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FEBS Letters 587 (2013) 3943–3948

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The crystal structure of novel chondroitin ODV-E66, a baculovirus envelope protein ⇑ Yoshirou Kawaguchi a, Nobuo Sugiura b, Koji Kimata c, Makoto Kimura a,d, Yoshimitsu Kakuta a,d,

a Laboratory of Structural Biology, Graduate School of System Life Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan b Institute for Molecular Science of Medicine, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan c Research Complex for the Medicine Frontiers, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan d Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan

article info abstract

Article history: Chondroitin have been known as pathogenic bacterial that degrade chondroitin. Received 5 August 2013 Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroi- Revised 1 October 2013 tin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteris- Accepted 15 October 2013 tics reflecting the life cycle of baculovirus. To understand ODV-E66’s structural basis, the crystal Available online 26 October 2013 structure was determined and it was found that the structural fold resembled that of polysaccharide Edited by Christian Griesinger lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discus- sion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus. Keywords: Polysaccharide lyase Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Chondroitin sulfate Interdomain movement b-Elimination Autographa californica

1. Introduction making it possible for these bacteria to access a target cell by degrading the chondroitin in its extracellular matrix [6]. Recently, Chondroitin, a sulfated glycosaminoglycan, exists as a compo- such enzymes have been studied as possible therapeutic tools for nent of extracellular matrix polysaccharides, which play an impor- dealing with infections and/or neural regeneration [6–8]. tant role in cell-to-cell associations [1]. Chondroitin is a linear We have previously reported an occlusion-derived virus enve- polysaccharide consisting of [–4-D-glucronic acid (GlcUA)-b-1-3- lope protein 66 (ODV-E66) from Autographa californica, a kind of

N-acetyl-D-galactosamine (GalNAc) b1–]n repeating disaccharide baculovirus, as a novel chondroitin lyase in virus [9]. ODV-E66 units and is frequently sulfated at the C-4 or C-6 positions of the has unique characteristics closely related to the baculovirus life GalNAc residue or at the C-2 position of the GlcUA residue [2,3], cycle, being released in an insect’s midgut after feeding on baculo- named chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan virus and degrading the host cells’ extracellular matrix to gain sulfate, respectively (also chondroitin sulfate A, C, and B, access to the cells. ODV-E66 is quite stable and retains its enzy- respectively). matic activity over a wide pH range (pH 4–9) and temperature Three main kinds of chondroitin lyases have been identified (30–60 °C). This character reflects that baculovirus exists in the that can degrade chondroitin [4,5]. Chondroitin AC lyase can field, awaiting ingestion by an insect. In addition, ODV-E66 has un- degrade non-sulfated chondroitin and chondroitin sulfates A and ique substrate specificities, only able to degrade non-sulfated C. Chondroitin B lyase can degrade chondroitin sulfate B, while chondroitin and chondroitin sulfate C, making it similar in this chondroitin ABC lyase can degrade all the above-mentioned respect to chondroitin AC lyase, except for the latter’s ability to chondroitins. These lyases are derived from pathogenic bacteria, degrade chondroitin sulfate A. This narrower range of substrate specificity might reflect the baculovirus’s host specificity. Baculovi- rus infects lepidopteran insect larvae midgut cells whose extracel- Abbreviations: ODV-E66, occlusion derived virus envelope protein 66; PL8, polysaccharide lyase 8 lular matrix, the peritrophic membrane, contains low proportions ⇑ Corresponding author at: Laboratory of Structural Biology, Graduate School of of chondroitin sulfate A [10–13]. Previous reports have shown that System Life Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, an odv-e66 gene is essential for baculovirus oral infection [14] and Japan. Fax: +81 92 642 2854. that the N-terminus region of ODV-E66 has an affinity for midgut E-mail address: [email protected] (Y. Kakuta).

