Crystallization of the virulent and benign subtilisin-like proteases from the ovine footrot pathogen Dichelobacter nodosus

Abstract

Dichelobacter nodosus is the principal causative agent of ovine footrot, a disease of significant economic importance to the sheep industry. D. nodosus secretes a number of subtilisin-like serine proteases which mediate tissue damage and presumably contribute to the pathogenesis of footrot. Strains causing virulent footrot secrete the proteases AprV2, AprV5 and BprV and strains causing benign footrot secrete the closely related proteases AprB2, AprB5 and BprB. Here, the cloning, purification and crystallization of AprV2, AprB2, BprV and BprB are reported. Crystals of AprV2 and AprB2 diffracted to 2.0 and 1.7 A resolution, respectively. The crystals of both proteases belonged to space group P1, with unit-cell parameters a = 43.1, b = 46.0, c = 47.2 A, alpha = 97.8, beta = 115.2, gamma = 115.2 degrees for AprV2 and a = 42.7, b = 45.8, c = 45.7 A, alpha = 98.4, beta = 114.0, gamma = 114.6 degrees for AprB2. Crystals of BprV and BprB diffracted to 2.0 and 1.8 A resolution, respectively. The crystals of both proteases belonged to space group P2(1), with unit-cell parameters a = 38.5, b = 89.6, c = 47.7 A, beta = 113.6 degrees for BprV and a = 38.5, b = 90.5, c = 44.1 A, beta = 109.9 degrees for BprB. The crystals of all four proteases contained one molecule in the asymmetric unit, with a solvent content ranging from 36 to 40%.

Figure 1

(a) Crystals of AprB2. The crystals are 80 µm in the longest dimension. (b) Crystals of AprV2. The crystals are 60 µm in the longest dimension. (c) Crystals of BprB. The crystals are 80 µm in the longest dimension. (d) Crystals of BprV. The crystals are 60 µm in the longest dimension.

Figure 2

Diffraction patterns from AprV2 (a), AprB2 (b), BprV (c) and BprB (d) crystals. The resolution limit at the edge of the plate is 2.0 Å for AprV2 and BprV, 1.6 Å for AprB2 and 1.8 Å for BprB.