Crystal structure of phospholipase A1 from Streptomyces albidoflavus NA297

Journal of Structural Biology 182 (2013) 192–196

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Structure Report

Crystal structure of phospholipase A1 from Streptomyces albidoflavus NA297 Kazutaka Murayama a,b,⇑, Kota Kano c, Yusaku Matsumoto c, Daisuke Sugimori c a

Graduate School of Biomedical Engineering, Tohoku University, Seiryo 2-1, Aoba, Sendai 980-8575, Japan RIKEN Systems and Structural Biology Center, Yokohama Institute, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan c Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan b

a r t i c l e

i n f o

Article history: Received 22 November 2012 Received in revised form 31 January 2013 Accepted 1 February 2013 Available online 13 February 2013 Keywords: Phospholipase A1 Metal-independent lipase Catalytic dyad Hydrophobic pocket

a b s t r a c t The metal-independent lipase from Streptomyces albidoflavus NA297 (SaPLA1) is a phospholipase A1 as it preferentially hydrolyzes the sn-1 acyl ester in glycerophospholipids, yielding a fatty acid and 2-acyllysophospholipid. The molecular mechanism underlying the substrate binding by SaPLA1 is currently unknown. In this study, the crystal structure of SaPLA1 was determined at 1.75 Å resolutions by molecular replacement. A structural similarity search indicated the highest structural similarity to an esterase from Streptomyces scabies, followed by GDSL family enzymes. The SaPLA1 active site is composed of a Ser-His dyad (Ser11 and His218), whereby stabilization of the imidazole is provided by the main-chain carbonyl oxygen of Ser216, a common variation of the catalytic triad in many serine hydrolases, where this carbonyl maintains the orientation of the active site histidine residue. The hydrophobic pocket and cleft for lipid binding are adjacent to the active site, and are approximately 13–15 Å deep and 14–16 Å long. A partial polyethylene glycol structure was found in this hydrophobic pocket. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction The phospholipase A (PLA) enzymes hydrolyze glycerophospholipids into free fatty acids and lysophospholipids, and are divided into two classes, A1 (PLA1) and A2 (PLA2), according to the hydrolysis products (2- and 1-acyl-lysophospholipids, respectively; Fig. 1) (Richmond and Smith, 2011). PLA1 was isolated from Streptomyces albidoflavus NA297 (Sugimori et al., 2012), and although most PLAs are metal-dependent enzymes (Kim et al., 1996; Sato et al., 2005; Scandella and Kornberg, 1971; Sugiyama et al., 2002; Tamori et al., 1979; Watanabe et al., 1999), the PLA1 from S. albidoflavus NA297 (SaPLA1) possesses metal ion-independent enzyme activity. The positional selective hydrolysis is estimated to be 63:37 between the sn-1 and sn-2 positions, respectively, for a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate substrate (Sugimori et al., 2012). According to a BLAST search (Altschul et al., 1997) of the Protein Data Bank, SaPLA1 shares 24% sequence identity with an esterase from Streptomyces scabies (SsEst) (Wei et al., 1995), in which the active site consists of the Ser-His catalytic dyad, a variation of the common serine protease catalytic triad. Mutagenesis of the corresponding amino acids (aa) in SaPLA1 indicated their importance for the enzymatic activity (Sugimori et al., 2012). Although the structures of SaPLA1 and SsEst are expected to be similar, the ⇑ Corresponding author at: Graduate School of Biomedical Engineering, Tohoku University, Seiryo 2-1, Aoba, Sendai 980-8575, Japan. Fax: +81 22 717 8460. E-mail address: [email protected] (K. Murayama). 1047-8477/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2013.02.003

