Crystal structure of an α/β serine hydrolase (YDR428C) from <i>Saccharomyces cerevisiae</i> at 1.85 Å resolution (2004)

In order to extend the structural coverage of eukaryotic genomes, we selected 288 open reading frames (ORF's) in the yeast genome with significant homology to mouse proteins. One of these, a hydrolase (YDR428C) from Saccharomyces cerevisiae, encodes a protein with a molecular weight of 29,859 Da (residues 1–261) and a calculated isoelectric point of 5.2. The ESTHER database of hydrolase enzymes (http://bioweb.ensam.inra.fr/esther)1 classifies it as a serine hydrolase belonging to the subfamily of hormone-sensitive lipase-like hydrolases, which has over 250 homologs in all kingdoms of life. Mutations in genes of this superfamily are associated with diseases such as autism, goiter, increased risk factor of late onset Alzheimer disease, and xenobiotic sensitivities, including hypersensitivity to acetylcholinesterase inhibitors.1 Here, we report the crystal structure of this putative α/β-serine hydrolase determined using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).2 The structure of YDR428C [Fig. 1(A)] was determined to 1.85 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes two protein monomers (residues 4–261 molecule A and residues 10–261 molecule B), two glycerol molecules, one chloride ion, and 454 water molecules. No electron density was observed for residues 2–3 in chain A and residues 1–9 in chain B. The Matthews' coefficient (Vm)3 for YDR428C is 2.10 Å3/Da and the estimated solvent content is 41.2%. The Ramachandran plot, produced by MolProbity,4 shows that 98% of the residues are in the favored regions and 2% are in additional allowed regions. Crystal structure of YDR428C. A: Stereoview of ribbon diagram of Saccharomyces cerevisiae YDR428C color coded from N-terminus (blue) to C-terminus (red) showing the domain organization. α-Helices H1–H12, and β-strands (β1–β7) are indicated. B: Diagram showing the secondary structure elements in YDR428C superimposed on its primary sequence. The α-helices, 310-helices, β-sheet strands (red A), β-bulges, and γ-turns are indicated. The disordered regions are depicted by a dashed line with the corresponding sequence in brackets. The final model of the YDR428C monomer consists of seven β-strands (β1–β7), with 1324567 topology, twelve α-helices (H1–H12) and four 310-helical segments (H2′, H4′, H7′, H9′) [Fig. 1(A,B)]. The total β-strand, α-helical, and 310-helical content is 16.1%, 53.3%, and 1.9%, respectively. YDR428C contains an α/β hydrolase fold5, 6 with a central, seven-stranded β-sheet, comprised of six parallel β-strands (β2–β7) that is preceded by an additional anti-parallel, N-terminal strand β1 [Fig. 1(A)]. The β-sheet is twisted and flanked by nine α-helices (H1–H5, H9–H12) [Fig. 1(A)]. An additional lid subdomain of three α-helices (H6–H8; residues 155–181) is inserted after the β5 strand [Fig. 1(A,B)]. Similar lid subdomains in several lipase structures have been shown to undergo a conformational change upon substrate binding.7 Further functional and structural studies of YDR428C will be needed to determine if this occurs with this lid subdomain. The α/β-hydrolase fold is usually similar to a scaffold for a catalytic triad of three residues: a nucleophile, His, and an acid residue. As for other α/β hydrolases, YDR428C contains a signature motif of 108-GXNuXG-112 (where Nu = nucleophile) with S110 as its “nucleophile elbow” with energetically-strained, main-chain torsional angles (ϕ = 63°, ψ = −134° for one subunit and ϕ = 54°, ψ = −130° for the other subunit) [Fig. 2(B)].5, 6 The three-dimensional structure of YDR428C reveals that the other two residues of the catalytic triad are composed of D211 and H243 [Fig. 2(A,B)], and could not have been predicted from sequence information alone due to a lack of consensus sequence. These active site residues are located at the bottom of a deep cleft. The residues that line this cleft are entirely hydrophobic (A39, W40, V111, I153, L156, L159, Y166, F169, L213, L214) and are likely to be important for substrate discrimination. A: Ribbon diagram of a superposition of YDR428C (blue) and P. fluorescens carboxylesterase (grey). Residues of the catalytic triad, shown in ball and stick, indicate the active site location. B: Same as A, but a close up view of the catalytic triad. Active site residues as observed in P. fluorescens carboxylesterase (PDB 1auo; grey; P. fluorescens residues shown in parenthesis) and their counterparts in YDR428C (blue) are shown in ball and stick. Hydrogen bond interactions and distances are indicated. C: Ribbon diagram of the YDR428C dimer. β7 strands and helices H10 and H12 forming part of the dimer interface and locations of active sites (arrow) are indicated. A structural similarity search, performed with the coordinates of YDR428C using the DALI server,8 indicates structural similarity to many other α/β hydrolases, but particularly to carboxylesterase from Pseudomonas fluorescens (PDB: 1auo).9 The RMSD is 2.5 Å over 184 aligned residues with 14% sequence identity [Fig. 2(A)]. Another close structural homolog is brefeldin A esterase from Bacillus subtilis (PDB: 1jkm),10 where the RMSD is 2.7 Å over 220 aligned residues with 11% sequence identity. Models for YDR428C homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=YDR428C. For P. fluorescens carboxylesterase, the active site contains a catalytic triad of residues S114, D168, and H199.9 Structurally similar active sites are also present in B. subtilis brefeldin A esterase.10 Interestingly, when