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

Abhin Kumar Megta, Airi Palva, Ingemar von Ossowski, Vengadesan Krishnan*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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 angstrom. 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 angstrom, alpha = beta = 90.0, gamma = 120.0 degrees. Obtaining an interpretable electron-density map via single-wavelength 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 (

Original languageEnglish
Pages (from-to)731-737
Number of pages7
JournalActa crystallographica section F : Structural biology communications
Volume75
DOIs
Publication statusPublished - Dec 2019
MoE publication typeA1 Journal article-refereed

Keywords

  • adhesion
  • probiotics
  • basal pilin
  • host-microbe interaction
  • sortase-dependent pili
  • Lactobacillus rhamnosus GG
  • SpaB
  • GRAM-POSITIVE BACTERIA
  • RHAMNOSUS GG
  • CRYSTALLOGRAPHIC ANALYSIS
  • FUNCTIONAL-ANALYSIS
  • CRYSTAL-STRUCTURE
  • ISOPEPTIDE BONDS
  • SUBUNIT
  • REVEALS
  • PROTEIN
  • SURFACE

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