Purification and crystallization of yeast glycosylphosphatidylinositol transamidase subunit PIG-S (PIG-S_71–467)

The PIG-S_71–467 subunit of the yeast GPI transamidase complex was cloned, overexpressed, purified and crystallized. The crystals belonged to space group C2 and diffracted to 3.2 Å resolution.

Abstract

The transfer of glycosylphosphatidylinositol (GPI) anchors onto eukaryotic proteins is catalyzed by the transamidase complex, which is composed of at least five subunits (PIG-K, PIG-S, PIG-T, PIG-U and GPAA1). Here, the recombinant protein PIG-S_71–467 from Saccharomyces cerevisiae, including residues 71–467 of the entire 534-residue protein, was cloned, expressed and purified to homogeneity. The monodisperse protein was crystallized by the vapour-diffusion method. A diffraction data set was collected to 3.2 Å resolution with 91.6% completeness. The crystals belonged to space group C2, with unit-cell parameters a = 106.72, b = 59.33, c = 124.3 Å, β = 114.19°, and contained two molecules in the asymmetric unit.

1. Introduction

Many eukaryotic proteins are attached to the extracellular side of the plasmalemma by glycosylphosphatidylinositol (GPI) lipid anchoring (Eisenhaber et al., 2003 ▶; Paulick & Bertozzi, 2008 ▶). Examples of GPI-anchored proteins include cell-surface receptors such as urokinase receptor, CD14 and CD16, enzymes such as alkaline phosphatase and 5′-nucleotidase, and carbonic anhydrase, and adhesion and antigen proteins such as Thy-1, DAF and MACIF (Haziot et al., 1988 ▶; Scallon et al., 1989 ▶; Eisenhaber et al., 2003 ▶). Defective GPI-anchor biosynthesis often has biological and medical implications, as in the case of paroxysmal nocturnal haemoglobinuria (Eisenhaber et al., 2003 ▶; Orlean & Menon, 2007 ▶). Disruption of GPI lipid-anchor biosynthesis in knockout experiments leads to embryonic lethality (Nozaki et al., 1999 ▶). The GPI lipid anchor is biosynthesized stepwise by a series of enzymes located in the endoplasmic reticulum (ER) membrane (Eisenhaber et al., 2003 ▶). Typically, the nascent substrate proprotein is exported into the ER after ribosomal synthesis via the signal peptide pathway. The so-called transamidase complex in the ER lumen recognizes a specific GPI-anchor attachment signal located at the C-terminus of protein substrates (Eisenhaber et al., 1998 ▶, 2004 ▶; Eisenhaber & Eisenhaber, 2010 ▶). Removal of the C-terminal pro­peptide and its replacement by a pre-synthesized GPI moiety are catalyzed by the GPI transamidase complex (Eisenhaber et al., 2003 ▶; Orlean & Menon, 2007 ▶). The GPI lipid anchor is a complex organic structure made up of a lipid-modified phosphatidylinositol, a tetrasaccharide with variable elaborations and phosphoethanolamine subunits (Paulick & Bertozzi, 2008 ▶). Although the general outline of the GPI lipid-anchor biosynthesis pathway is common among eukaryotes, there are distinctive taxon-specific differences that might be useful for the design of species-specific inhibitors, as described for the suppression of unicellular parasites such as trypanosomes (Izquierdo et al., 2009 ▶; Urbaniak et al., 2008 ▶).

The human and yeast transamidase complexes consist of at least five different proteins named PIG-K, PIG-T, PIG-U, PIG-S and GPAA1 in humans and Gpi8p, GpI16p, CDC91/GAB1, Gpi17p and GAA1 in yeast (Eisenhaber et al., 2003 ▶; Orlean & Menon, 2007 ▶). All five proteins are essential for GPI transamidase function. PIG-K shares sequence similarity with the cysteine protease of the C13 family and is responsible for C-terminal proteolytic processing of the substrate proteins (Maxwell et al., 1995 ▶; Ramalingam et al., 1996 ▶). It has been revealed that PIG-T is disulfide-linked to PIG-K (Ohishi et al., 2003 ▶) and probably ‘regulates the protein substrate’s access to the enzyme’s catalytic centre’ (Eisenhaber et al., 2003 ▶). The GPAA1 and PIG-U subunits have been suggested to be involved in GPI recognition (Vainauskas & Menon, 2004 ▶; Hong et al., 2003 ▶). PIG-S has been speculated to play a structural role in transamidase substrate-protein selectivity and/or may fix the hydrophobic tail of the substrate protein in the transition state (Eisenhaber et al., 2003 ▶).

