Interaction of Staphylococcus aureus fibronectin-binding protein with fibronectin: affinity, stoichiometry, and...

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 (Kd), 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., Hook, 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 (Kd, approximately 1 nm) and provided evidence for one or more lower affinity sites (Kd, approximately 0.5 microm). Bimodular D1-2 and D2-3 exhibit intermediate affinity sites with respective Kd values of 0.25 and 0.044 microm, as well as a low affinity site with a Kd value of 2.2-2.5 microm. 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 Kd values of approximately 2 microm, whereas the DuBA polypeptide possessing both segments and other intervening sites bound four molecules of N29 with much higher affinity (Kd, approximately 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 Kd of approximately 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.