New insights about pilus formation in gut-adapted Lactobacillus rhamnosus GG from the crystal structure of the SpaA backbone-pilin subunit

Priyanka Chaurasia, Shivendra Pratap, Ingemar von Ossowski, Airi Palva, Vengadesan Krishnan*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

Thus far, all solved structures of pilin-proteins comprising sortase-assembled pili are from pathogenic genera and species. Here, we present the first crystal structure of a pilin subunit (SpaA) from a nonpathogen host (Lactobacillus rhamnosus GG). SpaA consists of two tandem CnaB-type domains, each with an isopeptide bond and E-box motif. Intriguingly, while the isopeptide bond in the N-terminal domain forms between lysine and asparagine, the one in the C-terminal domain atypically involves aspartate. We also solved crystal structures of mutant proteins where residues implicated in forming isopeptide bonds were replaced. Expectedly, the E-box-substituted E139A mutant lacks an isopeptide bond in the N-terminal domain. However, the C-terminal E269A substitution gave two structures; one of both domains with their isopeptide bonds present, and another of only the N-terminal domain, but with an unformed isopeptide bond and significant conformational changes. This latter crystal structure has never been observed for any other Gram-positive pilin. Notably, the C-terminal isopeptide bond still forms in D295N-substituted SpaA, irrespective of E269 being present or absent. Although E-box mutations affect SpaA proteolytic and thermal stability, a cumulative effect perturbing normal pilus polymerization was unobserved. A model showing the polymerized arrangement of SpaA within the SpaCBA pilus is proposed.

Original languageEnglish
Article number28664
Number of pages17
JournalScientific Reports
Volume6
DOIs
Publication statusPublished - 28 Jun 2016
MoE publication typeA1 Journal article-refereed

Keywords

  • INTRAMOLECULAR ISOPEPTIDE BONDS
  • GRAM-POSITIVE BACTERIA
  • CRYSTALLOGRAPHIC ANALYSIS
  • STABILIZING ISOPEPTIDE
  • FUNCTIONAL-ANALYSIS
  • SPACBA PILUS
  • CROSS-LINKS
  • ADHESION
  • PROTEIN
  • CRYSTALLIZATION

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