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J. Biol. Chem., Vol. 279, Issue 41, 42945-42953, October 8, 2004

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Interaction of Staphylococcus aureus Fibronectin-binding Protein with Fibronectin

AFFINITY, STOICHIOMETRY, AND MODULAR REQUIREMENTS^*

Kenneth C. Ingham, Shelesa Brew, Dareyl Vaz¶, Daniel N. Sauder||, and Martin J. McGavin¶**

From the Department of Biochemistry, American Red Cross Holland Laboratory, Rockville, Maryland 20855, ^||Department of Dermatology, The Johns Hopkins University, Baltimore, Maryland 21287-0900, and Department of Laboratory Medicine and Pathobiology, University of Toronto and ^¶Department of Microbiology, Sunnybrook and Women's College Health Science Centre, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada

The repetitive D1, D2, and D3 elements of Staphylococcus aureus fibronectin-binding protein FnBPA each bind the N-terminal 29-kDa fragment (N29) of fibronectin with low micromolar dissociation constants (K_d), but in tandem they compose a high affinity domain, D1–3. An additional seven Fn-binding segments have been predicted in FnBPA in a region N-terminal of the D-repeats (Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Gurusiddappa, S., Kim, J. H., Pilka, E. S., Briggs, J. A., Gough, T. S., Höök, M., Campbell, I. D., and Potts, J. R. (2003) Nature 423, 177–181). We have evaluated the requirements for high affinity binding of N29 to the D-repeat domain and determined the affinity and stoichiometry of N29 binding to segments that are N-terminal of the D-repeats in the related FnBPB adhesin. We confirmed that D1–3 has two equivalent high affinity sites (K_d, 1 nM) and provided evidence for one or more lower affinity sites (K_d, 0.5 µM). Bimodular D1–2 and D2–3 exhibit intermediate affinity sites with respective K_d values of 0.25 and 0.044 µM, as well as a low affinity site with a K_d value of 2.2–2.5 µM. We also identified two binding domains that are N-terminal of the D-repeats, designated DuB and DuA. Segments internal to these domains individually bound N29 with similar K_d values of 2 µM, whereas the DuBA polypeptide possessing both segments and other intervening sites bound four molecules of N29 with much higher affinity (K_d, 10 nM). DuBAD, a larger polypeptide harboring all of the known or predicted binding motifs in FnBPB, bound seven to eight molecules of N29, with a K_d of 7 nM. Because most of the isolated binding segments display low affinity for N29 and lack motifs for binding of one or both of the ¹F1 and ⁵F1 modules in the N-terminal domain of Fn, we propose that high affinity is achieved in part as a consequence of self-interaction between bound molecules of N29.

Received for publication, June 22, 2004

^* This work was funded by the Aventis Pasteur-University of Toronto Research Program, Funded Research 72012558, and the Premiers Research Excellence Award (to M. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: S112 Dept. of Microbiology, Sunnybrook and Women's College Health Science Centre, 2075 Bayview Ave., Toronto, Ontario M4N 3M5, Canada. Tel.: 416-480-5831; Fax: 416-480-5737; E-mail: martin.mcgavin{at}sw.ca.

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