Extended Binding Site on Fibronectin for the Functional Upstream Domain of Protein F1 of Streptococcus pyogenes

Abstract

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with Kd values of 5.2 and 59 nm to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the 2FNI module had little effect, whereas mAb 7D5 to the 4FNI module in the fibrin-binding region, 5C3 to the 9FNI module in the gelatin-binding region, or L8 to the G-strand of 1FNIII module adjacent to 9FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to 2FNI-5FNI of the fibrin-binding domain and 8FNI-9FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of 2FNI-5FNI and 8FNI-9FNI.

Fibronectin Aggregation and Assembly

“…We found that domain III 2, destabilized by deletion of the G strand, bound to I 1-9, and the binding was completely inhibited by a bacterial adhesin (FUD) ( Table 1). Although there are no direct structural data for the FUD-I 1-9 complex, circular dichroism and modeling suggested that the FUD bound by ␤ strand addition (44). Therefore, we speculate that the binding of unfolded III 2 to I 1-9 may use a tandem ␤-zipper mechanism similar to known adhesins.…”

“…The FNBP of S. pyogenes also bound more tightly to I 1-9 than to I 1-5 (43,44,56). Because I 1-9 is stable against proteolysis in comparison with I 6 -9 (see "Experimental Procedures" for details), the longer I 1-9 may form a compact conformation that masks a protease-sensitive site.…”

Borrelia burgdorferi Protein BBK32 Binds to Soluble Fibronectin via the N-terminal 70-kDa Region, Causing Fibronectin to Undergo Conformational Extension

“…This extended interaction appears to be important for efficient invasion (11). Antibody inhibition studies indicate that the ␤-zipper formed by FUD may extend to encompass 9 FNI and the linker region between 9 FNI and 1 FNIII (16). Other studies have demonstrated that a peptide from FnBB of Streptococcus dysgalactiae uses the tandem ␤-zipper mechanism to bind to 1-2 FNI (13), as do FnZ (residues 370 -391) (17) and type I collagen (residues 778 -799) in binding to 8 FNI (18) (see Fig.…”

“…Integrins function as cell surface receptors for FN, and the conformational change allows pathogens coated with FN to undergo integrin-dependent host cell invasion (9 -11). Although FNBRs bind [1][2][3][4][5] FNI or [2][3][4][5] FNI, the "functional upstream domain" (FUD) comprising residues 423-471 of SfbI (16) and UR-FnZ-2 comprising residues 370 -428 of FnZ from Streptococcus equi subsp. zooepidemicus (17) bind to an extended site comprising modules [2][3][4][5] FNI and 8 FNI in a proposed extended tandem ␤-zipper (see Fig.…”

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

“…NOTE: KpnI and NheI sites were chosen to enable inserts to be ligated into a modified pET-28a vector with kanamycin resistance 25,26 . KpnI site had been added into the multiple cloning site.…”