Probing the Conformation of the Fibronectin III1–2 Domain by Fluorescence Resonance Energy Transfer (2008)

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III1 and III2, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III1–2. Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III1 and III2, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III1–2. Fibronectin (FN) 3The abbreviations used are: FN, fibronectin; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; GST, glutathione S-transferase; GdmCl, guanidinium chloride. 3The abbreviations used are: FN, fibronectin; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; GST, glutathione S-transferase; GdmCl, guanidinium chloride. is a 500-kDa modular dimeric protein and a major component of the extracellular matrix. It exists in the blood and other body fluids as a soluble compact molecule and undergoes cell-mediated assembly to form an insoluble three-dimensional fibrillar matrix (reviewed in Ref. 1Mao Y. Schwarzbauer J.E. Matrix Biol.. 2005; 24: 389-399Google Scholar). The process of FN matrix assembly has been implicated in embryonic development, wound healing, and cancer (2Ghosh K. Ren X.D. Shu X.Z. Prestwich G.D. Clark R.A. Tissue Eng.. 2006; 12: 601-613Google Scholar, 3Honest H. Bachmann L.M. Gupta J.K. Kleijnen J. Khan K.S. Br. Med. J.. 2002; 325: 301Google Scholar, 4Santimaria M. Moscatelli G. Viale G.L. Giovannoni L. Neri G. Viti F. Leprini A. Borsi L. Castellani P. Zardi L. Neri D. Riva P. Clin. Cancer Res.. 2003; 9: 571-579Google Scholar). FN is composed of type I–III modules, and sets of these modules comprise binding domains for cells and for other extracellular matrix components (see Fig. 1A). Three of these binding domains are essential for matrix assembly (1Mao Y. Schwarzbauer J.E. Matrix Biol.. 2005; 24: 389-399Google Scholar). Integrin receptor interactions with the cell-binding domain tether disulfide-bonded FN dimers to the cell surface, where FN-FN interactions involving the N-terminal assembly domain form dimers into fibrils. In addition to these essential domains, other FN-binding sites have been implicated in assembly. In particular, the III1–2 FN-binding domain plays a regulatory role in matrix assembly. Within this domain reside a cryptic FN-binding site in III1 and a site available for FN binding in the native form of III2 (5Aguirre K.M. McCormick R.J. Schwarzbauer J.E. J. Biol. Chem.. 1994; 269: 27863-27868Google Scholar, 6Hocking D.C. Sottile J. McKeown-Longo P.J. J. Biol. Chem.. 1994; 269: 19183-19187Google Scholar, 7Ingham K.C. Brew S.A. Huff S. Litvinovich S.V. J. Biol. Chem.. 1997; 272: 1718-1724Google Scholar, 8Sechler J.L. Rao H. Cumiskey A.M. Vega-Colon I. Smith M.S. Murata T. Schwarzbauer J.E. J. Cell Biol.. 2001; 154: 1081-1088Google Scholar). Recombinant FN lacking III1 is assembled into a matrix at wild-type levels, but that lacking the III1–2 domain results in short immature FN fibrils (8Sechler J.L. Rao H. Cumiskey A.M. Vega-Colon I. Smith M.S. Murata T. Schwarzbauer J.E. J. Cell Biol.. 2001; 154: 1081-1088Google Scholar). Peptides derived from the III1–2 domain or antibodies against III1–2 block matrix assembly by cultured cells (9Chernousov M.A. Faerman A.I. Frid M.G. Printseva O.Y. Koteliansky V.E. FEBS Lett.. 1987; 217: 124-128Google Scholar, 10Chernousov M.A. Fogerty F.J. Koteliansky V.E. Mosher D.F. J. Biol. Chem.. 1991; 266: 10851-10858Google Scholar, 11Morla A. Ruoslahti E. J. Cell Biol.. 1992; 118: 421-429Google Scholar). Furthermore, FN binding to this region is enhanced when FN is mechanically stretched (12Zhong C. Chrzanowska-Wodnicka M. Brown J. Shaub A. Belkin A.M. Burridge K. J. Cell Biol.. 1998; 141: 539-551Google Scholar). Taken together, these results suggest that conformational changes in the III1–2 domain may control its interactions during FN assembly. To more fully understand the roles of native and cryptic FN-binding sites in matrix assembly, the conformational dynamics of III1–2 must be characterized. One approach to this problem is to tag III1–2 with fluorescent probes, which, in conjunction with fluorescent resonance energy transfer (FRET), create a molecular conformational sensor. FRET involves the radiationless transfer of energy from an excited donor fluorophore to an acceptor fluorophore, a process that is very sensitive to the distance between the two fluorophores (13Miyawaki A. Dev. Cell.. 2003; 4: 295-305Google Scholar, 14Miyawaki A. Sawano A. Kogure T. Nat. Cell Biol.. 2003; 5: S1-S7Google Scholar, 15Tsien R.Y. Annu. Rev. Biochem.. 