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.10.021 3944 Y. Kawaguchi et al. / FEBS Letters 587 (2013) 3943–3948 proteins in a host insect, Heliothis virescens [15]. These reports sug- HKL2MAP software package [20]. The partial mode was built auto- gest the importance of ODV-E66 to infectivity of baculovirus which matically using ARP/wARP [21] and modified manually using COOT is now widely used as a protein expression system [16,17].To [22]. The structure was revised several times by alternately adjust- understand the relationship between these unique characteristics ing the model and refinements using Refmac [23]. The structure of of ODV-E66 and its structural information, the crystal structure ODV-E66 without iodine was solved by molecular replacement was determined. The structural information enables to discuss using the iodine derivative ODV-E66 coordinates as a search model the unique substrate specificity and the stability of ODV-E66, and and the program MOLREP [24]. The structure was then revised sev- the host specificity of baculovirus. eral times by alternately adjusting the model and refinements using COOT and Refmac. The resulting two structures were called 2. Materials and methods native and iodine derivative ODV-E66; the refinement statistics are summarized in Table 1. Stereochemical checks were carried 2.1. Preparation of native ODV-E66 crystal and X-ray data collection out using Molprobity [25]. The atomic coordinates and structural factors of the native and iodine derivative protein have been Procedures for expression, purification, crystallization, and deposited in the Protein Data Bank at Rutgers University under X-ray data collection of recombinant ODV-E66 was performed as accession code 3VSM and 3VSN, respectively. Each figure was de- previously reported [9,18]. In this study, ODV-E66 (67–704) is scribed using PYMOL program (Fig. 1 and Suppl. Fig. 2a). described as only ‘ODV-E66’. 2.4. Mutational analysis 2.2. Preparation of iodine derivative ODV-E66 crystal Bacterial pET15b vectors harboring the ODV-E66 mutants, A ODV-E66 crystal was soaked for 30 s in a pH 8.8 buffer, N236A, H291A, Y299F, R345A, and E395A were prepared using comprising 15% glycerol (v/v), 50 mM Tri-HCl, 100 mM NaCl, the PCR-based Takara PrimeSTAR mutagenesis kit (Takara Bio, 0.02 M citric acid, 0.08 M Bis–Tris propane, 18% PEG3350 (w/v), Inc., Otsu, Japan), with pET15b-ODV-E66 as the template; primer and 1 M iodine potassium, and then flash-cooled in nitrogen-gas sequences are shown in Supplementary Table 1. The PCR-amplified cryostream (Rigaku Corp., Tokyo, Japan) at À180 °C. All intensity pET15b-ODV-E66 mutant constructs were sequenced to confirm data were indexed, integrated, and scaled with the HKL-2000 soft- the anticipated mutations using the services of Operon Biotechnol- ware package [19]. Data collection statistics of native and iodine ogies (Huntsville, AL, USA). Both ODV-E66 wild type and mutants derivative ODV-E66 are summarized in Table 1. for mutational analysis were expressed and purified as previously reported [9,18]. The assay for measuring enzymatic activity is de- 2.3. Structure determination and refinement scribed as a Supplementary Method.

The initial phase of ODV-E66 structural determination was per- 3. Results and discussion formed using the iodine derivative data with single isomorphous replacement and an anomalous scattering (SIRAS)-method by the The crystal structures of native and iodine derivatized ODV- E66 were determined at 2.0 Å resolution. In the native ODV-E66 (PDB ID: 3VSM), a total of 632 of 638 residues (67–704) were Table 1 modeled and 615 water molecules found to be incorporated. In Data collection and refinement statistics. the iodine derivative ODV-E66 (PDB ID: 3VSN), a total of 631 of Data collection Native Iodine derivative 638 residues were modeled, and 606 water molecules and 76 io- dine ions were incorporated. As the r.m.s.d. value between native Space group P62 P62 Unit cell parameters a = b = 113.5 Å, a = b = 118.2 Å, and iodine derivative ODV-E66 was 0.88 Å from 631 Cas, the two c = 101.5 Å c = 100.1 Å structures resembled each other. The overall structure of native Wavelength (Å) 1.3 1.3 ODV-E66 confirmed the protein to be composed of two domains, No. of reflections an a-helix rich domain in the N-terminus side and a b-strand rich Observed/unique 791385/68227 550152/146886 domain in the C-terminus side (Fig. 1). The a-helix rich domain Redundancy 8.1 (7.9) 5.4 (5.3) contained eight a-helices and four short b-strands with 202 res- R 0.094 (0.901) 0.135 (0.714) sym idues of amino acids forming an a/a troid topology, whereas I/r (I) 25.26 (1.86) 28.13 (3.80) Completeness (%) 100.0 (100.0) 98.4 (96.8) the b-strand rich domain contained 31 b-strands and 12 short a-helices with 431 amino acids forming an anti-parallel Refinement statistics Resolution range (Å) 35.31–2.00 8.74–2.00 b-sandwich. These structural features were very similar to the well-characterized polysaccharide lyase 8 (PL8) family enzymes, No. of reflections Working set/test set 47690/2484 67619/3554 which includes chondroitin AC lyase (cAC lyase) and chondroitin Completeness (%) 99.95 97.84 ABC lyase (cABC lyase) [6], although the amino acid sequence Rcryst (%)/Rfree (%) 17.35/20.64 16.78/19.42 identities were <12%. The results of a VAST search [26] showed Root mean square deviation cAC lyase from Pedobacterium heparinum (Flavobacterium hepari- Bond length (Å) 0.006 0.0006 num) [27] to have the highest score, at 41.5. The structural Bond angles (°) 1.073 1.046 features of PL8 family enzymes are well defined [6,28],including Average B-factor (Å2)/No. of atoms the formation of an (a/a)5,6 troid domain in the N-terminus Protein 43.7/5094 32.2/5030 side and a b-sandwich domain in the C-terminus side, and corre- Water 29.4/615 22.2/606 Glycerol 73.4/12 47.6/48 sponded well with that of ODV-E66. Iodine 26.3/76 PL8 family enzymes are known as pathogenic, bacterial, poly- Ramachandran analysis saccharide lyases, which are capable of degrading on polysaccha- Favored (%) 95.2 95.9 rides in the extracellular matrix of host tissues and to contribute Allowed (%) 4.4 4.1 to their invasive abilities [29]. These enzymes recognize substrate Outlier (%) 0.3 0.0 polysaccharide between the a/a troid and b-sandwich domains. PDB ID 3VSM 3VSN Then, they catalyze a b-elimination reaction that results in Y. Kawaguchi et al. / FEBS Letters 587 (2013) 3943–3948 3945