Subunits of the GPI transamidase complex for which structures have been solved to date include low-resolution solution structures of PIG-S (yPIG-S_38–467) and PIG-K (yPIG-K_24–337) from Saccharomyces cerevisiae (Toh et al., 2011 ▶). yPIG-K_24–337 consists of two structural domains, an egg-like portion and a small globular segment, which are linked by a 1.9 nm stalk. yPIG-S_38–467 forms an elongated molecule with a larger domain of 10.1 nm in length and 9.1 nm in diameter and a smaller domain of 6.7 nm in length and 3.4 nm in width. The two domains of yPIG-S_38–467 are tilted relative to each other. Overall, the shape of yPIG-S_38–467 appears like an open-hand domain attached to a wrist made up by the smaller domain (Toh et al., 2011 ▶).

Sequence analysis of yeast PIG-S with the ANNIE suite (Ooi et al., 2009 ▶) revealed that the N-terminus (residues 9–29) and the C-­terminus (residues 473–493) contain transmembrane segments, whereby a major region between residues 30 and 472 is predicted to be exposed to the lumenal part of the endoplasmic reticulum. Based on sequence-analytic considerations including primary- and secondary-structure prediction, we generated a new construct, yPIG-S_71–467, with the first residue presumably starting from the second predicted helix after the transmembrane helix. In comparison to yPIG-S_38–467, the new recombinant protein yPIG-S_71–467 showed a higher degree of solubility and enabled us to grow three-dimensional crystals of yPIG-S_71–467 which diffracted to 3.2 Å resolution.

2. Materials and methods

2.1. Cloning and overexpression

The coding region for yPIG-S (amino acids 71–467; UniProt Q04080; EMBL U33007.1) was amplified by PCR using S. cerevisiae genomic DNA (strain AH104) as a template. The forward primer 5′-­CATGCCATGGCATGTGTTCATGACGCTATCCAA-3′ with an NcoI restriction site and the reverse primer 5′-CGAGCTCGTTAGAAGAAATTTTGTTGAACCAT-3′ with a SacI restriction site were used for amplification. The amplified products were ligated into pET9-d1-His3 vector (Grüber et al., 2002 ▶), which was then transformed into Escherichia coli cells (strain RG-2). To express the respective protein, consisting of the N-terminal amino-acid sequence HHH followed by the yPIG-S_71–467 protein sequence, liquid cultures were shaken in LB medium containing chloramphenicol (34 µg ml⁻¹), tetracycline (12.5 µg ml⁻¹) and kanamycin (30 µg ml⁻¹) for about 6 h at 310 K until an optical density OD₆₀₀ of 0.6–0.7 was reached. To induce the production of proteins, cultures were supplemented with isopropyl β-d-1-thiogalactopyranoside to a final concentration of 1 mM followed by overnight incubation at 293 K.

