Interaction of Staphylococcus aureus Fibronectin-binding Protein with Fibronectin

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., 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 (Kd, ∼1 nm) and provided evidence for one or more lower affinity sites (Kd, ∼0.5 μm). Bimodular D1–2 and D2–3 exhibit intermediate affinity sites with respective Kd values of 0.25 and 0.044 μm, as well as a low affinity site with a Kd 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 Kd values of ∼2 μm, whereas the DuBA polypeptide possessing both segments and other intervening sites bound four molecules of N29 with much higher affinity (Kd, ∼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 ∼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. 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., 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 (Kd, ∼1 nm) and provided evidence for one or more lower affinity sites (Kd, ∼0.5 μm). Bimodular D1–2 and D2–3 exhibit intermediate affinity sites with respective Kd values of 0.25 and 0.044 μm, as well as a low affinity site with a Kd 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 Kd values of ∼2 μm, whereas the DuBA polypeptide possessing both segments and other intervening sites bound four molecules of N29 with much higher affinity (Kd, ∼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 ∼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. Staphylococcus aureus is the leading overall cause of nosocomial infections and is able to infect virtually every tissue and organ system of the body (1Lowy F.D. N. Engl. J. Med. 1998; 339: 520-532Crossref PubMed Scopus (4469) Google Scholar, 2Sheagren J.N. N. Engl. J. Med. 1984; 310: 1368-1373Crossref PubMed Scopus (210) Google Scholar). The increasing incidence of multiply antibiotic-resistant S. aureus strains and the emergence of vancomycin-resistant, multiply antibiotic-resistant S. aureus (3Weigel L.M. Clewell D.B. Gill S.R. Clark N.C. McDougal L.K. Flannagan S.E. Kolonay J.F. Shetty J. Killgore G.E. Tenover F.C. Science. 2003; 302: 1569-1571Crossref PubMed Scopus (675) Google Scholar) have placed renewed interest on alternative means of prevention and control of infection. In this regard, members of the microbial surface components recognizing adhesive matrix molecules (MSCRAMM) 1The abbreviations used are: MSCRAMM, microbial surface components recognizing adhesive matrix molecules; Fn, fibronectin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; ECM, extracellular matrix. 1The abbreviations used are: MSCRAMM, microbial surface components recognizing adhesive matrix molecules; Fn, fibronectin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; ECM, extracellular matrix. family of adhesion proteins have come under intensive scrutiny due to their ability to promote adhesion to the extracellular matrix that surrounds and anchors cells in tissue (4Foster T.J. Höök M. Trends Microbiol. 1998; 6: 484-488Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar, 5Patti J.M. Allen B.L. McGavin M.J. Höök M. Annu. Rev. Microbiol. 1994; 48: 585-617Crossref PubMed Scopus (921) Google Scholar), thus representing attractive targets for therapeutic and vaccination strategies aimed at interfering with colonization. The fibronectin-binding proteins FnBPA and FnBPB are archetypal members of the MSCRAMM family and are encoded by tandem genes fnbA and fnbB (6Jonsson K. Signas C. Muller H.P. Lindberg M. Eur. J. Biochem. 1991; 202: 1041-1048Crossref PubMed Scopus (294) Google Scholar, 7Signas C. Raucci G. Jonsson K. Lindgren P.E. Anantharamaiah G.M. Höök M. Lindberg M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 699-703Crossref PubMed Scopus (306) Google Scholar). As exemplified by FnBPA, members of the MSCRAMM family display a modular architecture (Fig. 1A). Following the traditional domain nomenclature first used to describe FnBPA (7Signas C. Raucci G. Jonsson K. Lindgren P.E. Anantharamaiah G.M. Höök M. Lindberg M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 699-703Crossref PubMed Scopus (306) Google Scholar), the N terminus is a region of ∼500 amino acids designated the A domain, which is followed by the B-region composed of two 30-amino acid repeats, a short spacer designated C, and then the D-repeat domain composed of three complete 37- or 38-amino acid repeats and part of a fourth repeat. The C terminus of FnBPA and other MSCRAMMs is dedicated to anchoring the proteins within the peptidoglycan of the that the D-repeat domain of FnBPA a 29-kDa N-terminal domain (N29) of Fn, with affinity in the low (7Signas C. Raucci G. Jonsson K. Lindgren P.E. Anantharamaiah G.M. Höök M. Lindberg M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 699-703Crossref PubMed Scopus (306) Google Scholar). We that the and repeats each bound N29 with Kd values of μm, with a Kd of for the D3 and a polypeptide possessing the three D-repeats in tandem a high affinity of S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). this that the three tandem D-repeats to a that is for high affinity that the D1–3 domain in bound to N29 K. