title: New insights into the role of the glutamic acid of the E-box motif in group B Streptococcus pilus 2a assembly.
abstract: 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.
authors.affiliation: Novartis Vaccines and Diagnostics, via Fiorentina, 1 53100 Siena, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Cozzi
authors.forename: Annalisa
authors.initials: A
authors.lastname: Nuccitelli
authors.forename: Mariapina
authors.initials: M
authors.lastname: D'Onofrio
authors.forename: Francesca
authors.initials: F
authors.lastname: Necchi
authors.forename: Roberto
authors.initials: R
authors.lastname: Rosini
authors.forename: Francesca
authors.initials: F
authors.lastname: Zerbini
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Biagini
authors.forename: Nathalie
authors.initials: N
authors.lastname: Norais
authors.forename: Christian
authors.initials: C
authors.lastname: Beier
authors.forename: John L
authors.initials: JL
authors.lastname: Telford
authors.forename: Guido
authors.initials: G
authors.lastname: Grandi
authors.forename: Michael
authors.initials: M
authors.lastname: Assfalg
authors.forename: Martin
authors.initials: M
authors.lastname: Zacharias
authors.forename: Domenico
authors.initials: D
authors.lastname: Maione
authors.forename: C Daniela
authors.initials: CD
authors.lastname: Rinaudo
mesh_terms: D015153:Blotting, Western; D010861:Fimbriae, Bacterial / Q000502:physiology*; D018698:Glutamic Acid / Q000502:physiology*; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D013292:Streptococcus agalactiae / Q000502:physiology*
chemical_list: D018698:Glutamic Acid