Interaction of Staphylococcus aureus fibronectin-binding protein with fibronectin -: Affinity, stoichiometry, and modular requirements

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., 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 (K-d, similar to 1 nM) and provided evidence for one or more lower affinity sites (K-d, similar to 0.5 muM). Bimodular D1 - 2 and D2 - 3 exhibit intermediate affinity sites with respective Kd values of 0.25 and 0.044 muM, as well as a low affinity site with a K-d value of 2.2 - 2.5 muM. 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 similar to2 muM, whereas the DuBA polypeptide possessing both segments and other intervening sites bound four molecules of N29 with much higher affinity (K-d, similar to 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 similar to 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 (1)F1 and (5)F1 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.