1998; 67: 509-544Google Scholar). Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), are highly related to green fluorescent protein (GFP). Because the emission spectrum of CFP is well matched to the excitation spectrum of YFP, these two fluorophores have been widely used as a donor-acceptor pair in FRET studies (13Miyawaki A. Dev. Cell.. 2003; 4: 295-305Google Scholar, 14Miyawaki A. Sawano A. Kogure T. Nat. Cell Biol.. 2003; 5: S1-S7Google Scholar, 15Tsien R.Y. Annu. Rev. Biochem.. 1998; 67: 509-544Google Scholar). In this study, we describe a FRET conformational sensor designed to test the idea that intramolecular interactions between III1 and III2 sequester key FN-binding and assembly sites. We show that III1–2 with CFP and YFP fused to the N and C termini, respectively, displays a clear FRET signal, indicating that the attached fluorescent proteins and thus the ends of III1–2 are in close proximity. FRET data from III1–2 mutants support the presence of a stabilizing intermodule salt bridge that regulates FN-binding activity. Production and Purification of CFP/YFP Fusion Proteins and SDS-PAGE—The enhanced CFP gene (pECFP, Clontech, Mountain View, CA) with a Gly-Ser-Gly-Ser-Gly coding sequence at the 3′-end and BamHI and BglII restriction sites at the 5′- and 3′-ends, respectively, was generated by PCR and cloned into the pGEM vector (Promega, Madison, WI) to generate pGEM-CFP. The CFP BamHI-BglII fragment of pGEM-CFP was inserted into the BamHI site of pGEX-6P-2 (GE Healthcare) upstream of human III1–2 to generate the donor-only construct CFP-III1–2 in pGEX or CIII. The gene expressing III1–2-YFP with BamHI and EcoRI restriction sites at the 5′- and 3′-ends, respectively, was created by overlap extension PCR. Overlap PCR consisted of a first PCR with primers flanking the termini of the FN III1–2 domain in pGEX-6P-2 but excluding the stop codon at the 3′-end and primers flanking YFP in pEYFP (Clontech) including the YFP stop codon. The second PCR used the two PCR products from the first reaction and the aforementioned 5′-primer of the III1–2 domain and the 3′-primer of pEYFP. The final PCR product was cloned into the pGEM vector to generate pGEM-YFP, which was subsequently cloned into the BamHI and EcoRI sites of pGEX-6P-2 to generate the acceptor-only gene, III1–2-YFP or IIIY. The donor-acceptor construct, CFP-III1–2-YFP or CIIIY, was created by ligating the BamHI-CFP-BglII fragment from pGEM-CFP to the BamHI site of the acceptor-only construct IIIY. All of the constructs were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli DH12α. Mutants CIIIYK669A and CIIIYD767A, with alanine substitutions at FN residues Lys669 and Asp767, were created in a similar manner using mutant III1–2 constructs kindly provided by Dr. Iain Campbell (Oxford University). Mutant CIIIY in which both Lys669 and Asp767 were mutated to alanine (CIIIYK669A/D767A) was created by overlap extension using primers overlapping the single mutations in mutant III1–2 domains. Mutant CIIIYK672A was created by PCR with primers overlapping the mutation site and incorporating the coding sequence for the mutated residue. To create CIIIYK672A/D767A, we ligated pGEX-6P-2-CIIIYK672A at the BamHI and StuI sites and inserted this mutant fragment into the pGEX-6P-2-CIIIYD767A fragment ligated at the BamHI and StuI sites. Overlap PCR with primers corresponding to the Lys669 and Lys672 genes and Asp767 were used to create the pGEX-6P-2-CIIIYK669A/K672A/D767A triple mutant. The proteins were then purified by affinity chromatography on glutathione-Sepharose 4 Fast Flow (GE Healthcare) following the manufacturer's specifications. High concentration fractions were dialyzed into 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, and 1 mm dithiothreitol. Following dialysis, Precision protease (GE Healthcare) was used to cleave off GST, which was then separated from III1–2-containing proteins by affinity chromatography. Proteins were dialyzed into 50 mm Tris-HCl (pH 8.0), 50 mm NaCl, and 2 mm EDTA; flash