2.2. Protein purification

E. coli cells containing recombinant yPIG-S_71–467 were harvested from 2 l cultures by centrifugation at 8000g for 10 min at 279 K. The cells were lysed on ice in buffer A (50 mM Tris–HCl pH 8, 500 mM NaCl, 2 mM PMSF, 1 mM Pefabloc, 0.8 mM DTT) by sonication using an ultrasonic homogenizer (Bandelin, KE76 tip) for 3 × 1 min. After sonication, the cell lysate was centrifuged at 10 000g for 35 min at 277 K. The resulting supernatant was passed through a filter (0.45 mm pore size) and supplemented with Ni–NTA resin pre-equilibrated in buffer A. The His-tagged protein was allowed to bind to the matrix for 2 h at 277 K by mixing using a sample rotator (Neolab) and was eluted with an imidazole gradient (0–100 mM) in buffer A. Fractions containing the required protein were identified by SDS–PAGE (Laemmli, 1970 ▶), pooled and concentrated using a Millipore spin concentrator with a molecular-mass cutoff of 10 kDa. The sample was applied onto a Resource Q anion-exchange column (6 ml; GE Healthcare) and eluted at room temperature. Prior to injection, the sample was diluted ten times with buffer composed of 50 mM Tris–HCl pH 8 to a final NaCl concentration of 50 mM. Elution was performed with a linear gradient of buffer B (50 mM Tris–HCl pH 8 and 50 mM NaCl) and buffer C (50 mM Tris–HCl pH 8 and 1 M NaCl). Recombinant yPIG-S_71–467 eluted at 21–24% buffer C. Respective fractions were pooled, concentrated in a Millipore spin concentrator and simultaneously exchanged with buffer D (50 mM Tris–HCl pH 8 and 230 mM NaCl). The purity of the protein sample was analyzed by SDS–PAGE (Laemmli, 1970 ▶) stained with Coomassie Brilliant Blue R250. Protein concentration was determined by the bicinchoninic acid assay (BCA; Pierce, Rockford, Illinois, USA).

2.3. Circular-dichroism (CD) spectroscopy

Steady-state CD spectra were measured in the far UV (190–260 nm) using a Chirascan spectropolarimeter (Applied Photophysics). Spectra were collected using a 60 µl quartz cell (Hellma) with a path length of 0.1 mm at 293 K and a step resolution of 1 nm. Readings were obtained for an average of 2 s at each wavelength and the recorded ellipticity values were the average of three determinations for each sample. CD spectroscopy of recombinant yPIG-S_71–467 (2.0 mg ml⁻¹) was performed in a buffer consisting of 50 mM Tris–HCl pH 8 and 200 mM NaCl. The buffer spectrum was sub­tracted from the protein spectrum. CD values were converted to mean residue ellipticity in units of deg cm² dmol⁻¹ using the software Chirascan v.1.2 (Applied Photophysics). This baseline-corrected spectrum was used as an input for computer methods to obtain predictions of secondary structure. In order to analyze the CD spectrum, the following algorithms were used: Varselec (Manavalan & Johnson, 1987 ▶), Selcon (Sreerama & Woody, 1993 ▶), CONTIN (Provencher, 1982 ▶), K2D (Andrade et al., 1993 ▶) (all as incorporated into the program DICROPROT; Deléage & Geourjon, 1993 ▶) and CDNN (Böhm et al., 1992 ▶). The normalized root-mean-square deviation (NRMSD) was calculated according to Whitmore & Wallace (2004 ▶).

2.4. Crystallization conditions

The purified protein was concentrated to 10 mg ml⁻¹ in 50 mM Tris pH 8, 230 mM NaCl using a 10 kDa cutoff concentrator and stored at 193 K prior to crystallization. Preliminary screening for initial crystallization conditions was performed by the hanging-drop vapour-diffusion method using screens from Hampton Research at 296 K by mixing 1 µl droplets of concentrated protein solution with an equal volume of reservoir solution and equilibrating against 500 µl reservoir solution using 24-well Cryschem plates (Hampton Research, USA). Further screenings to find optimal crystallization conditions were performed by varying the protein concentrations, using different additives (Additive Screen from Hampton Research) and seeding method. Microseeding was performed by picking needle crystals into a seed bead (Hampton Research) containing 100 µl of well solution from the same drop, which was mixed for 90 s. Three different seed stocks were prepared from the seed solution by serial dilution (10⁻¹, 10⁻² and 10⁻³) in well solution and 0.2 µl seed stock was added to the protein drop. Finally, a good diffraction-quality plate-shaped native crystal was grown at 3 mg ml⁻¹ protein concentration in 0.1 M Tris–HCl pH 8.5, 2 M ammonium sulfate, 10 mM cadmium chloride.

