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 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.
Departments of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Wenjiang
W
Ma
Departments of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Hanqing
H
Ma
Departments of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Deane F
DF
Mosher
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism*; D005470:Fluorometry; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D012995:Solubility
D001426:Bacterial Proteins; D005353:Fibronectins; D008024:Ligands; D050505:Mutant Proteins; D010455:Peptides; C106209:p35 antigen, Borrelia