SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis

Abstract

The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The crystallization of SpaB remained particularly challenging until the implementation of a three-pronged crystallization approach involving C-terminal tail truncation, surface lysine methylation and magnesium additives. Ultimately, hexagonal crystals of SpaB were produced and were able to diffract to a resolution of 2.4 Å . This crystal form belonged to space group P6 5 22 or P6 1 22, with unit-cell parameters a = b = 51.53, c = 408.22 Å , = = 90.0, = 120.0 . Obtaining an interpretable electron-density map via singlewavelength anomalous diffraction (SAD) using iodide-derivative data sets did not succeed owing to the weak anomalous signal. As an alternative, attempts to provide phases by molecular replacement using the iodide-SAD data from SpaB and a collection of distant homology models (<28% sequence identity) are in progress. research communications Acta Cryst. (2019). F75, 731-737 Kumar Megta et al. SpaB 737

Clostridium perfringens Produces an Adhesive Pilus Required for the Pathogenesis of Necrotic Enteritis in Poultry

“…Our antiserum blocking results also suggest that both the tip and backbone pilin contribute to collagen binding, while the base pilin does not. Previous reports of other Gram-positive pili have also shown that pilins other than the tip adhesin may have adherent abilities and participate in host cell binding ( 22 , 23 ).…”

Crystal structure of the atypically adhesive SpaB basal pilus subunit: Mechanistic insights about its incorporation in lactobacillar SpaCBA pili

“…Full-length L. rhamnosus GG SpaB (GG-SpaB FL ) protein (residues 33–205) was solubly produced in Escherichia coli ( von Ossowski et al., 2010 ), essentially as done previously for the GG-SpaA ( Singh et al., 2013 ), GG-SpaC ( Kant et al., 2016 ), GG-SpaD ( Chaurasia et al., 2015 ), and GG-SpaE ( Mishra et al., 2017 ) pilins. Since the GG-SpaB FL protein (~20 ​kDa) did not yield diffraction quality crystals, a truncated version (GG-SpaB C-trun ) missing a flexible portion of the C-terminal region (residues 185–205), but containing N-terminal hexahistidine tagging was cloned and solubly produced in E. coli as described previously ( Kumar Megta et al., 2019 ). Homogeneous and pure GG-SpaB C-trun protein (residues 31–184) yielded hexagonal crystals after the surface lysine methylation (SLM) treatment and the inclusion of 0.2 ​M MgCl 2 as an additive ( Kumar Megta et al., 2019 ).…”

“…Since the GG-SpaB FL protein (~20 ​kDa) did not yield diffraction quality crystals, a truncated version (GG-SpaB C-trun ) missing a flexible portion of the C-terminal region (residues 185–205), but containing N-terminal hexahistidine tagging was cloned and solubly produced in E. coli as described previously ( Kumar Megta et al., 2019 ). Homogeneous and pure GG-SpaB C-trun protein (residues 31–184) yielded hexagonal crystals after the surface lysine methylation (SLM) treatment and the inclusion of 0.2 ​M MgCl 2 as an additive ( Kumar Megta et al., 2019 ). The SLM treatment was done as described previously ( Kumar Megta et al., 2019 ; Rayment, 1997 ).…”

“…Homogeneous and pure GG-SpaB C-trun protein (residues 31–184) yielded hexagonal crystals after the surface lysine methylation (SLM) treatment and the inclusion of 0.2 ​M MgCl 2 as an additive ( Kumar Megta et al., 2019 ). The SLM treatment was done as described previously ( Kumar Megta et al., 2019 ; Rayment, 1997 ). Briefly, GG-SpaB protein in HEPES buffer (50 ​mM HEPES pH 7.5, 250 ​mM NaCl) was concentrated to a final volume of 10 ​ml (~1 ​mg/ml) and then supplemented with aliquots of freshly prepared 1 ​M borane-dimethylamine complex (200 ​μl) (Sigma-Aldrich) and 36.5–38% formaldehyde solution (400 ​μl) (Sigma-Aldrich), followed by gentle shaking (100 ​rpm) at 4 ​°C for 2 ​h.…”

“…Eluted fractions of GG-SpaB protein were pooled and then concentrated to 40 ​mg/ml for crystallization trials. Here, crystals for X-ray diffraction experiments were obtained from lysine-methylated GG-SpaB C-trun protein (40 ​mg/ml in 20 ​mM Tris–HCl pH 8.0, 150 ​mM NaCl, 1 ​mM DTT) using a screening solution of 0.2 ​M MgCl 2 , 0.1 ​M MES pH 6.5, 30% (w/v) PEG 4000 ( Kumar Megta et al., 2019 ).…”