP-3F(a)Neu5Ac is represented by space ﬁlling spheres between the two Rossmann domains of the GT- B fold. The calcium ion is shown as a brown-colored sphere and the disulﬁde bond in the Ig-like domain is illustrated as sticks colored yellow.

biological relevance of this interaction remains to be determined Fig. 1A. The composition of a crystallographic asymmetric unit. The structure of a for P. damselae. D15Pd2,6ST(N) crystal soaked with CMP-3F(a)Neu5Ac is shown here in triclinic For the second and third domains of D15Pd2,6ST(N) (residues space group P1 with four monomers per asymmetric unit. Each monomer is colored 112–334 named as N-terminal GT-B domain and 335–497 named differently, the calcium ions are illustrated as a brown-colored spheres and the as C-terminal GT-B domain, respectively, equivalent to the bound CMP-3F(a)Neu5Ac molecules are shown as space ﬁlling spheres with white- colored carbon atoms. The ﬂuorine atoms are colored light blue. D112Pd2,6ST(N) construct), each contains an a/b/a fold resem- bling the Rossmann nucleotide-binding domain. Together, the domains form a structure found in the glycosyltransferase B (GT- twofold [18]. In contrast, deleting the Ig-like domain in the B) superfamily, which is composed of two Rossmann domains sep-

D112Pd2,6ST(N) actually decreased kcat/KM about twofold, mostly arated by a deep cleft that forms a substrate-binding site (Fig. 1B) by decreasing the kcat value [23]. [39,40]. In the D15Pd2,6ST(N) and D112Pd2,6ST(N) structures, the The Ig-like beta-sandwich domain contains a four-stranded N-terminal GT-B domain (residues 112–334) contains a central anti-parallel b-sheet on one side (topology b1–b6–b5–b3), covered seven-stranded parallel b-sheet (dark green, Topology b9-b8-b7- by a two-stranded anti-parallel b-sheet (b2–b4) and a single short b10-b11-b12-b13) flanked by seven a-helices on one side and four a-helix (between b3 and b4) on the other side of the beta-sand- helices (light green) on the other side of the central b-sheet. The wich (Fig. 1B). A disulfide bond is formed between Cys60 and C-terminal GT-B domain (residues 335–497) is a smaller Rossmann Cys89 on b3 and b5 respectively, which is also observed in the fold composed of a central six-stranded parallel b-sheet (dark blue, Ig-domain of the D16Psp26ST structure. Topology b16–b15–b14–b17–b18–b19) flanked by two a-helices In the D15Pd2,6ST(N) structure, the Ig-like domains from two on one side and five a-helices (light blue) on the other side. The adjacent monomers form a tight crystal contact across a non-crys- CMP-3F(a)Neu5Ac substrate analog interacts more with this tallographic 2-fold axis. The b1 strands from two monomers adopt C-terminal domain (see below). a dimeric anti-parallel b-sheet interaction resulting in a continuous Overall, the structure of D15Pd2,6ST(N) has a similar architec- eight-stranded b-sheet across the two Ig-like domains (Fig. 2A). ture to sialyltransferases from Photobacterium sp. JT-ISH-224 [18] This contact is observed between both the A–B monomers and and Photobacterium leiognathi [41,42], which contain a membrane the C–D monomers within the crystallographic asymmetric unit anchor, an N-terminal Ig-like domain, followed by a GT-B sialyl- (Fig. 1A). The b1 strand from each monomer is situated at the transferase domain. It is interesting to point out that the P. damse- non-crystallographic 2-fold axis where residues 24–29 formed lae sialyltransferase also contains a C-terminal PhoU domain that six main-chain hydrogen bonds together with two side-chain shares amino acid sequence similarity to phosphate regulatory hydrogen bonds between Thr26 and Ser28 (Fig. 2B). This Ig-domain proteins [43]. The D112Pd2,6ST(N) structure lacks the Ig-like dimerization is not observed for the D16Psp26ST structure, so the domain and simply contains the GT-B fold. Nevertheless, its 4724 N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729

Fig. 2B. A close-up view of the non-crystallographic 2-fold illustrating the dimer contacts between the Ig-like domains of the A and B-subunits in the asymmetric unit. Yellow dashed lines show hydrogen bonds with distances in Ångstroms shown in black texts. Six main-chain hydrogen bonds are formed between the side chains of Thr26 and Ser28 across the dimers.

