Synergistic Activity of the Ninth and Tenth FIII Domains of Human Fibronectin Depends upon Structural Stability

Abstract

The ninth and tenth FIII domains (FIII9–10) of human fibronectin act in synergy to promote cell adhesion via the interaction with integrin receptors. Here we describe the functional and structural properties of a set of recombinant FIII9–10 mutants containing various alanine substitutions within the key synergistic site, DRVPHSRN in FIII9, either alone or in combination with another substitution (Leu1408 to Pro), on the opposite face of FIII9, that increases stability and the functional capacity of FIII9–10. We show that the introduction of mutations into the synergistic sequence of FIII9–10 has a negative effect on the adhesion of baby hamster kidney fibroblasts and results in reduced ability of these ligands to recognize integrin α5β1. Conformational stability of the FIII9 domain in the synergy site mutants is likewise reduced in comparison with native FIII9. The Leu1408 to Pro substitution in mutant FIII9–10 proteins carrying substitutions in the synergy site results in a substantial recovery of the adhesive activity of the mutants and affinity to α5β1. In keeping with the enhancement of functional activity, the Leu1408 to Pro substitution in the FIII9–10 synergy site mutants also causes a significant increase in conformational stability of FIII9. These observations imply a strong positive correlation between the biological activity and conformational stability of the assessed FIII9–10 mutants and suggest that a Leu1408 to Pro substitution restores the biological activity of the mutants via their ability to restore their conformational stability. We conclude that domain stability may be a major determinant of the synergistic potential of FIII9. Our data underscore the value of using more than one approach in such structure-function studies and the requirement for validating the global structural integrity of protein ligands in which sequences that disrupt function have been perturbed. The ninth and tenth FIII domains (FIII9–10) of human fibronectin act in synergy to promote cell adhesion via the interaction with integrin receptors. Here we describe the functional and structural properties of a set of recombinant FIII9–10 mutants containing various alanine substitutions within the key synergistic site, DRVPHSRN in FIII9, either alone or in combination with another substitution (Leu1408 to Pro), on the opposite face of FIII9, that increases stability and the functional capacity of FIII9–10. We show that the introduction of mutations into the synergistic sequence of FIII9–10 has a negative effect on the adhesion of baby hamster kidney fibroblasts and results in reduced ability of these ligands to recognize integrin α5β1. Conformational stability of the FIII9 domain in the synergy site mutants is likewise reduced in comparison with native FIII9. The Leu1408 to Pro substitution in mutant FIII9–10 proteins carrying substitutions in the synergy site results in a substantial recovery of the adhesive activity of the mutants and affinity to α5β1. In keeping with the enhancement of functional activity, the Leu1408 to Pro substitution in the FIII9–10 synergy site mutants also causes a significant increase in conformational stability of FIII9. These observations imply a strong positive correlation between the biological activity and conformational stability of the assessed FIII9–10 mutants and suggest that a Leu1408 to Pro substitution restores the biological activity of the mutants via their ability to restore their conformational stability. We conclude that domain stability may be a major determinant of the synergistic potential of FIII9. Our data underscore the value of using more than one approach in such structure-function studies and the requirement for validating the global structural integrity of protein ligands in which sequences that disrupt function have been perturbed. glutathione S-transferase enzyme-linked immunosorbent assay baby hamster kidney guanidinium hydrochloride surface plasmon resonance Fibronectin, a widely expressed extracellular matrix protein, contains multiple modular regions that interact with various members of the integrin family of heterodimeric cell-surface adhesion receptors. The central cell binding domain, which is known to recognize α5β1, αvβ3, αIIbβ3 and at least eight other integrins, is particularly well characterized (for a review, see Ref. 1Johansson S. Svineng G. Wennerberg K. Armulik A. Lohikangas L. Front. Biosci. 