Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain

The crystallization of the precursor of a thermostable variant of papain and the collection of diffraction data to 2.6 Å resolution are reported.

Abstract

The crystallization of a recombinant thermostable variant of pro-papain has been carried out. The mutant pro-enzyme was expressed in Escherichia coli as inclusion bodies, refolded, purified and crystallized. The crystals belonged to space group P2₁, with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, β = 93.0°, and diffracted to 2.6 Å resolution using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 ų Da⁻¹, corresponding to a solvent content of 46%. Initial attempts to solve the structure using molecular-replacement techniques were successful.

1. Introduction

Papain-like cysteine proteases represent a major component of the lysosomal proteolytic enzymes and are involved in various biological functions (Dickinson, 2002 ▶); the enzymes from plant sources are often used in industry (Grzonka et al., 2007 ▶). The cysteine proteases are expressed as an inactive precursor in a pre-pro-enzyme form which contains a signal peptide (pre-region), an inhibitory pro-region and a mature catalytic domain. The pro-region is generally cleaved at acidic pH to produce the active enzyme (Brocklehurst et al., 1985 ▶). The pro-peptide parts of the cysteine proteases are essential for the correct folding, transport, maturation and regulation of activity of the enzyme (Winther & Sørensen, 1991 ▶; Baker et al., 1992 ▶; Wetmore et al., 1992 ▶; Fukuda et al., 1994 ▶). It has been observed that the pro-parts of cysteine proteases act as effective inhibitors of their cognate enzymes and also to some extent of other cysteine proteases of the same family (Mach et al., 1994 ▶; Taylor et al., 1995 ▶; Fox et al., 1992 ▶).

Knowledge of the three-dimensional structure of a pro-protease will help us to better understand the molecular basis of inhibition by the pro-region and its role in the activation and folding processes of the enzyme. Although the crystal structure of mature papain is known (Drenth et al., 1968 ▶), no report exists on the structure of pro-papain. Attempts to crystallize pro-papain in our laboratory were not successful, mainly because it precipitated during the concentration of the protein for crystallization trials. However, we could generate a thermostable variant (pro-pap-RSS) of pro-papain with three mutations in the interdomain region, K174R, G36S and V32S; the other functional properties of pro-pap-RSS (apart from the enhancement of thermostability) remained the same as those of the wild-type pro-papain (Choudhury et al., 2010 ▶). We have performed a preliminary X-­ray analysis of this variant of pro-papain with the aim of deciphering the three-dimensional structure of the pro-region and its interactions with the catalytic domain. A recombinant mutant of the variant was constructed by replacing the active-site cysteine residue Cys25 with an alanine (pro-pap-RSS-C25A; Fig. 1 ▶) for this study in order to avoid the autocatalytic activation process.

Figure 1.

A schematic representation of recombinant pro-pap-RSS-C25A expressed in E. coli using pET-30 Ek/LIC vector. (a) Recombinant pro-pap-RSS-C25A with a 44-amino-acid vector sequence with a hexahistidine tag at the N-terminus (black line), a 107-residue pro-region (blue bar) and a 212-residue mature catalytic domain (orange bar). The positions of the mutated residues are shown. (b) The amino-acid sequence of the pro and mature domains of pro-pap-RSS-C25A are shown in the corresponding colours, with the four mutations indicated in bold.

2. Materials and methods

2.1. Expression and purification

The thermostable variant of pro-papain, pro-pap-RSS, cloned in pET-30 Ek/LIC (Novagen, USA; Choudhury et al., 2010 ▶) was used to generate the pro-pap-RSS-C25A mutant following the standard protocol of the QuikChange site-directed mutagenesis kit (Stratagene, USA) with forward primer 5′-CAGGGTTCTTGTGGTAGTGCGTGGGCATTCTCAGCTGTTG-3′ and reverse primer 5′-CAACAGCTGAGAATGCCCACGCACTACCACAAGAACCCTG-3′. The PCR reaction involved 16 cycles of amplification using Pfu DNA polymerase (according to the manufacturer’s instructions) and the amplified product was transformed into Escherichia coli strain DH10B. The identity of the mutant clone was verified by automated DNA sequencing using the MegaBACE 1000 sequencing system (Amersham Biosciences, USA).

