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

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 FNI modules, ^2–5,8–9FNI, in the N-terminal 70-kDa region (FN70K) of FN. To facilitate further comparisons, FUD, Bbk32, two other polypeptides based on SfbI that target ^1–5FNI (HADD) and ^2–5FNI (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.