Fig. 2A. Dimeric contacts between two D15Pd2,6ST(N) monomers. A- and B-chain D15Pd2,6ST(N) monomers in the asymmetric unit are shown with the Ig-like domain mediating a dimeric contact. The ﬁrst strand of the Ig-like domain forms b- strand interaction across a non-crystallographic 2-fold resulting in a continuous eight-stranded b-sheet dimer interaction. A similar association is observed for the C- and D-chains in the asymmetric unit. structure and conformation are similar to those found in the D15Pd2,6ST(N) structure (see below).

3.2. Metal binding site

Fig. 3. The calcium binding site. A strong electron density is modeled as a calcium Strong electron density is observed at the loop between the first ion in the loop between the first helix and the b-strand of the C-terminal Rossmann a-helix of the C-terminal domain and b14 in all four monomers domain of the GT-B fold. Yellow dashed lines show coordination interactions with within the asymmetric unit of the D15Pd2,6ST(N) structures and distances in Ångstroms shown in black text. Water molecules are shown as small in the D112Pd2,6ST(N) structure. This density is consistent with red-colored spheres. The geometry of coordination is pentagonal bipyramidal. The calcium ion (shown as a brown sphere) is observed in all four monomers in the a divalent metal ion, which we modeled as a calcium ion based asymmetric unit for both the D15Pd2,6ST(N) ligand-free and the CMP- on results from the metal identification feature in PHENIX [44], 3F(a)Neu5Ac-bound structures as well as in the D112Pd2,6ST(N) structure. and the CheckMyMetal web server [45]. The Ca2+ ion is heptavalent and displays a pentagonal bipyramidal geometry. The ion is coordi- nated by main-chain oxygen atoms of Tyr345, Ser348, and Leu350, purification process. A similar divalent metal ion was also observed along with side chain oxygens of Asn352 and Asp395, and two in the D16Psp26ST structure, which was modeled as a Mg2+ ion water molecules (Fig. 3). All but one of the residues that coordinate [18]. The Ca2+ ion in D15Pd2,6ST(N) is not near the active site the Ca2+ ion come from the loop preceding strand b14. Asp395 is and may play a structural role. Nevertheless, the enzyme still dis- the first residue of neighboring strand b15. played activity in the presence of ethylenediaminetetraacetic acid As no divalent metal ions were included in the crystallization of (EDTA) [23]. Enzymatic activity assays in the presence of 10 mM of D15Pd2,6ST(N), the Ca2+ ion may be retained through protein ehtylene glycol tetraacetic acid (EGTA), which is more selective N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729 4725 than EDTA for calcium ions, revealed that D15Pd2,6ST(N) still retained more than 92% activity (data not shown), suggesting the ion is not crucial for sialyltransferase activity.