frozen; and stored at –80 °C. Prior to use, the fusion proteins were thawed at 4 °C and spun at 13,000 rpm for 5 min. The GFP and FN epitopes were immunodetected using mouse monoclonal antibodies JL-8 (BD Biosciences) and 5E6, respectively. JL-8 recognizes GFP and its variants such as CFP and YFP. Monoclonal antibody 5E6 was raised against a GST-III1–2 fusion protein and is specific for human III1–2. Proteins were separated by SDS-PAGE on a 10% polyacrylamide gel, transferred to Hybond ECL nitrocellulose membrane (Amersham Biosciences), and incubated with primary and antibody 5E6 was used at JL-8 was to the manufacturer's was used at a of were with in the region and were using a in a and at a of °C. of 2 and a of 50 were The of III1–2 and CIIIY were at of in and in the presence of and 2 guanidinium in were in the of and to which is expressed in Three were for FRET to FRET CFP/YFP fusion proteins were to in 50 mm Tris-HCl (pH 50 mm NaCl, and 2 mm were and matched using the protein the at and of by SDS-PAGE and In FRET were in a with a at °C. Prior to the proteins were thawed and at 4 °C. was by the protein in a with a the in the and the protein to excitation of was used for the FRET and the emission between and were by the emission in an and emission was a of and at and the CFP and YFP were with excitation of the FRET in the and presence of of at and for and respectively, were protease addition and excitation at for and at or for IIIY. To protease fusion proteins were with for using the of protease to was with 1 mm The products of proteolysis were separated on a polyacrylamide and with antibodies as of products were from a of molecular distance to molecular emission of CIIIY and were in the and presence of at of and 2 of CIIIY and its mutants at and was during excitation at in the and presence of N-terminal 70-kDa FN fragment using the following at and were at of 1 the 70-kDa fragment was to the and were for for binding and conformational emission data for the the addition of the 70-kDa fragment and for the at of were used to and final FRET FRET were from donor in the presence and of the 70-kDa fragment. FRET were from the emission of and P. S. Scholar). The FRET is in where and are the of the CFP emission in the donor-acceptor protein and respectively. of III1–2 of III1–2 into the by I. D. L.M. Campbell J.. was to the FRET The was to Lys672 and Asp767 other as in a salt In an III2 was with to III1 the the and residues were as and were and of a FRET in has a compact H. G. J. Biol. Chem.. but is into an form that in FN-FN interactions during We propose in soluble FN, III1 and III2 to sequester FN-binding sites that become exposed during FN extension and assembly. of the of III1–2 may to FN-binding sites is in Fig. and is based on the following of III1–2. FN type III are with a of and a of K. Ruoslahti E. J. Biol.. 2003; Scholar, I. Cell.. Scholar). The for III1 and III2 are and respectively, the that these modules may is a between III1 and III2 with the sequence the of and residues is of a P. J. Biol.. Scholar). The of a form of the of the is which is to the two modules to by intramolecular interactions III1–2 a compact which FN-binding sites are by (12Zhong C. Chrzanowska-Wodnicka M. Brown J. Shaub A. Belkin A.M. Burridge K. J. Cell Biol.. 1998; 141: 539-551Google Scholar, T. S. the domain and the FN-binding sites that the N of III1 and the C of III2 are close in To this we a conformational sensor. CFP and YFP were conjugated at the N and C termini of respectively, to create CIIIY We a between the FN modules and the fluorophores to a between the fluorophores and the III1–2 domain and to M. S. P. R.J. Biochem.. Scholar, Scholar). the of type III modules, a distance between N and C termini in this is The distance at which of CFP energy is transferred to YFP, as the is Biochem.. Scholar). The used in this is of FRET to or which to a distance of or between the donor and acceptor the distance between termini in is the distance for FRET to and be in In addition to the CIIIY we also created a donor-only with CFP conjugated to the N of III1–2 and a acceptor-only with YFP conjugated to the C fusion proteins were by SDS-PAGE and with antibodies specific to the III1–2 or GFP FN and GFP epitopes were in CIIIY, and and