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). MSCRAMMs A and C also repetitive motifs that bind N29 K. PubMed Google Scholar, P.E. McGavin M.J. Signas C. S. Höök M. Lindberg M. Eur. J. Biochem. PubMed Scopus Google Scholar, G. J. PubMed Scopus Google Scholar, S.R. PubMed Google Scholar), with the D-repeats of S. aureus a of amino acids as and amino acids in the are for binding M.J. S. Lindgren P.E. Lindberg M. Raucci G. Höök M. J. Full Text PDF PubMed Google Scholar). The N29 fragment is composed of tandem is a composed of two with A and a and C, and a PubMed Scopus Google Scholar). in each of the 1F1 and modules of N29 is the for a the MSCRAMM, which bound the with a Kd of U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). The two each of which a fourth to the in 1F1 and the a of amino acids with and in and this by that a in of these binding with the of additional and amino acids that are in binding M.J. S. Lindgren P.E. Lindberg M. Raucci G. Höök M. J. Full Text PDF PubMed Google Scholar), also the of and motifs in on these known and predicted the ability of a the of the to bind N29 with high affinity to all modules U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). by that three the larger each bind the respective or with low affinity (Kd, whereas the bound N29 with high affinity (Kd, and a the of intermediate affinity (Kd, μm). that motifs promote the binding of short segments with motifs for two modules display low affinity high affinity binding that all modules are in a tandem and the of a a in affinity for N29. this is well to the S. fibronectin-binding MSCRAMM, a of for the FnBPA and FnBPB of S. FnBPA predicted to Fn-binding segments on the of the first of these segments are N-terminal of the D-repeats and known to bind or well in of affinity and the D1–3 polypeptide N29 with high in the FnBPA and a of the D-repeat domain U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). the of the predicted binding segments lack motifs for binding of 5F1 and 1F1 to the that high affinity In this regard, the of to the for high affinity binding by the repetitive D-repeat domain and to binding segments that are N-terminal of the D-repeats are of high affinity D1–2 or D2–3 we that the affinity is with that for a the of S. that four modules but is of that of the D1–3 or as a to a of S. aureus we isolated two binding that are N-terminal of the designated and DuB binding segments and that individually bind N29 with low affinity as a DuBA display affinity that is with the D-repeats, their lack of and DuBA with D1–3 a DuBAD, that seven or eight high affinity binding sites for N29, to the of predicted binding are with segments in the in two high affinity we the high affinity binding with the and we that between bound molecules of N29 to high affinity and S. aureus has been M. R. McGavin M.J. J. PubMed Scopus Google Scholar, A. M. McGavin M.J. J. PubMed Scopus Google Scholar). strains or used for of and of of S. aureus and E. at in and in or with for of E. with and with for of with of for of at in a for and a with at for for the of at or followed by to of and in with and S. T. J. PubMed Scopus Google Scholar). cells to a of and of the cells achieved by the with for at followed by for as M. R. G.M. McGavin M.J. PubMed Scopus Google Scholar). with phosphate-buffered protein binding sites by with in for of and K. J. Lindberg M. 1998; PubMed Scopus Google Scholar) provided by and is that of a site of the promote of as N-terminal to the protein A of of S. aureus in K. Google Scholar, K. PubMed Scopus Google Scholar), which then in E. to a of binding of Fn, of for with of in of bound then with and with bound in and then with and used to E. then to a of on ECM, of in to of a a at with the with followed by of bound in low and in E. for a of both the of used to infect E. and cells on proteins identified by for the site with the of by the of for the and the protein to the provided by the for to S. aureus of and which the site of in with isolated a designated The site that is with the A of A site is provided by a of that in the in the is as glutathione protein the complete DuB domain as in and to a of the DuB the and sites of the the of protein a acid of the DuB with to that the site of and protein N-terminal amino acids of An harboring both and by of with and with and the the of protein acid at both the C terminus of the DuB a internal of the DuB domain by of with and with and the the of protein harboring a 38-amino acid internal of the DuB the and D1–3 domains of the FnBPB of S. aureus by of with and and of the fnbB (6Jonsson K. Signas C. Muller H.P. Lindberg M. Eur. J. Biochem. 