3.3. D15Pd2,6ST(N)—CMP-3F(a)Neu5Ac binary structure

Previous co-crystallization studies of sialyltransferases with their sugar nucleotide donor substrate, CMP-Neu5Ac, only led to the observation of CMP bound in the active site, due to the cleavage of Neu5Ac during crystallization [2,13]. To prevent the hydrolysis of the donor sugar to identify amino acid residues that may interact with the Neu5Ac moiety, inert analogs of CMP-Neu5Ac, such as CMP-3F(a)Neu5Ac, have been synthesized and used in crystallization. An electronegative fluorine substitution at the C-3 of Neu5Ac in CMP-Neu5Ac destabilizes the oxocarbenium ion-like transition state for the reaction and hence slows down the enzyme turnover rate. Previously, sialyltransferases from P. multocida and C. jejuni have been co-crystallized with CMP-3FNeu5Ac to identify residues involved in binding of the nucleotide and the sugar components of the sialyltransferase donor [14,16]. Here, crystals of P. damselae D15Pd2,6ST(N) were soaked with CMP-3F(a)Neu5Ac and a data set was collected (Table 1). The resulting CMP-3F(a)Neu5Ac-soaked structure (Fig. 4A) Fig. 4B. The substrate analog CMP-3F(a)Neu5Ac binding site of D15Pd2,6ST(N). The reveals clearly defined electron density of CMP-3F(a)Neu5Ac in a protein is illustrated as a cartoon. The side chain and the main chain atoms of catalytic site between the two nucleotide binding domains of all important residues are drawn as sticks. CMP-3F(a)Neu5Ac is drawn as sticks with four monomers within the crystallographic asymmetric unit. The white-colored carbon atoms with some atoms labeled in red text. Yellow dashes structure (Fig. 4B) identifies similar interactions between the show potential hydrogen bonds between the substrate analog and the protein or enzyme and the substrate as seen in previous reported structures ordered water molecules (red spheres). The fluorine atom in the sialic acid is colored cyan. Trp361 is labeled. It has been observed to swing down and interact complexed with a CMP moiety [18]. The CMP moiety interacts with the sialic acid in other sialyltransferases. almost exclusively with the C-terminal nucleotide-binding domain through a total of ten hydrogen bonds. The cytosine base interacts through N4 with two hydrogen bonds to the main-chain carbonyl hydrogen-bonding interactions with the base have also been oxygens of residues Gly357 and Lys399. The side chain amine observed in the previous CMP-bound structures belonging to the group of Lys399 hydrogen bonds to O2 of the cytosine ring. These GT-B group [2,18,19]. Additionally, as seen in other GT-B sialyltransferase structures with CMP bound, a conserved Glu side chain makes a bidentate interaction with both the 20 and 30 hydroxyls of the ribose ring (Fig. 4B). The phosphate group hydrogen bonds to main chain nitrogen of Ser446 and side chain of Ser445. Surprisingly, conserved His401 that normally interacts with the CMP phosphate group in other sialyltransferases [16], is seen to form an ion-pair with the carboxylate group of the sialic acid moiety. Also unexpectedly, conserved Arg150 of the N-terminal domain, which typically ion-pairs with the sialic acid carboxylate group [16], interacts with the 20-OH of the ribose ring here in the D15Pd2,6ST(N) structure, and represents the only interaction between CMP-3F(a)Neu5Ac and the N-terminal Rossmann domain. For the sialic acid moiety of CMP-3F(a)Neu5Ac in the D15Pd2,6ST(N) structure, the majority of contacts are with ordered water molecules (Fig. 4B). Only O8 and O9 of the sialic acid are in hydrogen bonding distance to carbonyl oxygen of Ala444 of the C-terminal domain, and no interactions are observed to the N-terminal Rossmann domain. These unique interactions are likely due to the binding of CMP-3F(a)Neu5Ac to the sialyltransferase in an open conformation. Because CMP-3F(a)Neu5Ac was soaked into ligand-free D15Pd2,6ST(N) crystals to obtain this binary structure, crystal packing and numerous lattice contacts, especially between the C-terminal domain of the GT-B fold, impedes the substrate- induced large conformational changes seen in other sialyltransfe-

Fig. 4A. The original electron density map around the donor analog after soaking rases that were obtained by co-crystallization with substrate D15Pd2,6ST(N) crystals with CMP-3F(a)Neu5Ac prior to data collection. The analogs [16,18]. We have previously shown large conformational electron density is calculated from the original molecular replacement solution changes in PmST1 upon binding to CMP and CMP-3F(a)Neu5Ac using the three ligand-free structure domains as search models. The electron [2,16]. In PmST1, distances between loops that close in over the density is contoured at 1r displayed with the ﬁnal reﬁned structure. Similar substrate-binding site move from 30 Å separation to 15 Å density is seen for all four active sites in the asymmetric unit. CMP-3F(a)Neu5Ac is shown with white-colored carbon atoms. Fluorine and phosphorous atoms are [2,16]. The equivalent loop distances in the D15Pd2,6ST(N) move colored cyan and orange respectively. only from 27 Å apart in the ligand-free structure to 25 Å in 4726 N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729

