Cloning, purification, crystallization and preliminary crystallographic analysis of a penicillin-binding protein homologue from Pyrococcus abyssi

The crystallization of a hypothetical penicillin-binding protein from the archaeon P. abyssi in space group C2 by hanging-drop vapour diffusion is reported.

Abstract

The genome of the hyperthermophilic archaeon Pyrococcus abyssi contains a gene (pab0087) encoding a penicillin-binding protein (PBP) homologue. This sequence consists of 447 residues and shows significant sequence similarity to low-molecular-weight PBPs and class C β-lactamases. The Pab0087 protein was overexpressed, purified and crystallized. Diffraction data from two different crystal forms were collected to 2.7 and 2.0 Å resolution. Both crystals belong to space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 Å, β = 117.36° and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81°, respectively. The asymmetric unit contains four and eight molecules, respectively, with fourfold non-crystallographic symmetry.

1. Introduction

In bacteria, the last step in cell-wall biosynthesis is catalyzed by d,d-­transpeptidases. These enzymes are members of the family of penicillin-binding proteins (PBPs), wich are separated into two groups: high-molecular-weight PBPs and low-molecular-weight PBPs (Ghuysen, 1991 ▶). PBPs are the essential target of β-lactams, which are analogues of their natural substrate d-Ala:d-Ala (Tipper & Strominger, 1965 ▶). This class of antibiotics acts by inactivating PBPs through acylation of their catalytic serine.

Production of β-lactamases is the major resistance mechanism that has emerged as a result of intensive use of β-lactam antibiotics. These enzymes catalyze the irreversible hydrolysis of the β-lactam ring, thus yielding inactive antibiotics. Based on their primary sequence, β-­lactamases are separated into four molecular classes: A, B, C and D (Ambler, 1980 ▶). Classes A, C and D are active serine enzymes and follow a catalytic pathway similar to that of PBPs. Class B β-lactamases are zinc metalloenzymes.

Low-molecular-weight PBPs and β-lactamases are both members of the penicillin-recognizing protein family (PRPs) and exhibit the same overall structural fold: they are composed of a single domain containing two regions, one α/β and one all-helical, with the catalytic site lying between them. PRPs also possess three conserved sequence motifs close to the active-site serine which are directly or indirectly involved in substrate recognition and in the catalytic pathway (S-X-X-K containing the active serine, Y/S-X-N and K/H-T-G).

Pyrococcus abyssi is a hyperthermophilic archaeon with an optimal growth temperature of 369 K (Erauso et al., 1993 ▶) and its genome has been completely sequenced (Cohen et al., 2003 ▶). Analysis of the genome revealed the presence of an amino-acid sequence homologous to those of low-molecular-weight PBPs and class C β-lactam­ases (Lecompte et al., 2001 ▶). This protein of 50.4 kDa, called Pab0087, shows an amino-acid sequence identity of 20–25% with PBPs and class C β-lactamases and possesses the three conserved catalytic elements. Currently, little information is available about the archaeal cell wall (Kandler & König, 1998 ▶), but the homology of Pab0087 to PBPs suggest that it may play a role in the biosynthesis of a pseudopeptidoglycan in archaea. Thus, the three-dimensional structure of Pab0087 will provide for the first time data about a non-bacterial PRP member and will further our understanding of the evolution of the PRP family as well as of archaeal cell-wall biosynthesis.

Here, we describe the cloning, expression, purification and crystallization of the P. abyssi Pab0087 protein. Preliminary X-ray analyses are reported for two crystal forms, both belonging to space group C2.