1997; 2: 126-146Crossref PubMed Scopus (266) Google Scholar). This region, spanning type III domains FIII8, FIII9, and FIII10, has become the focus of intensive research as a model for the universal and ubiquitous RGD-driven receptor-ligand interactions. The RGD tripeptide motif, residing on a protruding, flexible loop within the FIII10 module (2Main A.L. Harvey T.S. Baron M. Boyd J. Campbell I.D. Cell. 1992; 71: 671-678Abstract Full Text PDF PubMed Scopus (421) Google Scholar, 3Leahy D.J. Aukhil I. Erickson H.P. Cell. 1996; 84: 155-164Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar), is essential for receptor binding and downstream cell adhesion events such as pp125FAK-, Rho-, and Rac-dependent signaling, cytoskeletal reorganization, and acquisition of spread morphology (4Guan J.L. Trevithick J.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (473) Google Scholar, 5Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1180) Google Scholar, 6Hotchin N.A. Kidd A.G. Altroff H. Mardon H.J. J. Cell Sci. 1999; 112: 2937-2946PubMed Google Scholar). Although it is presently not clear by which mechanism an RGD-containing ligand initiates a conformational change in the integrin headpiece and transmittal of the adhesive signal across the plasma membrane, the recent solution of the crystal structure of integrin αvβ3 in complex with an isolated RGD peptide by Xiong et al. (7Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1387) Google Scholar) is an important first step toward elucidating the structural basis of the FIII10-integrin interaction. The FIII9 domain adjacent to FIII10 is known to act in synergy with the latter in promoting integrin-mediated cell adhesion, drastically increasing the activity of a single FIII10 domain but not having any activity on its own (8Mardon H.J. Grant K. FEBS Lett. 1994; 340: 197-201Crossref PubMed Scopus (81) Google Scholar). A number of sites within FIII9 have been proposed to be critical for its synergistic effect. The most well studied of these is the DRVPHSRN sequence or the so-called “synergy site,” the larger part of which is located on a surface loop of FIII9 lying approximately in plane with the RGD loop (3Leahy D.J. Aukhil I. Erickson H.P. Cell. 1996; 84: 155-164Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 9Copié V. Tomita Y. Akiyama S.K. Aota S. Yamada K.M. Venable R.M. Pastor R.W. Krueger S. Torchia D.A. J. Mol. Biol. 1998; 277: 663-682Crossref PubMed Scopus (133) Google Scholar) and thus being potentially accessible for contact with the integrin dimer. Previous studies have demonstrated that integrins binding to the FIII9–10 domain pair have a common requirement for the intact RGD motif, but they display distinct preferences for individual residues within the synergy site of FIII9 or may not need this site at all. For instance, while high affinity α5β1 binding is most sensitive to substitutions for Arg1379 (10Aota S. Nomizu M. Yamada K.M. J. Biol. Chem. 1994; 269: 24756-24761Abstract Full Text PDF PubMed Google Scholar), the most critical residues for αIIbβ3 appear to be Asp1373 and Arg1374 (11Bowditch R.D. Hariharan M. Tominna E.F. Smith J.W. Yamada K.M. Getzoff E.D. Ginsberg M.H. J. Biol. Chem. 1994; 269: 10856-10863Abstract Full Text PDF PubMed Google Scholar), whereas αvβ3 and α3β1are indifferent to the presence of the FIII9 domain (11Bowditch R.D. Hariharan M. Tominna E.F. Smith J.W. Yamada K.M. Getzoff E.D. Ginsberg M.H. J. Biol. Chem. 1994; 269: 10856-10863Abstract Full Text PDF PubMed Google Scholar, 12Akiyama S.K. Aota S. Yamada K.M. Cell Adhes. Commun. 1995; 3: 13-25Crossref PubMed Scopus (54) Google Scholar). Other amino acids in the vicinity of the classical synergy site (notably Arg1369, Arg1371, Thr1385, and Asn1386) have also been shown to contribute to the synergistic activity of FIII9 (13Redick S.D. Settles D.L. Briscoe G. Erickson H.P. J. Cell Biol. 2000; 149: 521-527Crossref PubMed Scopus (161) Google Scholar), although no integrin specificity has been determined for these residues. Taken together, these findings support the notion that each distinct integrin-ligand interaction, despite exhibiting some overall topological similarities, has its idiosyncratic characteristics, which define the precise conditions for an efficient and specific binding to the synergistic FIII9–10 module pair. Biophysical studies have revealed that in comparison with FIII10, the FIII9 domain is thermodynamically rather unstable (14Spitzfaden C. Grant R.P. Mardon H.J. Campbell I.D. J. Mol. Biol. 1997; 265: 565-579Crossref PubMed Scopus (92) Google Scholar) and displays much slower folding kinetics (15Plaxco K.W. Spitzfaden C. Campbell I.D. Dobson C.M. J. Mol. Biol. 1997; 270: 763-770Crossref PubMed Scopus (109) Google Scholar). Coupling to the neighboring FIII modules within fibronectin clearly plays an important role in stabilizing FIII9, since it exhibits around 3-fold higher conformational stability in module pairs FIII8–9 and FIII9–10 than on its own (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and altering its distance from FIII10 by introducing interdomain linkers of varying length critically reduces its stability (14Spitzfaden C. Grant R.P. Mardon