On-Off Kinetics of Engagement of FNI Modules of Soluble Fibronectin by β-Strand Addition

PLoS One. 2015 Apr 28;10(4):e0124941. doi: 10.1371/journal.pone.0124941. eCollection 2015.


Intrinsically disordered sequences within bacterial adhesins bind to E-strands in the β-sheets of multiple FNI modules of fibronectin (FN) by anti-parallel β-strand addition, also called tandem β-zipper formation. The FUD segment of SfbI of Streptococcus pyogenes and Bbk32 segment of BBK32 of Borrelia burgdorferi, despite being imbedded in different adhesins from different bacteria, target the same 2-5,8-9 FNI modules, 2-5,8-9 FNI, in the N-terminal 70-kDa region (FN70K) of FN. To facilitate further comparisons, FUD, Bbk32, two other polypeptides based on SfbI that target 1-5 FNI (HADD) and 2-5 FNI (FRD), and mutant Bbk32 (ΔBbk32) were produced with fluorochromes placed just outside of the binding sequences. Unlabeled FUD competed ~ 1000-fold better for binding of labeled Bbk32 to FN than unlabeled Bbk32 competed for binding of labeled FUD to FN. Binding kinetics were determined by fluorescence polarization in a stopped-flow apparatus. On-rates for FUD, Bbk32, HADD, and FRD were similar, and all bound more rapidly to FN70K fragment than to full length FN. In stopped-flow displacement and size exclusion chromatographic assays, however, k off for FUD or HADD to FN70K or FN was considerably lower compared to k off of FRD or Bbk32. FUD and Bbk32 differ in the spacing between sequences that interact with 3FNI and 4FNI or with 5FNI and 8FNI. ΔBbk32, in which 2 residues were removed from Bbk32 to make the spacing more like FUD, had a k off intermediate between that of Bbk32 and FUD. These results indicate a "folding-after-binding" process after initial association of certain polypeptide sequences to FN that results in formation of a stable complex and is a function of number of FNI modules engaged by the polypeptide, spacing of engagement sites, and perhaps flexibility within the polypeptide-FN complex. We suggest that contributions of SfbI and BBK32 adhesins to bacterial pathogenicity may be determined in part by stability of adhesin-FN complexes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • Binding, Competitive
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Fibronectins / chemistry*
  • Fibronectins / metabolism*
  • Fluorometry
  • Kinetics
  • Ligands
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Binding
  • Protein Stability
  • Protein Structure, Secondary
  • Solubility


  • Bacterial Proteins
  • Fibronectins
  • Ligands
  • Mutant Proteins
  • Peptides
  • p35 antigen, Borrelia