1991; 202: 1041-1048Crossref PubMed Scopus (294) Google Scholar). The and then with and sites provided by the and that the at the of the is by the site by the fragment the and sites of and protein The D-repeats by with a that is the and of and of the S. aureus fnbB (6Jonsson K. Signas C. Muller H.P. Lindberg M. Eur. J. Biochem. 1991; 202: 1041-1048Crossref PubMed Scopus (294) Google Scholar). with and the the of protein by with the system whereas all other with by affinity on as Full Text PDF PubMed Scopus Google Scholar) with for of E. A. C. Biochem. PubMed Scopus Google Scholar), the 29-kDa N-terminal fragment that as S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). and of glutathione the and harboring the tandem and repeats or and D3 repeats, of the FnBPA of S. aureus as M. R. G.M. McGavin M.J. PubMed Scopus Google Scholar). proteins with to the binding domains and then by M. R. G.M. McGavin M.J. PubMed Scopus Google Scholar). and with by the of of each a with the values of and as determined by on a and with as M.J. C. K. PubMed Google Scholar). as S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). or by with a of in for at by on The of determined as PubMed Scopus Google Scholar). The of and on the of determined by amino acid and D3 to the and provided with the then to the affinity which then with and and then in as determined by and to to by the affinity in with of a of in PBS, and of with and binding of to of at with of in and with the of for at on with and to a on a the followed by of for and with on a with a are as the values of or a of the value determined in the of in and at of the N-terminal 29-kDa fragment (N29) to a the the at with at as S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). The in to one of for the and to a binding of S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). for the D1–2 and to a the and the respective dissociation constants of sites and and of N29. Because the of with the of N29 to the is for of or of the much higher In this to a that the of bound and the stoichiometry as S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). of N29 or with of DuBA or DuBAD, for and then a protein The at The by at nm. of by of FnBPA and FnBPB and the of segments are in A and segments the Fn-binding D-repeats with both and the ability of the to Fn-binding isolated on segments within the C domain of FnBPA, with a binding domain designated S. J. Höök M. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar). domain amino acids of FnBPA is designated DuA. both identified a of possessing or within a of FnBPB that has been domain amino acids of is designated of to the high between the of FnBPA and FnBPB (6Jonsson K. Signas C. Muller H.P. Lindberg M. Eur. J. Biochem. 1991; 202: 1041-1048Crossref PubMed Scopus (294) Google Scholar), the amino acid of (Fig. is in the two proteins but as fnbA on the of The is within the C domain that the repetitive and of A acid internal of is to the of the S. (Fig. and also to predicted Fn-binding of FnBPA U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). The amino acid of DuB S. aureus (Fig. with known of FnBPB S. aureus and with A acid N-terminal of DuB in is in and additional amino acids that are in FnBPB of S. aureus A acid internal of DuB is to amino acids and of the FnBPA which to the and repeats (Fig. to the nomenclature (7Signas C. Raucci G. Jonsson K. Lindgren P.E. Anantharamaiah G.M. Höök M. Lindberg M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 699-703Crossref PubMed Scopus (306) Google Scholar) and are known to predicted Fn-binding segments and U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). The amino acids of DuB are to in both FnBPA and FnBPB of S. aureus and the C terminus of DuB (Fig. with the N terminus of that these domains are is in the amino acid of the DuBA polypeptide the FnBPB that is used in this with of Fn-binding on with the predicted Fn-binding segments of of and