2.5. X-ray data collection and analysis

The crystal was quick-soaked in a cryoprotectant solution con­sisting of 20% glycerol in mother liquor and flash-cooled in liquid nitrogen at 100 K. Single-wavelength data sets were collected from yPIG-S_71–467 crystals at 140 K on beamline 13B1 at the National Synchrotron Radiation Research Centre (NSRRC, Hsinchu, Taiwan) using an ADSC Quantum 315 CCD detector. The diffraction data were indexed, integrated and scaled using the HKL-2000 suite of programs (Otwinowski & Minor, 1997 ▶). Data-collection statistics are given in Table 1 ▶.

Table 1. Statistics of crystallographic data collection and processing for yPIG-S_71–467 .

Values in parentheses are for the highest resolution shell.

Wavelength (Å)	1.5417
Space group	C2
Unit-cell parameters (Å, °)	a = 106.72, b = 59.33, c = 124.3, α = γ = 90.0, β = 114.19
Resolution range (Å)	50–3.2 (3.31–3.20)
No. of unique reflections	10981 (1066)
Total No. of reflections	19587
Completeness (%)	91.6 (92.1)
Rmerge† (%)	13.2 (40.8)
Multiplicity	2.8 (2.6)
Mean I/σ(I)	6.9 (2.4)
Mosaicity (°)	0.93
Wilson B factor (Å2)	50.1

^† R _merge = × 100%, where 〈I(hkl)〉 is the average intensity of reflection hkl and I _i(hkl) is the intensity of the ith measurement of reflection hkl.

3. Results and discussion

3.1. Protein characterization

SDS–PAGE of recombinant yPIG-S_71–467 revealed a prominent band of about 50 kDa found entirely within the soluble fraction, which was purified by affinity and ion-exchange chromatography (Fig. 1 ▶ a). The higher migration of the recombinant protein (sequence-based predicted mass of 45.2 kDa, including the His tag) in gel electrophoresis may be attributed to high hydration of the protein in solution, which is characteristic for extended molecules with large specific accessibility to solvent, and supports the recently determined elongated shape of yeast PIG-S_38–467 of 16.8 nm in length (Toh et al., 2011 ▶). Based on secondary-structural predictions from the amino-acid sequence of yPIG-S_71–467, the secondary-structure content was 49% α-helical, 17% β-sheet and 34% random coil. The secondary structure of this protein was determined from the circular-dichroism spectrum (Fig. 1 ▶ b), resulting in a secondary-structure composition of 52% α-helix, 18% β-sheet and 30% random coil.

Figure 1.

(a) SDS–PAGE (17% total acrylamide and 0.4% cross-linked acrylamide) of the purified recombinant yPIG-S_71–467; lane 1 contains protein marker (labelled in kDa). (b) Far-UV CD spectrum of yPIG-S_71–467 (2 mg ml⁻¹).

3.2. Crystallization and preliminary X-ray analysis

In the initial screening, small needle-shaped crystals were obtained after several days in condition No. 4 of Crystal Screen from Hampton Research (0.1 M Tris pH 8.5, 2.0 M ammonium sulfate) at 293 K. Attempts to optimize this condition by further screening identified an additive (cadmium chloride hydrate) that produced a cluster of needles. Microseeding with these needle crystals using a seed bead (Hampton Research) yielded good diffraction-quality plate-shaped single crystals. Finally, optimized native crystals were grown at 3 mg ml⁻¹ protein concentration in 0.1 M Tris–HCl pH 8.5, 2 M ammonium sulfate, 10 mM cadmium chloride. A typical crystal is shown in Fig. 2 ▶ with dimensions of 0.08 × 0.22 × 0.02 mm. These crystals diffracted to 3.2 Å resolution (Fig. 3 ▶) and belonged to space group C2, with unit-cell parameters a = 106.72, b = 59.33, c = 124.3 Å, β = 114.19°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was 39% and V _M was 2.1 ų Da⁻¹ (Matthews, 1968 ▶). Research is in progress to determine the three-dimensional structure of yPIG-S_71–467.

Figure 2.

Crystals of yPIG-S_71–467. The crystals are approximately 0.08 × 0.22 × 0.02 mm in size.

Figure 3.

Diffraction pattern of yPIG-S_71–467. The ring indicates 3 Å resolution.