H.J. Campbell I.D. J. Mol. Biol. 1997; 265: 565-579Crossref PubMed Scopus (92) Google Scholar). The relationship between the thermodynamic properties of FIII9 and its synergistic integrin-dependent biological activity, however, remains little studied despite its likely important implications for understanding the mechanism of integrin-ligand complex formation. A previous investigation has shown that the decrease in FIII9 stability due to the insertion of the above-mentioned linkers between FIII9 and FIII10 coincides with the reduced adhesive activity of the domain pair and impaired induction of intracellular signaling (17Grant R.P. Spitzfaden C. Altroff H. Campbell I.D. Mardon H.J. J. Biol. Chem. 1997; 272: 6159-6166Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). More recently, we were able to demonstrate a link between the enhancement of biological function of the FIII9–10 pair by the FIII8 module and its positive influence on the structural stability of FIII9 (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In the latter study we also probed the role of the FIII9 synergy site in stabilizing the FIII8–9 pair within the FIII8–9–10 module tandem, documenting a negative impact of perturbing the synergy sequence in this three-domain context. Here, we have specifically addressed the question whether, and to what extent, do specific amino residues within the synergistic DRVPHSRN loop contribute to the global stability of FIII9 and this to the functional of the FIII9–10 domain pair. We a combination of and biological to show that the structural properties of various FIII9–10 synergy site mutants to their integrin of the type FIII9–10 domain pair into the has been (8Mardon H.J. Grant K. FEBS Lett. 1994; 340: 197-201Crossref PubMed Scopus (81) Google Scholar). substitutions into the type FIII9–10 were the The mutations were to the synergistic DRVPHSRN sequence of Arg1379 to mutant to and to mutant as in of mutant containing the substitution of with has been (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). mutations in the synergy site were in FIII9 by Leu1408 with Pro and The latter substitution also into the type FIII9–10 in mutant The sequence of mutants using of of for mutant FIII9–10 substitutions in the of of Leu1408 with in a type and mutant FIII9–10 were expressed in as glutathione S-transferase proteins and as (8Mardon H.J. Grant K. FEBS Lett. 1994; 340: 197-201Crossref PubMed Scopus (81) Google Scholar). For FIII proteins were by of the proteins as (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). α5β1 in and surface plasmon resonance studies from human as (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). with α5β1 and FIII9–10 ligands were in the presence of as (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). were in and binding in the of ligand from the binding of the proteins to with for each ligand and from the for data from the were by using a results expressed as the of at least or interaction using a were at using the as resonance on to the A of of FIII9–10 proteins in were at to a of For integrin α5β1 for at and at a of the and were in containing and The of integrin were on a protein as a The were with a of were by using the global of the using a least were for each interaction. S.D. Cell and were with baby hamster kidney fibroblasts as (8Mardon H.J. Grant K. FEBS Lett. 1994; 340: 197-201Crossref PubMed Scopus (81) Google Scholar). Cell by with crystal as (8Mardon H.J. Grant K. FEBS Lett. 1994; 340: 197-201Crossref PubMed Scopus (81) Google Scholar). The data expressed as the of at least were on recombinant FIII9–10 in guanidinium hydrochloride in as (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). were at on a using an of at from to from the were for a mechanism as A Scholar). in the the effect of perturbing the synergy site of FIII9 on integrin α5β1 receptor binding were with type FIII9–10 ligand and mutants and various substitutions within the DRVPHSRN sequence The of the interaction at the of the shown in to increase as more substitutions were from for the type to for mutants and and for mutant on This of high affinity α5β1 binding on the intact synergy site in with previous studies (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Aota S. Yamada K.M. A. Y. Mardon H.