A recombinant clone with the desired mutation was transformed into E. coli strain BL21 (DE3) and a single colony was cultured in LB medium with 50 µg ml⁻¹ kanamycin at 310 K to an OD₆₀₀ of 0.6–0.8 and then induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 5 h with vigorous shaking. The cells were harvested by centrifugation, resuspended in a buffer con­sisting of 20 mM Tris–HCl pH 8.0, 1% Triton X-100, 5% sucrose and 2.5 mM DTT and lysed by sonication. The recombinant protein was expressed as inclusion bodies and the expressed protein was solubilized in urea, purified on an Ni–NTA (Novagen, USA) column and refolded using the dilution method as described previously (Choudhury et al., 2009 ▶).

After refolding, the protein was concentrated and purified further by gel-filtration chromatography on a Sephacryl S-100 (Amarsham, USA) column that had been pre-equilibrated with 20 mM Tris–HCl pH 8.0, 150 mM NaCl and 1% glycerol. The protein was eluted in the same buffer and the peak fractions were collected. The purity of the protein was checked by SDS–PAGE analysis. Around 300 mg refolded and purified protein was obtained from 1 l bacterial culture.

2.2. Crystallization and data collection

The data were processed and scaled using HKL-2000 (Otwinowski & Minor, 1997 ▶) and the data-collection and processing statistics are summarized in Table 1 ▶.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

Data-collection parameters
X-ray source	BM14, ESRF, Grenoble, France
Wavelength (Å)	0.9785
Temperature (K)	100
Oscillation range (°)	1
Crystal-to-detector distance (mm)	279.95
Data-integration statistics
Space group	P21
Unit-cell parameters (Å, °)	a = 42.9, b = 74.8, c = 116.5, β = 93.0
Mosaicity (°)	1.04
Resolution range (Å)	50–2.6 (2.64–2.60)
Observed reflections	157456
Unique reflections	21685
Multiplicity	7.3 (6.9)
Completeness (%)	97.0 (94.6)
Rmerge† (%)	5.7 (68.2)
〈I/σ(I)〉	28.4 (2.0)

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

3. Results and discussion

Recombinant pro-pap-RSS-C25A was expressed in inclusion bodies in E. coli. The purity and molecular weight (41 kDa) of the refolded purified protein were checked by SDS–PAGE (Fig. 2 ▶) and it was concentrated to 5 mg ml⁻¹ for crystallization trials. Diffraction-quality crystals (of dimensions ∼300 × 200 × 100 µm) were obtained at 293 K after about 70 d (Fig. 3 ▶) using the hanging-drop vapour-diffusion method in condition No. 37 of Crystal Screen (Hampton Research, USA). The pro-pap-RSS-C25A crystals diffracted to 2.6 Å resolution (Fig. 4 ▶) and belonged to space group P2₁, with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, β = 93.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 ų Da⁻¹ with a solvent content of 46% (Matthews, 1968 ▶). A model of a chimeric pro-protease structure was generated from the structures of mature native papain (PDB code 9pap; Kamphuis et al., 1984 ▶) and the pro-domain of pro-caricain (PDB code 1pci; Groves et al., 1996 ▶), which has 89% identity and 98% similarity in amino-acid sequence to the pro-region of pro-pap-RSS-C25A. This model, with mismatched residues mutated to alanine, was subsequently used as a search model in molecular-replacement calculations using MOLREP (Vagin & Teplyakov, 2010 ▶) from the CCP4 program suite (Winn et al., 2011 ▶). The cross-rotation function for this model was calculated for the entire resolution range and the first two peaks in the output appeared as distinct solutions. These two peaks became more prominent in the translation calculations, with an overall correlation coefficient of 56.0% and an R factor of 45.3%. This solution revealed good crystal packing and no clashes between symmetry-related molecules. Further analysis is in progress.

Figure 2.

15% SDS–PAGE analysis of purified pro-pap-RSS-C25A. Lane M, protein molecular-weight markers (kDa); lane 1, purified mutant protein after gel-filtration chromatography. The gel was stained with Coomassie Brilliant Blue R-250.

Figure 3.

A crystal of pro-pap-RSS-C25A. The approximate crystal dimensions are 300 × 200 × 100 µm.

Figure 4.

Diffraction of a pro-pap-RSS-C25A crystal. The resolution at the edge of the plate is 2.6 Å.