Fig. 5B. A superposition of the D15Pd2,6ST(N) structure (green) onto V. Photobac- Fig. 5A. A superposition of all four monomers of the CMP-3F(a)Neu5Ac-bound terium sp. JT-ISH-224 sialyltransferase (D16Psp26ST) (blue). The donor substrate D15Pd2,6ST(N) structures seen in the triclinic P1 asymmetric unit. Each monomer analog CMP-3F(a)Neu5Ac bound in D15Pd2,6ST(N) is shown in space ﬁlling spheres is colored differently and only the GT-B folds are aligned, illustrating a small shift in with green-colored carbon atoms. The CMP of the D16Psp26ST structure is not Ig-like domain orientations. The subunits GT-B domains are superimposed with shown for clarity, but lies very close to the CMP moiety of CMP-3F(a)Neu5Ac bound rmsd ranges 0.21–0.32 Å for 374 equivalent a-carbons. in D15Pd2,6ST(N). Only the N-terminal Rossmann domains of the GT-B fold are aligned (rmsd 0.95 Å) illustrating the differences in closure around the active site the CMP-3F(a)Neu5Ac-binary structure. Additionally, in the previ- (C-terminal Rossmann domain swings down as shown by arrow) upon binding ously reported structure of the homologous D16Psp26ST [18] activated donor. The D15Pd2,6ST(N) structure with CMP-3F(a)Neu5Ac bound resembles, more closely, other GT-B sialyltransferases in the ligand-free state (not which contains an N-terminal Ig-like domain as D15Pd2,6ST(N), shown). The alignment also illustrates the different orientations of the Ig-like CMP binding in the active site resulted in a closed conformation domains of two structures. of the enzyme where the distance between equivalent loops are 14 Å apart [18]. Thus, the D15Pd2,6ST(N) binary structure with CMP-3F(a)Neu5Ac bound presented here provides a unique This aspartate residue hydrogen bonds with the O3 of lactose in glimpse of sugar nucleotide donor binding prior to a conforma- the PmST1 ternary structure, leading to the formation of an tional change. This also suggests that the conformational change a2–3-linkage with sialic acid catalyzed by the enzyme. It also (closure of the two Rossmann domains) is not required for donor hydrogen bonds to the O6 of lactose in the D16Psp26ST ternary substrate binding. structure, allowing the formation of the a2–6-sialyl linkage cata- The previously determined sialyltransferase structures with lyzed by the enzyme. In the D15Pd2,6ST(N) structure here, nucleotide bound in the closed conformation also reveal a big Asp229 is over 10 Å away from the sialic acid anomeric carbon movement of a conserved tryptophan (Trp) residue of the C-termi- because the conformation of D15Pd2,6ST(N) is in the open state. nal domain. In the ligand-free structure, this Trp residue (Trp270 of Therefore, the D15Pd2,6ST(N) binary structure with CMP- PmST1) is buried in the hydrophobic core of the C-terminal Ross- 3F(a)Neu5Ac bound reported here likely represents a conformation mann domain. Upon binding to nucleotide, Trp270 residue pops of a pre-Michaelis complex structure when the donor substrate out of the core and swings down where the indole nitrogen hydro- binds prior to a conformational change. gen bonds with sialic acid, which helps to shape the acceptor sugar binding site [16]. In the D15Pd2,6ST(N) structure here, Trp361 3.4. Structural comparisons (equivalent to Trp270 of PmST1) remains buried in the C-terminal hydrophobic core (Fig. 4B), suggesting CMP-3F(a)Neu5Ac binding Superposition of the four monomer structures found in the alone is not enough to release Trp361. In the D16Psp26ST struc- triclinic asymmetric unit shows very similar structures with the ture, which is in the closed conformation with CMP and lactose largest differences coming from the relative orientation of bound, homologous Trp365 is released from the C-terminal core the N-terminal Ig-like domain with respect to the GT-B sialyltrans- and interacts with lactose [18]. ferase domain (Fig. 5A). Overall, the root-mean-square deviations The conformational closure upon binding to donor substrate (rmsd) between the four CMP-3F(a)Neu5Ac-bound structures does not only help to shape the acceptor binding site, but also posi- within the asymmetric unit range from 0.35 to 1.07 Å for all 474 tion catalytically important residues in close proximity to the a-carbons. Aligning only the GT-B domains of each monomer acceptor substrate to catalyze the sialic acid transfer. Speciﬁcally, reveals that the GT-B domains are more closely aligned with rmsd a conserved aspartic acid in the N-terminal domain serves as a cat- range 0.21–0.32 Å for 374 equivalent a-carbons. alytic base to deprotonate the acceptor hydroxyl to create a better Comparing the binary D15Pd2,6ST(N) structure to the ternary nucleophile to attack the anomeric carbon of CMP-Neu5Ac [16]. D16Psp26ST structure co-crystallized with CMP and lactose (also N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729 4727

