Abstract
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, koff for FUD or HADD to FN70K or FN was considerably lower compared to koff 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 koff 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.
Highlights
Fibronectin (FN) is a glycoprotein found in vertebrates as a disulfide-linked dimer
Cell surface-attached proteins expressed by different bacteria, including FNBPA of Staphylococcus aureus, SfbI of Streptococcus pyogenes, FNZ of Streptococcus dysgalactiae, and BBK32 of Borrelia burgdorferi, contain intrinsically disordered regions that bind to FN through an unusual mechanism called tandem β-zipper formation[12,13,14]
Certain sequence motifs can be paired with specific FN dimer includes type 1 (FNI) modules, isothermal titration calorimetric (ITC) studies of effects of mutations on a polypeptide based on SfbI failed to identify major “hot spots” and suggested that the binding energy is distributed across the interface[16]
Summary
Cell surface-attached proteins expressed by different bacteria, including FNBPA of Staphylococcus aureus, SfbI of Streptococcus pyogenes, FNZ of Streptococcus dysgalactiae, and BBK32 of Borrelia burgdorferi, contain intrinsically disordered regions that bind to FN through an unusual mechanism called tandem β-zipper formation[12,13,14]. Such proteins engage the FN70K domain comprising the N-terminal 19FNI and 1-2FNII modules of FN (Fig 1A) and form β–strands with the E-strands of sequential FNI modules in an interaction that can have low nM affinity[14, 15]. Much more needs to be understood about tandem β-zipper formation and its role in bacterial pathogenesis
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