Crystallization and preliminary crystallographic analysis of Achromobacter protease I mutants

Achromobacter protease I (API), a lysine-specific serine protease, exhibits an order of magnitude higher activity than bovine trypsin, while its optimum pH is in the alkaline region at approximately pH 9. Two single point mutants have previously been reported to improve the optimum pH range; determination of the tertiary structure of API mutants would help in understanding these mechanisms.

Abstract

Achromobacter protease I (API), a serine protease, shows an order of magnitude higher activity than bovine trypsin. The optimum pH of mutant enzymes with His210 replaced by Ser (H210S) and Trp169 replaced by Phe (W169F) has been shown to shift from approximately pH 9 (wild-type enzyme) to approximately pH 7 while retaining high activity. The mutants were crystallized by the hanging-drop vapour-diffusion technique with 2 M ammonium sulfate as the precipitant. The space group of the W169F mutant crystal was P1, with unit-cell parameters a = 42.6, b = 34.7, c = 69.5 Å, α = 91.8, β = 97.5, γ = 89.9°, while the space group of the H210S mutant crystal was P2₁, with unit-cell parameters a = 42.4, b = 34.2, c = 67.7 Å, β = 97.6°. Diffraction data were collected from W169F and H210S crystals to resolutions of 2.0 and 2.3 Å, respectively.

1. Introduction

Achromobacter protease I (API) consists of 268 amino-acid residues with a low sequence similarity to other serine proteases (approximately 10%). API has several unique properties that are distinct from those of other trypsin-type serine proteases, such as a relatively higher protease activity than bovine trypsin, a wide pH optimum ranging from 8.5 to 10.7, restricted Lys specificity, including the Pro–Lys bond, and structural stability against denaturation with 0.1% SDS and 4 M urea (Masaki, Fujihashi et al., 1981 ▶; Masaki, Tanabe et al., 1981 ▶). Because of these unique properties, API has been an effective tool for peptide fragmentation in protein-sequence analysis (Morihara et al., 1986 ▶; Sakiyama & Masaki, 1994 ▶). However, its property of having its optimum pH shift in the alkaline region is a disadvantage, because a neutral pH (typically pH 6–7) is preferred when a protease cleaves a substrate protein to peptide fragments (Tsunasawa et al., 1989 ▶).

X-ray crystallographic analysis of API (PDB code 1arb; Tsunasawa et al., 1989 ▶) revealed that the apparent tertiary structure of the protein is similar to that of bovine trypsin. However, a careful structural comparison between API and trypsin-type serine proteases showed that API has a critically different arrangement of aromatic amino acids around the active site (PDB code 1arb). We previously reported the contribution of the electrostatic interaction Asp113–His210, which is supported by Trp169, and the optimum pH shift of the His210 mutant from about pH 9 (in the wild-type enzyme) to approximately pH 7 while retaining high activity. In particular, we previously reported that the k _cat/K _m of H210S is two times higher than that of wild-type API (Shiraki & Sakiyama, 2002 ▶). In contrast, the k _cat/K _m of W169F is similar to that of wild-type API (Shiraki, Norioka, Li & Sakiyama, 2002 ▶). The molecular mechanism has not been elucidated, but we have hypothesized that the structure of the aromatic stacking between His210 (in the substrate-binding S1 site) and Trp69 (in close proximity to subsite S1) plays an important role in the high catalytic potency of API. In this article, we present preliminary X-ray studies of the API mutants. The H210S mutant crystallized in space group P2₁, whereas the W169F mutant crystallized in space group P1.

2. Methods and results

2.1. Protein expression and purification

Single-stranded DNA for mutagenesis was obtained from plasmid pKYN200 (Ohara et al., 1989 ▶). Mutagenesis was performed according to the uracil-DNA-mediated method (Kunkel, 1985 ▶). The mutagenic primers were 5′-CGGCGCCGCCGCCGAAGGCCACGAACG-3′ for the W169F mutant and 5′-CGACGGGCCCCCGGACAGCTG­GCCGAGCAC-3′ for the H210S mutant. Escherichia coli strain JM109 was used for preparation of the plasmid and phage DNA. The mutant genes were identified by DNA sequencing and then sub­cloned into the expression vector pKYN200. Transformants of E. coli strain JA221 cells were grown on Luria–Bertani medium supplemented with 50–100 µg ml⁻¹ ampicillin. The expressed mutated APIs were purified by affinity chromatography on hen egg-white ovo­mucoid. A periplasmic fraction prepared from 1 l culture medium was passed through a DEAE-cellulose column (3.6 × 50 cm) equilibrated with 10 mM Tris–HC1 pH 9.0. After the pH of the unadsorbed fraction had been adjusted to 8.0 by the addition of glacial acetic acid, the fraction was applied onto an ovomucoid Sepharose 4B column (chicken ovomucoid content 0.3 mmol per millilitre of wet gel; 1.6 × 25 cm) equilibrated with 50 mM Tris–HCl pH 8.0. After the column had been successively washed with 50 mM Tris–HCl pH 8.0 and distilled water, the adsorbed mutated API was eluted with 10 mM ammonium acetate pH 3.0. The eluted solution was immediately neutralized carefully by adding diluted ammonium hydroxide since API is unstable below pH 4.0 (Norioka et al., 1994 ▶). The amount of mutant protein obtained from 1 l culture medium was 0.2–0.4 mg.