the to the of predicted Fn-binding segments in the and DuB of these domains the of binding segments in the FnBPA adhesin. A of proteins harboring segments of DuB and for of binding to of with the (Fig. binding of with value of nm. the N terminus or the that is in both FnBPA and FnBPB as in the to provided similar values of and whereas both and segments of nm. of with protein which the acid internal of DuB that to the and repeats of this a for more whereas on the of to the Fn-binding of S. (Fig. 1A). affinity or a polypeptide on each (Fig. and with affinity matrix the D3 which the affinity of the D-repeats and for N29 S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). each of the three binding domains a for the N29 fragment of of or by with N29 provided Kd values of and μm, with for the D3 S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). The value for binding to N29 lower the value of as of binding to the DuB the that proteins are as and this promote binding that are of higher that of the isolated binding domain I. G. J. Full Text Full Text PDF PubMed Scopus Google Scholar). of Bimodular D-repeat that a polypeptide of the tandem D1, D2, and D3 repeats a Kd of for the N29 fragment of Fn, with for the repeats S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). The with the D1–3 the of two equivalent high affinity binding the three bind with low the of this we of D1–2 and each of which two repeats (Fig. As with of the to a but a with a that the each low and intermediate affinity binding D1–2 Kd values of 0.25 and μm, whereas D2–3 values of 0.044 and μm. Because the and repeats are in amino acid and similar Kd values of for binding to the N29 fragment individually S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar), is that the two repeats in affinity for N29. in the Kd of for the D3 with the high affinity value of 0.044 for the D2–3 is in Because the higher affinity Kd value of the value of for the D1–3 domain S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar), we the of the of to higher of N29 (Fig. these to a to that used but additional for the evidence for a site in this as well with a Kd value of μm, whereas the higher affinity of to value of nm. The stoichiometry for the site but the of high affinity sites to the value of We that the D1–3 polypeptide two equivalent high affinity binding sites (Kd, nm) and at one lower affinity site (Kd, whereas the two one with affinity that is intermediate between D1–3 and D-repeats, and a affinity is with of the of a in affinity for the D-repeat domain, as repeats are to two and three tandem We to a DuBA the FnBPB as in The of the to the a Kd of with (Fig. affinity is of higher and higher of the A and all three D-repeats a lower Kd nm) with higher stoichiometry The and with the in each the of the is by at values and the this we that the stoichiometry for DuBA between and whereas that of is between and the of stoichiometry in the of Fn-binding additional by of N29 in with Fn-binding Because of the of and the low of in DuBA and DuBAD, their at is with the N29 the of N29 to in the of of the are in the N29 fragment in a is with increasing of with the of representing of the The of N29 used in these at μm, to the lower of DuBA and the of the N29 is the of the of DuBA to that of N29 (Fig. is that the is or more at a for DuBA and for (Fig. The a of by to the on the at of and to values of for DuBA and for DuBAD, in with the of DuBA and on of the N29 fragment of as in for of DuBA and DuBAD, and the of the for N29 fragment the of the of DuBA or to that of N29, with the at μm. In more one at a the are as The are to all with We have of the requirements for high affinity binding of the 29-kDa N-terminal fragment of (N29) to the FnBPB of S. In to the D-repeats, two binding domains which we designated DuB and DuA. of these domains to the of two binding and which to Fn-binding segments that predicted to in FnBPA U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). The amino acid of these and most of the other Fn-binding segments that predicted to within FnBPA are in and to have the of most of these As in the DuBA polypeptide equivalent of the predicted FnBPA binding the is to the predicted FnBPA segments and and this is followed by complete of the predicted Fn-binding segments and and a of Fn-binding The of three complete binding segments and a of a fourth is in with the stoichiometry values of and in of N29 binding to In to these the polypeptide a complete FnBPA followed by segments and a of the C terminus of the D3 repeat. additional