crystal structure of SaPLA1 is needed to determine its molecular mechanism. In the present study, the crystal structure of SaPLA1 was solved at 1.75 Å resolutions by the molecular replacement method. This is the first report of the detailed structure of PLA1. The catalytic center was found to be a Ser-His dyad, and a hydrophobic pocket adjacent to the catalytic site was identified as a putative substrate binding site. The positional hydrolytic selectivity is discussed, based on these structural characteristics. 2. Materials and method 2.1. Protein expression and purification The protein sample was prepared as previously described (Sugimori et al., 2012). Briefly, SaPLA1, produced by transformed S. lividans, was purified from a 48 h culture supernatant, which was mixed with a saturated ammonium sulfate solution (80% by mass), and centrifuged. The resulting precipitate was resuspended in 20 mM Tris–HCl buffer (pH 9.0) containing 1.5 M ammonium sulfate, loaded onto a Toyopearl Phenyl-650 M column (2.5  4 cm, Tosoh Bioscience GmbH, Tessenderlo, BE), and eluted with a linear gradient of 1.5–0 M ammonium sulfate. The active fractions were pooled, and the buffer was replaced with 20 mM MES-NaOH buffer (pH 6.0), using a Vivaspin 20 centrifugal concentrator (10 kDa cut off; GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The bufferexchanged fraction was then loaded onto a HiTrap SP column (5 mL column volume, CV; GE Healthcare Bio-Sciences AB). After

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OH

R3

O R3

O

sn-1

O

R1

sn-2

O

R2

P O

O –

O

PLA1

O

O

P O

R2

O

O O

O

–

O O

PLA2

O R3

O

P O

R1

OH O –

Fig. 1. Catalytic reactions and products of PLA1 and PLA2.

at 20 °C, and the data were analyzed by the algorithms included in the instrument’s software. Measurements were performed with a protein concentration of 1.0 mg/mL, in 20 mM Tris-HCl buffer (pH 9.0) containing 20 mM NaCl.

Table 1 Crystal parameters, data collection and refinement statistics. Crystal characteristics Space group Unit cell parameters (Å) Molecules in asymmetric unit

P3121 a = b = 67.6, c = 96.1 1

Data collections Wavelength (Å) Resolution range (Å) Redundancy Unique reflections Completeness (%)a I/r (I) Rsym

BL-1A 1.0 37.1–1.75 10.9 26008 100 (100) 29.5 (5.8) 0.105 (0.618)

Refinement statistics Resolution range (Å) Unique reflections R-factor/Free R-factorb No. of protein atoms No. of water molecules RMS deviation from ideal geometry Bond lengths (Å) Bond angles (deg.) Average isotropic B-value (Å2) Ramachandran plot Most favored regions (%) Allowed regions (%)

2.3. Crystallization and data collection In house 1.5418 37.0–2.20 5.8 13250 99.8 (98.6) 17.7 (4.3) 0.085 (0.382)

37.1–1.75 25973 0.186 (0.208)/0.223 (0.206) 1700 218

0.009 1.50 18.4 90.9 9.1

a Numbers in parentheses refer to the highest resolution shell 1.81–1.75 Å (BL1A), 2.28–2.20 Å (in-house). b Free R-factor was calculated using 5% of reflections omitted from refinement. Numbers in parentheses refer to the highest resolution shell 1.86–1.75 Å.

elution with a linear gradient of 0–1 M NaCl in the same buffer, the active fractions were pooled, and the buffer was exchanged to 20 mM Tris–HCl buffer (pH 9.0) through concentration and resuspension, as above. The enzyme solution was then applied to a HiTrap Q column (CV 5 mL; GE Healthcare Bio-Sciences) and eluted with a linear gradient of 0–1 M NaCl in the same buffer. Fractions exhibiting high specific activity were pooled and concentrated to 12.0 mg/mL, and the buffer was replaced with 20 mM Tris-HCl buffer (pH 9.0) containing 20 mM NaCl during the concentration step. 2.2. Dynamic light scattering measurements The hydrodynamic radius and size distributions of SaPLA1 were measured using a Zetasizer NanoZ instrument (Malvern Instruments, Ltd., Worcestershire, UK), based on dynamic light scattering