fusion proteins were of the binding that the N-terminal 70-kDa FN fragment to CIIIY and III1–2 Fig. indicating that FN binding was by the addition of CFP and YFP. The of FN type III modules and of CFP and YFP are In CIIIY and III1–2 were both by a at of a The of the at that CIIIY and III1–2 have similar CIIIY has a of which is the III1–2 at the of GFP, which has very similar to CFP and YFP M. 2002; is M.A. FEBS Lett.. 2002; Scholar). of the of two GFP and III1–2 a of which is with for the of CIIIY from its CFP, YFP, and III1–2. is a at in the for which to may be to an domain such as the that may be by the from CFP and YFP in the CIIIY FRET in studies of CIIIY a FRET between the CFP and YFP CFP excitation in CIIIY resulted in emission at and corresponding to CFP and YFP emission respectively. The of YFP emission excitation at that the CFP and YFP in the CIIIY construct are close for To this we designed to the close of the FRET sites for in III1–2 A. Ruoslahti E. J. Cell Biol.. 1992; 118: 421-429Google the CFP/YFP fluorophores are to protease (reviewed in Ref. M. 2002; Scholar). The addition of to CIIIY resulted in a of YFP emission and a of the CFP emission by at changes are highly with energy transfer in the CIIIY The transfer in CIIIY, using 1 (see a FRET of in similar of FRET was using and 50 of CIIIY, indicating that FRET was of protein concentration Because GFP fluorescence is sensitive to changes in the M. 2002; which may with during we fluorescence emission in CIIIY and in the with during of CIIIY proteolysis a in FRET with in the presence of the protease protease addition to control the emission of the donor-only and the emission of excitation to We also in the acceptor in when YFP at products were the to FRET in Fig. with antibodies with molecular of and CIIIY products to generated by of the donor-only and acceptor-only were The fragment is the of CFP or YFP, at the between III1–2 and the fluorophore, the fragment fluorophore and a of III1–2. the of FRET and the distance between the donor and acceptor fluorophores from CIIIY results demonstrate the presence of intramolecular FRET in In a second we CIIIY with this has been to intramolecular interactions and FN from a compact to an M.S. S.A. J.. Scholar). In 2 GdmCl, fluorescence emission and FN in an conformation, but the its modules M.S. S.A. J.. Scholar). the other GFP fluorophores are and fluorescent at of to K. M. I. D. T. 2003; Scholar). We the of and 2 on FRET in The emission of CIIIY in was by a of the YFP emission with its emission in a the of CIIIY and III1–2 in were with in a and indicating that the of III1–2 was by the concentration of was from to 2 the YFP emission was eliminated from the emission of CIIIY excited at The of CIIIY and III1–2 in 2 were from in and and were by a in the to a of The of with the of FRET in the the of intramolecular interactions between the FN modules in III1–2. and FRET studies in the and presence of are of III1–2 in the a compact III1–2 domain in the of a III1–2 domain with in the FN modules in GdmCl, and a fully III1–2 domain with of in 2 in III1–2 FRET and FN of III1–2 is to be by a salt bridge between Lys669 in III1 and Asp767 in III2 I. D. L.M. Campbell J.. Scholar). We used FRET to the of CIIIY mutants with alanine residues in of Lys669 and Asp767 FRET in was by an in the acceptor of the donor data or the indicating that the N and C termini are in the mutant. of changes in FRET in in the presence of the presence of intramolecular FRET a FRET CIIIY Furthermore, the of on was similar to its on CIIIY, a in FRET of protease addition (see Fig. the of proteolysis in CIIIY and were that the mutations major the in FRET from in CIIIY to in the that or both of these mutations conformational changes in III1–2 that the distance between the N and C termini. of the single mutant acceptor emission similar to that of as by the acceptor of the donor data In the emission of CIIIYK669A was with that of wild-type mutation of Asp767 III1–2 conformation, the mutation Furthermore, the single mutant results show that the FRET of and is to of III1–2 sequence from a highly Lys672 I. D. L.M. Campbell J.. Scholar). CIIIY FRET a in the with that in The in FRET changes with mutation of Lys672 or Asp767 that these two residues are in is by the