title: New insights about pilus formation in gut-adapted Lactobacillus rhamnosus GG from the crystal structure of the SpaA backbone-pilin subunit.
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 non-pathogen 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.
authors.affiliation: Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad-121 001, India.|Department of Biotechnology, Manipal University, Karnataka, 576104, India.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Chaurasia
authors.affiliation: Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad-121 001, India.
authors.forename: Shivendra
authors.initials: S
authors.lastname: Pratap
authors.affiliation: Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Ingemar
authors.initials: I
authors.lastname: von Ossowski
authors.affiliation: Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Airi
authors.initials: A
authors.lastname: Palva
authors.affiliation: Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad-121 001, India.
authors.forename: Vengadesan
authors.initials: V
authors.lastname: Krishnan
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D036781:Fimbriae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010861:Fimbriae, Bacterial; D052201:Lacticaseibacillus rhamnosus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D000072417:Protein Domains; D055550:Protein Stability
chemical_list: D036781:Fimbriae Proteins