Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG

Arjun K. Mishra, Abhin Kumar Megta, Airi Palva, Ingemar von Ossowski, Vengadesan Krishnan*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 angstrom were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 angstrom. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 angstrom resolution.

Original languageEnglish
Pages (from-to)321-327
Number of pages7
JournalActa crystallographica section F : Structural biology communications
Volume73
DOIs
Publication statusPublished - Jun 2017
MoE publication typeA1 Journal article-refereed

Keywords

  • adhesion
  • probiotics
  • basal pilin
  • host-microbe interaction
  • sortase-dependent pili
  • Lactobacillus rhamnosus GG
  • SpaE
  • GRAM-POSITIVE BACTERIA
  • BACKBONE-PILIN SUBUNIT
  • CRYSTALLOGRAPHIC ANALYSIS
  • FUNCTIONAL-ANALYSIS
  • CRYSTAL-STRUCTURE
  • SPACBA PILUS
  • ADHESION
  • PURIFICATION

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