Fig. 1. Overall structure of ODV-E66. Crystal structure of ODV-E66 (PDB ID: 3VSM) described as a cartoon mode. Right figure indicates 90°-turn around vertical axle of left figure structure; N, N-terminus and C, C-terminus of ODV-E66; a/a troid domain, orange; and b-sandwich domain, light orange. scission of the glycosidic bond adjacent to a carboxylate group, Fig. 2). Thus, ODV-E66 should move between domains to exert forming a new reducing end and an unsaturated sugar at the its activity. non-reducing end. This catalytic reaction is performed by five Considering the ’s movement, an active model of ODV- catalytic residues, with Asn or Asp as a neutralizer for the car- E66 was constructed and the putative catalytic residues deter- boxyl group in substrate uronic acid and His, Tyr, Arg, and Glu mined to be indeed catalytic residues (Fig. 3a). The model was as catalytic bases/acids [28]. formed by superposing ODV-E66 on a cAC lyase derived from P. The structural alignment, based on structural information of heparinum [38], with the a/a troid and b-sandwich domains sepa- ODV-E66 and structurally characterized PL8 proteins [27,30–33] rated. The model was constructed by superimposing all atoms of using the MATRAS program [34], is described in Fig. 2. According Asn236 in ODV-E66 and Asn175 in cAC lyase, while His291, to this alignment, ODV-E66 possessed all five residues correspond- Tyr299, Arg345, and Glu395 in ODV-E66 were superposed on ing to the catalytic residues in PL8, as Asn236, His291, Tyr299, His225, Tyr234, Arg288, and Glu371 in cAC lyase. In this model, Arg345, and Glu395 (Fig. 2). Notably, a mutational analysis showed Trp182, Tyr183, and His184 were suitably overlapped with the that the mutated ODV-E66 forms N236A, H291A, Y299F, R345A, corresponding residues of cAC lyase, Trp126, Trp127, and His128, and E395A lose their chondroitin degrading activity, suggesting and interacting with the sugar chain of a substrate chondroitin that these five residues were indeed essential for catalysis (Suppl. [38]. Thus, this ODV-E66 model, shown in Fig. 3a, was concluded Fig. 1). to represent a model of active ODV-E66, with relative positions For these residues to be regarded as catalytic residues, an inter- of this model’s catalytic residues also conserved, as in other PL8 domain movement should occur between the aa troid and b-sand- proteins exerting catalytic activity (Fig. 3b). wich domains. This should occur because the distances between The present ODV-E66 model along with mutational analysis Asn236 in the aa troid domain and the other four putative cata- indicated that the reaction mechanism of ODV-E66 was similar lytic residues existing in the b-sandwich domain was 5 Å longer to that of other PL8 lyases [31,38]. In the model, Asn236 and than other PL8 family’s corresponding residues when they exert His291 neutralized the carboxyl group of substrate glucuronic acid their activity. In the PL8 family, interdomain movements con- and Tyr299 abstracted a proton, as a catalytic base at C-5 of glucu- trolled by substrate binding have been observed in hyaluronate ronic acid (Fig. 4). At this stage, Arg345 interacted with O-4 binding lyases derived from Streptomyces coelicolor A3(2), Streptococcus C-4 of glucuronic acid and assisted the role of Tyr299; Glu395 sup- pneumonia, and Streptococcus agalactiae [30,35–37]. In addition, a ported the reaction by interacting with His291 and Arg345. Then, slight interdomain difference in position was observed between the glycosidic bond was broken, producing a new reducing end native and iodine-derivatized ODV-E66 crystal structures (Suppl. and generating unsaturated sugar. Actually, in the active ODV-E66