triple mutant which also FRET with CIIIY and The mutant a FRET that was similar to that of its was with that of CIIIY FRET are from the donor and the in a conformational similar to that of the single mutant. The for the mutant that may be an in the acceptor the FRET in CIIIYK672A/D767A, as well as the in acceptor emission in single mutant and in an between Lys672 and Asp767 in III1–2. To the conformational that the FRET the FN-binding of we the of CIIIY and its mutants in the presence and of the N-terminal 70-kDa FN-binding fragment (see Fig. 1A). of Asp767 or in with Lys672 in FRET similar to The addition of the 70-kDa fragment to a of FRET in CIIIYD767A, and but on the FRET in CIIIY and CIIIYK669A In the presence of the 70-kDa the FRET of CIIIYD767A, and by and respectively. in FRET with mutation of Asp767, or both support the that these two residues a salt bridge in CIIIY and that of this salt bridge conformational changes in III1–2. The changes in FRET by the 70-kDa fragment suggest that the mutations a FN-binding site in III1–2 and that binding to this site conformational changes were with indicating that this is in these intermodule The III1–2 domain of FN, two FN-binding plays a regulatory role in matrix assembly. a sensor of we have that ends of III1–2 are in close in as are other type III C. 1994; Scholar). The compact of III1–2 was disrupted by mild that separated N and C termini but the of the modules. The of CIIIY was also by mutation of Lys672 in III1 or Asp767 in of the 70-kDa FN matrix assembly fragment to mutant CIIIY a in FRET, that FN-FN interactions the of the III1–2 results into the distance between the ends of III1–2 and the of intramolecular interactions in this domain that in a compact We have used FRET to a distance between the two fluorophores and to an of the distance between the N and C termini of III1–2. FRET depends on the of the CFP and YFP fluorophores with to the of the two fluorophores is is to an of the distance between from the distance of the FRET pair J. of Scholar). FRET of in CIIIY to a distance between CFP and YFP that is or of III1–2 based on of the modules, III1 K. Ruoslahti E. J. Biol.. 2003; Scholar, M. D. Campbell K. S. 2003; and III2 I. D. L.M. Campbell J.. a distance of between the N and C termini I. D. L.M. Campbell J.. with the from FRET these two are in with to a compact of III1–2. salt bridge between Lys669 in III1 and Asp767 in III2 has been by I. D. L.M. Campbell J.. Scholar). the between III1 and III2 was disrupted in mutant with the native the other a mutation interactions between III1 and III2 I. D. L.M. Campbell J.. Scholar). We have also using FRET that of Asp767 with alanine a on III1–2 and on binding to the 70-kDa FN fragment. In on or 70-kDa fragment we a in the distance between N and C termini. similar changes in the distance between the domain ends and in 70-kDa fragment binding were with the The on III1–2 and FN binding of and but that Lys672 and Asp767 similar roles in N and C termini to other and suggest that these two residues form a salt FRET data of the distance between the ends of III1–2 and the residues that are in be with to into III1–2 with a I. D. L.M. Campbell J.. we propose two III1–2 In both a salt bridge has a and a compact III1–2 is The major is in the of III2 to The first in Fig. is similar to that of I. D. L.M. Campbell J.. with N and C termini in but with III2 to a In the second the of the modules has been III2 was to its in Fig. using as a N and C termini in the in this The of the region are between the two is overlap in the residues that the intermodule and Lys669 is in the of residues in III2 in both a for and to the interactions between these modules. The of the 70-kDa fragment with the single and mutants to an in the distance between the N and C termini of III1–2. an between the matrix assembly domain of FN and III1–2. III1–2 has both native and cryptic FN-binding this in may in of binding site for FN or for other extracellular matrix components in III1–2. The III1–2 domain has been implicated in of FN fibrils. The of binding sites 70-kDa fragment binding may be for of matrix. the to to more FN molecule the that III1–2 may as a for FN fibrils. We the with Iain and We Dr. for with the studies and Dr. for the to into III1–2. with