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and demonstrated the and effect of individual synergistic residues in efficient receptor The results by were using surface plasmon resonance the of of the complex formation. The for the interaction by this a increase introduction of mutations into the synergy loop of FIII9, being for native FIII9–10 and for mutant on This from the slower and of the integrin from the FIII9–10 as with the type not The between the from these and the data affinity of the least mutant type and is likely due to the assay with fibronectin ligands and whereas integrin and fibronectin were for in studies the integrin to the ligand in whereas the in the of which may have to the of the of individual FIII9–10 show a clear of binding affinity on the native structure of the synergy loop and suggest that this synergy requirement is of the of of the FIII9–10 ligand to the studies in have shown that a single Leu1408 to Pro within the FIII9–10 domain pair is able to a significant increase in structural stability to the native FIII9 domain an intact synergy loop der C. H. H. J. in Scholar). We to the functional of this substitution in the of type FIII9–10 as well as synergy site mutants and with integrin binding with the various FIII9–10 mutants and α5β1 revealed that mutant to the integrin with higher affinity than the type and the substitution in mutant in a enhancement of function A in α5β1 binding capacity also with mutants and in comparison with mutants and determined the integrin binding properties of the FIII9–10 we to the ability of these fibronectin to support integrin-mediated adhesion of which integrins α5β1 and αvβ3 (17Grant R.P. Spitzfaden C. Altroff H. Campbell I.D. Mardon H.J. J. Biol. Chem. 1997; 272: 6159-6166Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), as a more of biological activity of cell to revealed that type FIII9–10 and mutant were adhesive for than mutants and the latter and of the type activity, at a of Cell a in the of cell adhesion, more sensitive to in the synergy the of cell at the to and of type FIII9–10 activity for and while the for of that of the type The FIII9–10 mutants carrying an substitution were in these to this able to the adhesion promoting in the FIII9–10 synergy site The adhesive properties of mutant were in comparison with the native FIII9–10 at previous Cell on mutants and to type being and more efficient with and at to however, reduced in comparison with The of likewise by the substitution The activity of mutants and with and and at Cell on mutant at the as that by mutant Previous with fibronectin spanning domains FIII8 FIII10 a role for the FIII9 synergistic sequence in to the structural stability of the FIII8–9 pair (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In this we the correlation between the functional activity of FIII9–10 various mutations in the synergy site and their thermodynamic from with FIII9–10 mutants using as a in and of the FIII9–10 domain pair is known to by a well for of for the individual FIII9 and FIII10 domains (14Spitzfaden C. Grant R.P. Mardon H.J. Campbell I.D. J. Mol. Biol. 1997; 265: 565-579Crossref PubMed Scopus (92) Google Scholar, H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). the stability of FIII10 not on the presence of FIII9 (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), the first step of the to the of FIII9 (14Spitzfaden C. Grant R.P. Mardon H.J. Campbell I.D. J. Mol. Biol. 1997; 265: 565-579Crossref PubMed Scopus (92) Google Scholar), of and is shown in for The a domain mechanism as by and A Scholar). FIII9 in mutant the properties as in type the FIII9 module in mutant an effect that more for mutant The of to domain from for type FIII9 to for and to for and the negative in the of to the type from to These results thus demonstrate that the thermodynamic stability of FIII9 within the FIII9–10 pair is sensitive to specific amino substitutions within the synergy for FIII9 in mutants the Leu1408 to Pro substitution were a substantial to the for mutants as with their The increase in conformational stability of FIII9 as by the and for mutant pairs and for the first step of the type and mutant FIII9–10 domain to potential with the individual for the region, the of were from an value of to potential with the individual for the region, the of were from an value of A Scholar). in a the thermodynamic stability of the FIII9–10 mutants with their functional activity by correlation from due to its high The results of the in a strong correlation between the structural stability of the FIII9–10 proteins and biological activity correlation The study toward elucidating the structural basis for the synergistic biological activity of FIII9 in the FIII9–10 module pair of the of a of we have a distinct role for the DRVPHSRN synergy sequence within the FIII9 domain in the overall conformational stability of FIII9. Our findings demonstrate perturbing the native of the FIII9 synergy site, for efficient integrin α5β1 binding and cell adhesion, to impaired domain the Leu1408 to Pro for of biological activity of the synergy site and a strong correlation between the global structural integrity and synergistic function of the FIII9–10 pair. We have alanine within the synergy site to the structural of residues to a key role in integrin-dependent adhesive (10Aota S. Nomizu M. Yamada K.M. J. Biol. Chem. 1994; 269: 24756-24761Abstract