Purified SaPLA1 was crystallized by the hanging-drop vapor-diffusion method, at a final protein concentration of 12 mg/mL. After initial screening trials using a PEGRx screening kit (Hampton Research, Aliso Viejo, CA, USA), the crystals were grown by equilibrating a mixture containing 1 lL of protein solution and 1 lL of reservoir solution (1.8 M ammonium sulfate, 3% polyethylene glycol monomethyl ether (PEGMME 2000), and 0.1 M Hepes buffer, pH 7.5). Rod-shaped crystals appeared within a few days and grew to 0.1  0.1  0.5 mm. Data collections were conducted using crystals transferred into Paratone N (Hampton Research), as a cryoprotectant, for 1 min before flash-cooling in a 110 K nitrogen stream. Diffraction data sets were collected with in-house Cu Ka radiation (1.5418 Å), using R-AXIS IV++ imaging-plate detectors mounted on FR-E super-brilliant X-ray generators (Rigaku Corp., Tokyo, Japan), and on the beamline BL1-A (wavelength 1.0 Å) at the Photon Factory (Tsukuba, Japan). All diffraction data were integrated and scaled using the HKL2000 program suite (Otwinowski and Minor, 1997), and the data collection statistics are presented in Table 1.

2.4. Structure determination and refinement The crystal structure of SaPLA1 was determined by the molecular replacement method with MOLREP, in the CCP4 program suite (Winn et al., 2011). The search model was a homology-modeled structure generated by the SWISS-MODEL Server (Arnold et al., 2006), with the structure of SsEst (PDB: 1ESC) as the template structure. The phasing and initial refinement was conducted with diffraction data collected by the in-house diffractometer. The initial model was manually modified, using the O molecular modeling program (Jones et al., 1991). The structure was refined using CNS (Brunger et al., 1998). A PEGMME partial structure was fitted to the unidentified electron densities that could not be traced as the peptide chain. The final refinement data were resolved to 1.75 Å, and the refinement statistics are provided in Table 1. The stereochemical quality of the final model was assessed using PROCHECK (Laskowski et al., 1993), and the protein structural figures (Figs. 2a and c, 3, 4a and b) were generated using the MolFeat program (Fiatlux Co., Tokyo, Japan). The atomic coordinates have been deposited in the Protein Data Bank, with the accession code 4HYQ.

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Fig. 2. (a) Ribbon representation of the SaPLA1 structure. Secondary structural elements are colored blue (helices) and pink (strands); aa residues in the active site are indicated as stick models. (b) Structure-based alignment between SaPLA1 and SsEst. Secondary structural elements are shown on the sequence. The residues highlighted by red boxes are involved in the active site. The three disulfide bonds are indicated by red lines. (c) Superimposed structures of SaPLA1 (pink) and SsEst (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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+LV 1δ

6HU

2γ

1ε

2

6HU

tering measurements revealed an estimated molecular mass, based the hydrodynamic radius (2.38 nm), of 25.5 kDa (calculated as 24.2 kDa for the mature form), suggesting that SaPLA1 is monomeric in solution. The mature enzyme included 236 aa residues, which were traced from Ala1 to Ala236 in the electron density maps. The crystal structure possessed ten a-helices and five bstrands, with the latter forming a parallel b-sheet. The b-sheet included many hydrophobic residues and thus formed a hydrophobic core, although the sheet was asymmetrically located in the molecule, and was flanked by a2, a3, a6 and a10 (Fig. 2a). The mature form of the N-terminus started with alanine, which was stabilized by two hydrogen bonds, Ala1(N)–Asn42(O) and Ala1(O)–Ala46(N), in addition to van der Waals contacts between the Cb side chain and the surrounding residues. All six cysteines in the sequence formed three disulfide bonds, Cys28–Cys53, Cys94–Cys102, and Cys152–Cys199. Two of these disulfide bonds linked loop structures, and one (Cys94–Cys102) linked a-helices (a4 and a5, Fig. 2b).

Fig. 3. Active site structure. Electron densities are contoured at 1.0 r, and hydrogen bonds are indicated by dashed lines.

3.2. Comparison with SsEst 3. Results and discussion 3.1. Overall structure The crystal structure of SaPLA1, determined by the molecular replacement method and refined to 1.75 Å resolution, contained one molecule in the asymmetric unit (Fig. 2a). Dynamic light scat-

