New insights into the role of the glutamic acid of the E-box motif in group B Streptococcus pilus 2a assembly

Roberta Cozzi, Annalisa Nuccitelli, Mariapina D'Onofrio, Francesca Necchi, Roberto Rosini, Francesca Zerbini, Massimiliano Biagini, Nathalie Norais, Christian Beier, John L. Telford, Guido Grandi, Michael Assfalg, Martin Zacharias, Domenico Maione, C. Daniela Rinaudo

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Group B Streptococcus pili are covalently linked structures assembled via a sortase-catalyzed transpeptidation mechanism involving specific residues and motifs. A sequence element containing a conserved glutamic acid, called the E-box, has been described to be involved in pilus formation. Although it is known that the glutamic acid is involved in stabilizing the internal isopeptide bonds, its role in pilus assembly still needs to be investigated. Using site-specific mutagenesis and complementation studies of knockout strains, we found that the E-box glutamic residue of the backbone and the major ancillary proteins is essential for pilus protein polymerization. NMR analysis revealed that the mutation of this residue seriously affected the folding of the protein. By contrast, the mutation of the lysine involved in the same isopeptide bond did not engender a structural destabilization, and the native fold was preserved. Moreover, molecular dynamics simulations on the E-box-containing domain of the backbone protein showed that the E-box glutamic acid is necessary to maintain the appropriate dryness of the domain core and that its mutation favors an unfolded state. The data provide the first direct evidence that the E-box has an additional and key role in maintaining the correct protein fold independently of isopeptide bond formation.

Original languageEnglish
Pages (from-to)2008-2018
Number of pages11
JournalFASEB Journal
Issue number5
StatePublished - May 2012
Externally publishedYes


  • Backbone protein
  • Isopeptide bonds
  • Molecular dynamic simulations
  • NMR spectroscopy


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