superimposed with the P. fluorescens carboxylesterase, three YDR428C residues, S110, D211, and H243, closely overlap with S114, D168, and H199 of the active site with an RMSD of 0.18 Å (all atoms) [Fig. 2(B)]. The same YDR428C residues superimpose with S202, D308, and H338 of the B. subtilis brefeldin A esterase active site with an RMSD of 0.25 Å. The arrangement of the YDR428C catalytic triad is consistent with those of other catalytic triads found in other lipases and esterases.5, 6 Thus, the YDR428C catalytic mechanism and reaction may be similar to those of carboxylesterase or brefeldin A esterase. The noncrystallographic symmetry in the YDR428C crystal results in dimer formation through extensive association of the C-terminal β-strands β7 (residues F233, K234, L235, Y236, L237) of each subunit in an antiparallel fashion leading to a formation of a fourteen-stranded, highly curved β-sheet [Fig. 2(C)]. Additional interactions are formed by the C-terminal α-helices H10 (residues L216, N220, I223, S224, Q227) and H12 (residues D258, N259, C261) from each subunit. This extensive dimer interface accounts for a buried surface area of 1803 Å2 for each monomer.11 The two subunits are believed to act independently because of the distance between their respective active sites (∼36 Å). In support of YDR428C being a crystallographic dimer, the homologous B. subtilis brefeldin A esterase is also a homodimer in solution.10 Interestingly, the structure to YDR428C with equivalent secondary structures forming the dimer interface with the exception being that one of the subunits of brefeldin A esterase is shifted in the plane of the C-terminal β-strands relative to that in the YDR428C dimer. Despite the similarity to brefeldin A esterase, the YDR428C dimerization might still be due to crystal packing. Thus, further functional studies will be needed to confirm its oligomerization state. The YDR428C structure reported here represents a putative hormone-sensitive lipase from yeast, whose structure has been determined by X-ray crystallography using the MAD method. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of the equivalent protein in mammals. YDR428C (TIGR: YDR428C; Swissprot: S69709) was amplified by PCR from genomic DNA from Saccharomyces cerevisiae using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of YDR428C. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag consisting of MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in selenomethionine-containing medium using the E. coli methionine auxotrophic strain DL41. Bacteria were lysed by sonication in lysis buffer (50mM K2HPO4, pH 7.8, 300 mM NaCl, 10% glycerol, 5 mM imidazole, Roche EDTA-free protease inhibitor tablets) with 0.5 mg/ml lysozyme. Immediately after sonication, the cell debris was pelleted by ultracentrifugation at 60,000 g for 20 min (4°C). The soluble fraction was applied to a gravity flow metal chelate column (Talon resin charged with cobalt; Clontech) equilibrated in lysis buffer. The column was then washed with 7 column- volumes (CV) of wash buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 10% glycerol, 10 mM imidazole) and eluted with 3 CV of elute buffer (25 mM, Tris 7.8, 300 mM NaCl, 150 mM imidazole). The protein was then buffer exchanged into 10 mM Tris (pH 7.8), 150 mM NaCl and concentrated to 13 mg/mL by centrifugal ultrafiltration (Orbital). The protein was either frozen in liquid nitrogen for later use or used immediately for crystallization trials. The protein was crystallized using the nanodroplet vapor diffusion method12 with standard JCSG crystallization protocols.2 The crystallization solution contained 17% PEG MME 2000, 10% glycerol, and 100 mM HEPES at pH 7.0. Thecrystals were indexed in the monoclinic space group P21 (Table I). Anomalous diffraction data were collected at the Advanced Photon Source (APS, Argonne, USA) on beamline NE-CAT-8BM at wavelengths corresponding to the inflection point (λ1), high energy remote (λ2), and peak (λ3) of a selenium MAD experiment using the ADSC data collection environment (Table I). The data sets were collected at 100 K using a Quantum 315 CCD detector. Data were integrated, reduced, and scaled using HKL2000.13 Data statistics are summarized in Table I. The structure was determined by using the CCP4 suite14 and SOLVE/RESOLVE.15 Structure refinement was performed using REFMAC5,14 O,16 and Xfit.17 Refinement statistics are summarized in Table I. The final model includes two protein monomers, residues 4–261 for molecule A, and two histidine residues (−1 and 0) of the purification tag, and residues 10–261 for molecule B, 454 water molecules, two glycerol molecules, and a chloride ion. No electron density was observed for residues 2–3 and the first 10 residues of N-terminal expression and purification tag of molecule A, as well as residues 1–9 and the entire tag of molecule B. Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,4 SFcheck 4.0,18 and WHAT IF 5.0.19 Protein quaternary structure analysis used GRASP.11 Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/) and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of YDR428C have been deposited with the PDB and are accessible under the code 1vkh. This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). Data collection was conducted at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source, supported by award RR-15301 from the National Center for Research Resources at the National Institute of Health. Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under contract No. W-31-109-ENG-38.