A structural similarity search on the DALI server (Holm and Rosenstrom, 2010) indicated that the highest structural similarity was to SsEst (Wei et al., 1995), with a Z-score of 27.7, as expected from the BLAST search. The second structure was assigned to the GDSL family enzymes (PDB: 3RJT), with a much lower Z-score of 17.3. A structural comparison between SaPLA1 and SsEst was performed on the MATRAS server (Kawabata, 2003). The secondary

a

b W210 V208 V95

hydrophobic pocket/cleft

Y217

Y12 F145

90°

α 4 α 7

Y142

α 5 F87

L171

hydrophilic

hydrophobic

Fig. 4. Surface representations of SaPLA1. The surface models are colored according to the hydrophobicity, from magenta (hydrophilic) to yellow (hydrophobic) in five steps. (a) Upper panel: side view of the cross section of the surface model around the hydrophobic pocket. The SaPLA1 structure is shown as a ribbon model (blue), and the polyethylene glycol structure is depicted as a stick model. Lower panel: top view of the surface model. (b) Hydrophobic amino acids in the pocket and the cleft. The hydrophobic residues are drawn as stick models. The polyethylene glycol structure is depicted as a stick model, with the electron density contoured at 1.0 r. For interpretation of color in this figure, the reader is referred to the web version of this article.

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structural elements superimposed well (Fig. 2c), with an r.m.s.d. of 2.08 Å for the common Ca carbons. However, the structure-based alignment revealed eight deletions in SaPLA1, comprising 63 aa residues, as compared to SsEst (Fig. 2b). The three deletions between a3 and b3, a4 and a5 and b4 and a7 included more than ten residues and formed short loop structures. Due to these deletions and the fewer residues, the molecular surface area of SaPLA1 (9,587 Å2) is more compact than that of SsEst (12,745 Å2).

Japan). We would like to thank the beamline staff at BL-1A of the Photon Factory for assistance during data collection. We also acknowledge the support of the Biomedical Research Core of Tohoku University, Graduate School of Medicine. This work was partly supported by the Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References

3.3. Catalytic site and hydrophobic pocket The SaPLA1 active site included Ser11, as the nucleophile, and His218 (Fig. 3), but lacked the Glu/Asp commonly found in conventional serine protease catalytic triads. In the structure of SaPLA1, the carbonyl oxygen of Ser216 hydrogen bonded with the d-nitrogen of His218, forming a catalytic dyad, as a variation of the catalytic triad. This dyad system has also been observed in the structure of SsEst, although the main chain carbonyl oxygen is provided by Trp280 (Wei et al., 1995). The hydrogen bond distances for Ser(Oc)-His(e2) and His(d1)-Ser/Trp(O) within the active site are 2.65 and 2.87 Å (for SaPLA1), and 2.71 and 2.74 Å (for SsEst). The histidine residue in SaPLA1 is asymmetrically located. Although asymmetrical hydrogen bond distances were also observed in the structure of a-chymotrypsin (PDB: 4CHA), the histidine residue (His57) was closer to the aspartate residue (Asp102); i.e., Ser(Oc)-His(e2): 2.82 Å and His(d1)-Asp(Od1): 2.61 Å. An analysis with the pocket/cavity detection program fpocket (Le Guilloux et al., 2009) revealed that a deep pocket existed at the C-terminal side of the b-sheet, and this pocket is observed in the present surface model (Fig. 4a). The pocket involved many hydrophobic residues, including Tyr12, Phe87, Tyr142, Phe145 and Leu171 (Fig. 4b). In addition, the hydrophobic cleft is located along with a4. The cleft contains hydrophobic amino acids, including Val95, Met91, Val208, Trp210 and Tyr217 (Fig. 4b). The hydrophobicity of the pocket’s interior and the cleft are depicted in Fig. 4a. Although some hydrophilic residues are located near the opening of the pocket (magenta), the inside of the pocket and the cleft are hydrophobic (yellow). The pocket depth was estimated as 13–15 Å, which is sufficient for accepting a C14–C16 aliphatic chain. The cleft spans 14–16 Å, and Trp210 blocks the end of the cleft. In the crystal structure, the hydrophobic pocket was occupied by the partial structure of polyethylene glycol [(CH2–CH2–O)n, n = 5] (Fig. 4b). This polyethylene glycol was probably derived from the low molecular weight PEGs in the crystallization solution. This structure is the first step towards better understanding of the sn-1 preference of SaPLA1. Acknowledgements We thank Associate Professor Chiaki Ogino (Kobe University, Japan) for providing the expression vector. The synchrotron radiation experiments were performed at the Photon Factory (Tsukuba,

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