Fig. 2. Selected sequence alignment of ODV-E66 based on structural information. Multiple alignment of ODV-E66 with chondroitin lyases and/or hyaluronate lyases categorized into PL8, described using MATRAS program. Selected sequence, position of key residues; aligned lyases can degrade chondroitin sulfate A except for ODV-E66; numbers along alignment, amino acid number of ODV-E66; aligned residues, white on black, essential for catalysis for aligned lyases. The Arg or His, white on gray, positioned at interaction with sulfate group of chondroitin sulfate. The pdb code in figure indicated as below: 1cb8, chondroitin AC lyase derived from Pedobacterium heparinum; 1rw9, chondroitin AC lyase derived from Arthrobacter aurescens; 2wda, derived from Streptomyces coelicolor A3(2); 1hn0, chondroitin ABC lyase derived from Proteus vulgaris; and 2q1f, chondroitin ABC lyase derived from Bacteroides thetaiotaomicron WAL2926. 3946 Y. Kawaguchi et al. / FEBS Letters 587 (2013) 3943–3948

Fig. 3. Active ODV-E66 model with cAC lyase from P. heparinum. (A) The ODV-E66 model; orange, cAC lyase, blue; right figures enlarged active sites of left figure; and substrate chondroitin in figure derived from ES complex structure of cAC lyase from P. heparinum. (B) The ODV-E66 model superposed on other PL8 enzymes based on catalytic residues. Relative position of each catalytic residue shown; each protein’s pdb ID as in Fig. 2: 1cb8, blue; 1rw9, red; 2wda, pink; 1hn0, magenta; and 2q1f, cyan. model, the calculated pKa value of Tyr299 was decreased from 10.5 sulfate group of chondroitin sulfate A directly interacts with to 9.4 due to Arg345, calculated by MOE [39]. His561 [41]. On the other hand, ODV-E66, which cannot degrade Using this active ODV-E66 model, a portion of ODV-E66’s un- chondroitin sulfate A, did not have these residues (Suppl. Fig. 3b) ique characters can be discussed. In terms of substrate specificity, and the nearest residue, Asn346 of ODV-E66, was far from the sul- chondroitin lyases able to degrade chondroitin sulfate A have fate group of chondroitin sulfate A (6.6 Å). The absence of this key either an Arg or His residue in their active sites (Fig. 2, gray and residue appears to disallow ODV-E66 from degrading chondroitin Suppl. Fig. 3a). In cAC lyase from P. heparinum, the sulfate group sulfate A. of chondroitin sulfate A is directly associated with Arg292 and, Baculovirus host specificity and the stability of ODV-E66 could interestingly, an R292A mutant of cAC exhibits low chondroitin also be discussed using this model. Comparing active ODV-E66 sulfate A activity [40]. In cABC lyase from Proteus vulgaris, the model with other PL8 enzymes, there were two structurally Y. Kawaguchi et al. / FEBS Letters 587 (2013) 3943–3948 3947

Fig. 4. Schematic diagram indicating ODV-E66’s reaction mechanism. Five catalytic residues described around non-sulfated chondroitin disaccharide, and dashed line, hydrogen bond network. different regions; Region 1 (amino acid 74–105) and Region 2 ments on BL38B1 at SPring-8 were performed with the approval (amino acid 350–365) as Suppl. Figs. 4 and 5. These regions might of the Japan Synchrotron Radiation Research Institute (JASRI; Pro- contribute to the unique characters of baculovirus and ODV-E66. posal No. 2011A2014). This work was supported by JSPS KAKENHI First, host specificity of baculovirus might derive from Region 1. Grant Number 25440029. Region 1 possesses sequences reported to interact with midgut proteins in a host insect, Heliothis virescens [15] (Suppl.Fig.4, Appendix A. Supplementary data red and Suppl. Fig. 5). Thus, the unique conformation of this re- gion might contribute to host specificity. Second, stability of Supplementary data associated with this article can be found, in ODV-E66 might derive from Region 2 and a large number of the online version, at http://dx.doi.org/10.1016/j.febslet.2013. hydrophobic interactions in ODV-E66. Region 2 was an only loop 10.021. which was shorter than in other PL8 lyases (Suppl. Fig. 4). Region 2 consisted of 16 amino acids, while there are 63 amino acids on References average corresponding region in other PL8 enzymes (Suppl. Figs. 4 and 5). Because a solvent-exposed surface loop is known to be a [1] Gandhi, N.S. and Mancera, R.L. (2008) The structure of glycosaminoglycans and factor contributing to instability [42], the truncated loop of Re- their interactions with proteins. Chem. Biol. Drug Des. 72, 455–482. gion 2 might have correlated with high stability of ODV-E66. In [2] Esko, J. D. and Linhardt, R.J. (2009) Proteins that Bind Sulfated terms of hydrophobic interactions, a large number of aromatic- Glycosaminoglycans. 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