Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.
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 K(d) 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 (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI 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 (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI 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 (2)FNI-(5)FNI and (8)FNI-(9)FNI.
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Lisa M
LM
Maurer
Bianca R
BR
Tomasini-Johansson
Wenjiang
W
Ma
Douglas S
DS
Annis
Nathan L
NL
Eickstaedt
Martin G
MG
Ensenberger
Kenneth A
KA
Satyshur
Deane F
DF
Mosher
D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018829:Adhesins, Bacterial; D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins; D011994:Recombinant Proteins