2. Materials and methods

2.1. Cloning, expression and purification

The predicted open reading frame encoding the hypothetical β-­lactamase Pab0087 was amplified by PCR from genomic DNA of P. abyssi. The PCR-amplified fragment was cloned into the pPCR-Script Cam SK(+) cloning vector (Stratagene). The DNA was subcloned into pET29a (Novagen). Escherichia coli BL21-CodonPlus (DE3) RIL (Stratagene) cells freshly transformed with pET29a/pab0087 were grown at 310 K overnight in LB medium containing 30 µg ml⁻¹ kanamycin and 40 µg ml⁻¹ chloramphenicol. At an OD₆₀₀ of 0.6, IPTG was added to a final concentration of 1 mM and cells were grown at 310 K for 3 h. Following harvesting, the cell pellet was resuspended in 50 mM Tris–HCl buffer pH 8.5 and lysed by sonication. The cell lysate obtained by centrifugation at 27 000g for 20 min was heated at 363 K for 15 min and then centrifuged for 1 h at 27 000g. The supernatant was treated with spermine at a final concentration of 14 mM. After an additional centrifugation for 15 min at 12 000g, the supernatant was filtered (Minisart 0.45 µm, Sartorius) and concentrated (Amicon Ultra 10K 15 ml, Millipore). This extract was injected onto a 5 ml Q-Sepharose Hi-Trap column pre-equilibrated in 50 mM Tris–HCl buffer pH 8.5. The protein Pab0087 was eluted with a linear gradient of 0–1 M NaCl and eluted at 300 mM. Fractions containing Pab0087 were pooled, concentrated and then injected onto a Superdex 75 HR 10/30 gel-filtration column (Amersham Biosciences) pre-equilibrated in 50 mM Tris–HCl buffer pH 8.5, 300 mM NaCl. The protein Pab0087 was eluted with an apparent molecular weight of approximately 112 kDa, indicating that Pab0087 is a dimer under these conditions.

2.2. Crystallization

The protein was concentrated to 12 mg ml⁻¹ in 50 mM Tris–HCl buffer pH 8.5, 300 mM NaCl using an Amicon Ultra 10K 4 ml (Millipore). Crystals of Pab0087 were grown by vapour diffusion in 2 µl hanging drops comprising equal volumes of protein and reservoir solution at 291 K. After 2–3 d incubation, crystals were observed in two conditions containing 300 mM NaCl, 200 mM KSCN, 8% PEG 20K, 8% PEG 550 MME pH 8.4 (solution No. 23, Clear Strategy Screen 1 from Molecular Dimension Ltd) for crystal form 1 and 20 mM CaCl₂, 30% MPD pH 4.6 (solution No. 1, Crystal Screen from Hampton Research) for crystal form 2. The average dimensions of these crystals were 250 × 150 × 20 µm (Fig. 1 ▶ a) and 200 × 40 × 40 µm (Fig. 2 ▶ b), respectively.

Figure 1.

(a) Crystal form 1 of Pab0087 obtained in 300 mM NaCl, 200 mM KSCN, 8% PEG 20K, 8% PEG 550 MME pH 8.4. The crystal size was 250 × 150 × 20 µm. (b) Crystal form 2 of Pab0087 obtained in 20 mM CaCl₂, 30% MPD pH 4.6. The crystal size was 200 × 40 × 40 µm.

Figure 2.

Self-rotation function calculated using crystal form 1 data with orthogonalization code 1 (a, c*^a, c*). The integration radius is 25 Å and the resolution limits are 12–3 Å. (a) χ = 90° section. The peak indicates that the fourfold non-crystallographic axis is parallel to c*. (b) χ = 180° section. This plot shows the twofold crystallographic axis parallel to b, the fourfold non-crystallographic axis and three twofold non-crystallographic axis (one parallel to a and two orthogonal to c*).