orientation between the two C-terminal Rossmann domains. Each individual domain superimposes with rmsd of 0.95 Å and 1.37 Å for the N-terminal and C-terminal Rossmann domains respectively, and an rmsd of 1.95 Å for the overall GT-B fold between the D15Pd2,6ST(N) and the D16Psp26ST. If all three domains are included, the rmsd is 2.49 Å, which also includes the effect due to the difference in orientation between the N-terminal Ig-like domain and the overall GT-B domain (Fig. 5B). In spite of crystal packing contacts, which prevent large conformational changes upon substrate binding during the crystal soaking, a slight closure between the two Rossmann domains upon substrate binding is identified when comparing the binary complex to the ligand-free structure (Fig. 5C). The loop containing Trp361 closes in about 1.8 Å on the substrate, which compares to a 7.4 Å a-carbon movement observed in PmST1 upon substrate binding [16]. The rmsd between the GT-B domains alone of the binary complex and the ligand-free structure is 0.64 Å for 374 equivalent a-carbons, and the superposition reveals a slight movement in orientation between the Ig-like domain and the GT-B fold (Fig. 5C). Comparing the D15Pd2,6ST(N) to the D112Pd2,6ST(N) lacking the Ig-like domain reveals greater conformational flexibility between the two Rossmann-like domains. The ligand-free D112Pd2,6ST(N) structure is actually in a slightly more closed conformation compared to the substrate-bound D15Pd2,6ST(N) structure (Fig. 5D). This is likely because the truncated D112Pd2,6ST(N) Fig. 5C. A superposition between the ligand-free (tan) and CMP-3F(a)Neu5Ac- crystallizes in a different space group thus creating different lattice bound (green) structures of D15Pd2,6ST(N). The alignment shows a small closure between the two domains of the GT-B fold upon soaking in CMP-3F(a)Neu5Ac contacts. While this structure is slightly more closed, it still does (white-colored carbon spheres). Crystal packing and lattice contacts may prevent not resemble the completely closed structure seen in other GT-B the larger closure seen in other GT-B sialyltransferases [2,18]. folded sialyltransferases co-crystallized with CMP or CMP-Neu5Ac analogs. However, comparing all three Pd2,6ST structures suggests there is some inherent flexibility between the two Rossmann-like domains of the GT-B sialyltransferases even in the absence of substrates.

4. Conclusions

The crystal structures of D15Pd2,6ST(N) have provided additional insights into the reaction mechanism and the function of conserved residues important in catalyzing the transfer of sialic acids. D15Pd2,6ST(N) is found to contain three separate domains. The first domain at the N-terminus adopts an Ig-like fold. The second and third domains are each a Rossmann fold bearing a deep catalytic binding cleft in between, a characteristic of members in the GT-B structural group. Soaking the donor substrate analog CMP-3F(a)Neu5Ac into the crystal prior to data collection provided a structure with a potential pre-Michaelis complex of sugar nucleotide bound in the enzyme. This represents a stage before the induction of a conformational closure between the two Rossmann domains. It is interesting to note that unlike reported GT-B sialyltransferase structures, other glycosyltransferases with GT-B folds, that are not sialyltransferases, do not exhibit large closures between the two Rossmann domains. Most likely this is due to the presence of the C-terminal ‘‘backbone’’ that traverses both the C-terminal and N-terminal Rossmann domains in non- Fig. 5D. A superposition of the D112Pd2,6ST(N) construct lacking the Ig-like domain and substrates (red) and the ligand-free (tan) and CMP-3F(a)Neu5Ac-bound sialyltransferase glycosyltransferases with GT-B folds, such as (green) structures of D15Pd2,6ST(N). The alignment shows the most closed peptidoglycan glycosyltransferase MurG [46], T4 phage b-glucosyl- structure occurs in the ligand free D112Pd2,6ST(N). This is likely due to crystal transferase [47], and UDP-glucosyltransferase GtfB [48]. The contacts illustrating the inherent flexibility between the two Rossmann-like significance for the large conformational changes of the GT-B domains. The CMP-3F(a)Neu5Ac seen in the D15Pd2,6ST(N) binary structure is drawn as white-colored carbon spheres. sialyltransferases is unknown. However, many of these enzymes display substrate promiscuity and alternate activities such as catalyzing the formation of different (a2–3 and a2–6) sialyl linkages containing an N-terminal Ig-like domain) clearly shows the struc- [6,7,49,50], or having even sialidase or trans-sialidase activities tural conformational change that occurs upon binding substrates [6–8]. Therefore the large conformational changes may be impor- to generate a productive complex. As shown in Fig. 5B, superposing tant for these biological functions. Nevertheless, the hypothesis the N-terminal Rossmann domains reveals a large difference in remains to be tested. 4728 N. Huynh et al. / FEBS Letters 588 (2014) 4720–4729

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