2.2. Crystallization

The API mutants H210S and W169F were prepared as described previously (Shiraki & Sakiyama, 2002 ▶; Shiraki, Norioka, Li & Sakiyama, 2002 ▶; Shiraki, Norioka, Li, Yokota et al., 2002 ▶). The API mutants were crystallized using the hanging-drop vapour-diffusion method. Hanging drops were obtained by mixing 5.0 µl protein solution (10–20 mg ml⁻¹ in 10 mM Tris–HCl pH 8.0) and 5.0 µl reservoir solution and were equilibrated against 1 ml reservoir solution consisting of 2 M ammonium sulfate and 5% 2-propanol in 10 mM Tris–HCl pH 8.0 at room temperature; the crystals reached dimensions of above 0.2 × 0.2 × 0.1 mm after one week.

2.3. X-ray diffraction study

Crystals were mounted and sealed in a glass capillary with a small amount of mother liquor. X-ray diffraction data for the W169F and H210S mutant crystals were collected on R-AXIS IIc and R-AXIS IV detectors, respectively, using a rotating-anode generator at room temperature. The W169F crystal, which belonged to space group P2₁ with unit-cell parameters a = 42.6, b = 34.7, c = 69.5 Å, α = 91.8, β = 97.5, γ = 89.9°, diffracted to 2.0 Å resolution. The H210S crystal, which belonged to space group P2₁ with unit-cell parameters a = 42.4, b = 34.2, c = 67.7 Å, α = γ = 90.0, β = 97.6°, diffracted to 2.3 Å resolution. Data were processed using the DENZO and SCALEPACK programs (Otwinowski, 1992 ▶) for the W169F mutant crystal and the MOSFLM (Leslie, 1992 ▶) and SCALA programs from the CCP4 package (Collaborative Computational Project, Number 4, 1994 ▶) for the H210S mutant crystal. The data-processing statistics are shown in Table 1 ▶.

Table 1. Data-collection and diffraction data statistics.

Values in parentheses are for the highest resolution shell.

	H210S	W169F
Detector	R-AXIS IV	R-AXIS IIc
Wavelength (Å)	1.5418	1.5418
Crystal-to-detector distance (mm)	120	100
Oscillation angle (°)	1.8	1.5
Total oscillation range (°)	183.6	180
Space group	P21	P1
Unit-cell parameters
a (Å)	42.4	42.6
b (Å)	34.2	34.7
c (Å)	67.7	69.5
α (°)	90.0	91.8
β (°)	97.6	97.5
γ (°)	90.0	89.9
Resolution (Å)	41.88–2.30 (2.42–2.30)	68.89–2.00 (2.07–2.00)
Multiplicity	3.6 (3.6)	1.9 (1.8)
Completeness (%)	99.8 (99.3)	86.5 (71.0)
Rmerge†	0.089 (0.165)	0.085 (0.271)
〈I/σ(I)〉	14.4 (8.2)	10.8 (2.6)

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

Assuming the presence of one molecule of the H210S mutant in the asymmetric unit, the V _M value (Matthews, 1968 ▶) was calculated to be 1.7 ų Da⁻¹, corresponding to a solvent content of 26.2%. The V _M value calculation for the W169F mutant (V _M = 1.8 ų Da⁻¹, corresponding to a solvent content of 29.4%) indicated the probable presence of two molecules in the asymmetric unit, designated chains A and B. The crystal structure was determined using the molecular-replacement (MR) method. The MR calculation was performed using MOLREP (Vagin & Teplyakov, 1997 ▶) from the CCP4 package. The structure of API (PDB code 1arb) was used as the search model. The R factor and correlation coefficient for the H210S mutant obtained from the MR calculations were 33.8% and 69.2%, respectively. The R factor and correlation coefficient for the W169F mutant obtained from the MR calculations were 34.8% and 69.5%, respectively. Structure determination and refinement are currently under way.