Full Text PDF PubMed Google Scholar, R.D. Hariharan M. Tominna E.F. Smith J.W. Yamada K.M. Getzoff E.D. Ginsberg M.H. J. Biol. Chem. 1994; 269: 10856-10863Abstract Full Text PDF PubMed Google Scholar, S.D. Settles D.L. Briscoe G. Erickson H.P. J. Cell Biol. 2000; 149: 521-527Crossref PubMed Scopus (161) Google Scholar). In the in biological activity for a single Arg1379 mutant is to that by et al. (13Redick S.D. Settles D.L. Briscoe G. Erickson H.P. J. Cell Biol. 2000; 149: 521-527Crossref PubMed Scopus (161) Google Scholar). of function the and of the DRVPHSRN sequence is also in with the latter substitutions of amino acids the DRVPHSRN site have been shown to have an effect in combination with Arg1379 in (13Redick S.D. Settles D.L. Briscoe G. Erickson H.P. J. Cell Biol. 2000; 149: 521-527Crossref PubMed Scopus (161) Google Scholar), although it is not clear to what these substitutions or global we show that the in the FIII9–10 mutants not the of the synergy site but also have a strong negative effect on the thermodynamic stability of the FIII9 that the synergy site on a surface loop from the domain it to the overall of FIII9, as synergy site mutants carrying multiple substitutions at much than the The that this effect is not for the single to the of more between the and the synergy loop in stability to FIII9. The structure-function correlation with the synergy site mutants is by the that a single for on the other of the FIII9 module in to a contact with either the synergy loop or the integrin the effect of the synergy site in of biological function and structural of the FIII9–10 pair. influence on integrin is most likely via global of the FIII9 In a previous we studied the influence of the FIII8 module within the FIII8–9–10 domain the stability of FIII9, a link between enhancement of synergistic biological activity and of structural integrity of an FIII9–10 pair containing a synergy site (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). the basis of these we that the requirement for the DRVPHSRN sequence may be critical for integrin binding in the of an isolated FIII9–10 pair. we show that a Leu1408 to Pro substitution within FIII9–10 a effect the need for an Taken together, these studies imply that any strong influence an increase in the global stability of FIII9, be it a single critical amino substitution or a domain, may the requirement for an intact synergy site in efficient integrin This of receptor to an important role in the of The latter notion may be in the of the that some integrins appear to synergy site binding as a function of their For instance, α5β1 may its on the synergy sequence in the presence of or Aota S. van Yamada K.M. D.J. van J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), while and recognize FIII9 L. Aota S. Yamada K.M. R. A. R. 1999; Google Scholar). the requirement for the synergy site may to a on the specific overall of the fibronectin ligand and the integrin studies synergy in (11Bowditch R.D. Hariharan M. Tominna E.F. Smith J.W. Yamada K.M. Getzoff E.D. Ginsberg M.H. J. Biol. Chem. 1994; 269: 10856-10863Abstract Full Text PDF PubMed Google Scholar) or in the of a FIII (10Aota S. Nomizu M. Yamada K.M. J. Biol. Chem. 1994; 269: 24756-24761Abstract Full Text PDF PubMed Google Scholar) have shown that mutations in the DRVPHSRN sequence integrin binding and cell adhesion in the of a native FIII9 This that while conformational stability of FIII9 may be an important function of the synergy it is likely that this is not the mechanism this its synergistic potential and as proposed by a number of previous and specific between residues in the synergy site and the ligand binding of the receptor also for biological (16Altroff H. van der Walle C.F. Asselin J. Fairless R. Campbell I.D. Mardon H.J. J. Biol. Chem. 2001; 276: 38885-38892Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), of these of may not be and need to be into the and of interactions. The specific stability of the FIII9–10 domain pair appear to the conditions in which domain is For instance, while in this and other studies show that the FIII9 module is thermodynamically much than FIII10, using of the module pair that FIII9 may be more than FIII10 A. K. V. Sci. S. A. 2001; PubMed Scopus Google Scholar). The between the data may be by of the that is a that the of rather than the by that the the of the fibronectin and that the FIII9 domain in it be to the synergy site mutants in the study the to stability of FIII9 and which specific most to by In this strong in support of the that domain stability is an important determinant of integrin synergistic biological potential of the FIII9 Our that amino substitutions in the thermodynamic stability of FIII9 thus the requirement for structural of modular proteins in receptor-ligand interactions. We to Grant for the mutants and and to van der for with