Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB

Abstract

The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB(BR)) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB(BR) buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB(BR) in each buffer. While both sets of conditions supported crystal growth in space group P2(1)2(1)2(1), the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Å for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Å for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall R(merge) value of 0.04 and an R(merge) value of 0.33 in the highest resolution shell.

Figure 1

Overall architecture of GspB. GspB is comprised of a signal peptide (SP), a short serine-rich region (SRR1), a unique basic region (BR) that is responsible for carbohydrate binding, a second, longer, serine-rich region (SRR2) and a cell-wall-anchoring domain (CWAD). A recently published structural study of the Fap1 adhesin from S. parasanguinis identified the structural elements of both the serine-rich repeats and the unique region in that protein (Ramboarina et al., 2010 ▶). The repeat region of GspB is likely to form a super-helical fibril like that observed in the repeat region of Fap1.

Figure 2

Purification of GspB_BR for crystallization. The gel shows two separate concentrations of the GST-GspB_BR fusion protein after affinity purification and two concentrations of purified isolated GspB_BR(246–601) after size-exclusion chromatography. Lane 1, Kaleidoscope Ladder (Bio-Rad; labeled in kDa); lanes 2 and 3, 4 µg GST-GspB_BR; lane 4, 1 µg GST-GspB_BR; lane 5, 4 µg of GST and GspB_BR after factor Xa cleavage; lane 6, 4 µg GspB_BR after size-exclusion chromatography; lane 7, 1 µg GspB_BR after size-exclusion chromatography; lane 8, dissolved crystals grown in 26% Jeffamine-ED 2001 and 0.05 M HEPES pH 7.5.

Figure 3

Crystals of GspB_BR from S. gordonii strain M99 grown using the hanging-drop vapor-diffusion method. (a) Crystals of GspB_BR grown from 25% PEG 3350, 0.15 M ammonium acetate, 0.1 M HEPES pH 7.5 and 10 mM spermidine. (b) Crystals of GspB_BR grown from 34% Jeffamine ED-2001, 0.15 M KCl and 0.1 M HEPES pH 7.5.

Figure 4

Typical diffraction for each of the crystal forms. (a) Diffraction image for crystals grown using PEG 3350 as the precipitant. This diffraction image was collected on the SSRL 11-1 beamline and diffraction to 1.8 Å resolution can be observed. (b) Typical diffraction image for crystals grown using Jeffamine ED-2001 as the precipitant. This diffraction image was collected on the LS-CAT ID-21-G beamline and diffraction to 1.3 Å resolution can be observed.