complete known or predicted binding segments that are in for a of more stoichiometry of to the of binding segments that predicted to in the FnBPA on the of motifs U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). We have that the and segments each bind N29 with low whereas the larger DuBA polypeptide that these and other segments affinity or are by the D-repeats, two tandem D-repeats display affinity for N29 that is intermediate between repeats, and the D1–3 The affinity of the D1–2 and D2–3 with repeats by the by U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar) to the of high affinity N29 binding (Kd, nm) to acid the of the S. the affinity of D1–3 with the and the high affinity of the DuBA domain are more to by this As in the high affinity (Kd, of the is to a of motifs that promote of a that the modules in the N29 domain of high affinity segments and that bound of modules with much lower affinity (Kd, μm). the that motifs in each of these three segments are for the of most with the which bound with a Kd of μm. to two and which motifs in the of S. and are known to 1F1 and bound in tandem to the with a Kd of U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). The is that segments that bind two modules are to display low micromolar Kd and high affinity binding that all modules in a tandem The of a is the of which the and a of affinity for N29 with (Fig. Because FnBPA of S. aureus U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar), the ability of the D1–3 polypeptide to bind N29 with affinity with that of for by that binding of of the of binding motifs that are or predicted to modules binding of N29 to the D1–3 polypeptide and is at the of The 1F1 which is to by a PubMed Scopus Google Scholar), is as the of N29 with D1–3. D1–3 is to of motifs to bind two molecules of N29 in the as by two equivalent high affinity sites (Kd, ∼1 nm). The C terminus of D1–3 by is to bind a of N29 by a which for the lower affinity binding site Kd of that in of D1–3 with N29. is by that this bind with a Kd of S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar) but with or to the low affinity sites by the D1–2 and D2–3 (Kd, and which we to the respective segments and that motifs for binding of (Fig. The higher affinity with Kd of 0.25 for D1–2 and 0.044 for by the of the to segments and of that four (Fig. In of this the Kd of 0.044 for D2–3 is to that of the of S. which has the of four binding motifs and a Kd of for N29 U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). the additional in affinity that all three repeats are on this D1–3 in D1–2 or The is that D1–3 has two sites complete segments and in which to that between bound N29 molecules additional to the in this that the N29 domain of a in the of in the extracellular matrix I. J. Sci. 2003; PubMed Scopus Google Scholar). The seven predicted Fn-binding segments that are N-terminal of the domain also lack a by the in also lack a 5F1 the in binding to this are in the of of this that binding of N29 to these segments to three modules the ∼2 values for and are of similar to of other and that are known or predicted to bind two the DuB and domains are in tandem as the DuBA we binding of N29 with a Kd of ∼10 and stoichiometry of in with of binding segments and in this of the DuB and domains in the of segments that the of the tandem which as the affinity two D-repeats are to one we as means of binding 1F1 we are to this in affinity to between bound molecules of N29. In the of motifs in binding of we that the of motifs for binding of in of the known or predicted segments U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar). is to a of or in the binding segments and also in of the FnBPA segments of S. in a to (Fig. A for or in binding of is in that two and FnBPA both bound with respective Kd values of and U. J.M. S. Höök M. 2003; PubMed Scopus Google Scholar), similar to other that are known or predicted to bind two these the respective and are and is that the Kd values for on the of the is by that the which is known to a (Fig. with N29 S. McGavin M.J. J. 1994; Full Text PDF PubMed Google Scholar). is in and and we have that the affinity of these segments that of other that bind two to determined a in these and other between the and motifs to a and of the MSCRAMM the with N29, in the of to binding of which has been to and PubMed Scopus Google Scholar). The of in predicted 5F1 and binding segments with the of whereas the and are by and are to these other of these and the of additional

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