2.3. X-ray diffraction analysis

Crystals were mounted in a cryoloop and flash-frozen in the presence of 30% glycerol for crystal form 1 or directly from the drop for crystal form 2. Diffraction data were collected using a MAR 345 detector on a Micromax 7 rotating-anode generator at the Institut de Biologie et Physico-Chimie, Paris and using a MAR CCD detector at beamline FIP-BM30A of the European Synchrotron Radiation Facility, Grenoble, respectively. The crystal form 1 data set was collected to 2.7 Å resolution with a 360° sweep and that for form 2 was collected to 2.0 Å resolution with a 180° sweep. Data were processed with XDS and scaled with XSCALE (Kabsch, 1988 ▶). Autoindexing and consideration of systematically absent reflections revealed that the two crystal forms belong to the monoclinic space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 Å, β = 117.36° and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81°, respectively. Data-collection statistics of the two crystal forms from protein Pab0087 are reported in Table 1 ▶.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

	Pab0087 form 1	Pab0087 form 2
X-ray source	Micromax 7 rotating anode	FIP-BM30A
Space group	C2	C2
Unit-cell parameters (Å, °)	a = 160.59, b = 135.74, c = 113.02, β = 117.36	a = 166.99, b = 131.27, c = 189.41, β = 113.81
Wavelength (Å)	1.5418	0.97976
Resolution range (Å)	50–2.7 (2.8–2.7)	20–2.0 (2.12–2.0)
No. of observations	517326 (52727)	934274 (141608)
Unique reflections	58345 (5933)	244085 (38374)
Completeness (%)	98.8 (98.3)	97.0 (95.4)
Redundancy	8.86 (8.88)	3.83 (3.69)
〈I/σ(I)〉	24.1 (6.8)	26.58 (5.93)
Rsym† (%)	7.9 (33.3)	3.9 (32.2)
Subunits per ASU	4	8
VM (Å3 Da−1)	2.71	2.37
Unit-cell volume (Å3)	2.2 × 106	3.8 × 106

^† R _sym = , where I is the intensity of an individual reflection and 〈I〉 is its mean value.

Using the program MOLREP (Vagin, 1997 ▶) from the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶), self-rotation functions were computed for each crystal form to test for the presence of non-crystallographic symmetry. A self-rotation function of the form 1 data set indicates the presence of a crystallographic twofold axis, three twofold axes and one perpendicular fourfold axis, suggesting that the protein is a tetramer in the crystal (Figs. 2 ▶ a and 2 ▶ b). Four molecules per asymmetric unit correspond to a Matthews coefficient (Kantardjieff & Rupp, 2003 ▶) of 2.71 ų Da⁻¹ and 53.4% solvent content. Self-rotation functions of the form 2 data set reveal the presence of a crystallographic twofold axis, two fourfold axes and several twofold axes generated by combination of the crystallographic twofold and fourfold axes (Figs. 3 ▶ a and 3 ▶ b). Thus, there are two non-parallel tetramers in the asymmetric unit. Eight molecules per asymmetric unit, totalling 403.2 kDa, correspond to a Matthews coefficient of 2.37 ų Da⁻¹ and 48.1% solvent content.

Figure 3.

Self-rotation function calculated using crystal form 2 data with orthogonalization code 1 (a, c*^a, c*). The integration radius is 25 Å and the resolution limits are 12–3 Å. (a) χ = 90° section. (b) χ = 180° section.

3. Conclusion

The Pab0087 protein from P. abyssi has been crystallized in two forms. Analysis of the X-ray data from these crystals revealed that Pab0087 crystallized in space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 Å, β = 117.36° for form 1 and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81° for form 2. Calculation of self-rotation functions revealed the presence of one tetramer per asymmetric unit in crystal form 1 and two tetramers in crystal form 2. As the PRPs fold is highly conserved, we are attempting to solve the structure of Pab0087 by molecular replacement using AMoRe (Navaza, 1994 ▶) with the available structures of similar PRPs as the starting model. In parallel, the production of selenomethionine-substituted protein and a search for heavy-atom derivatives are in progress. We have already used form 2 crystals for soaking in solution containing holmium (HoCl₃; Heavy-Atom Screen M2 from Hampton Research). Analyses of the diffraction data from this heavy-atom derivative and other derivatives will allow us to solve the structure by MIR/MAD methods.