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The hairpin structure of the 6F11F22F2 fragment from human fibronectin enhances gelatin binding

2001; Springer Nature; Volume: 20; Issue: 7 Linguagem: Inglês

10.1093/emboj/20.7.1519

1460-2075

Andrew R. Pickford, Steven P. Smith, David Staunton, Jonathan Boyd, Iain D. Campbell,

Peptidase Inhibition and Analysis

Article2 April 2001free access The hairpin structure of the 6F11F22F2 fragment from human fibronectin enhances gelatin binding Andrew R. Pickford Andrew R. Pickford Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Steven P. Smith Steven P. Smith Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author David Staunton David Staunton Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Jonathan Boyd Jonathan Boyd Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Iain D. Campbell Corresponding Author Iain D. Campbell Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Andrew R. Pickford Andrew R. Pickford Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Steven P. Smith Steven P. Smith Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author David Staunton David Staunton Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Jonathan Boyd Jonathan Boyd Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Iain D. Campbell Corresponding Author Iain D. Campbell Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Author Information Andrew R. Pickford1, Steven P. Smith1, David Staunton1, Jonathan Boyd1 and Iain D. Campbell 1 1Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QU UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1519-1529https://doi.org/10.1093/emboj/20.7.1519 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The solution structure of the 6F11F22F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous 6F1 and 2F2 modules. The buried surface area between 6F1 and 2F2 (∼870 Å2) is the largest intermodule interface seen in fibronectin to date. The dissection of 6F11F22F2 into the 6F11F2 pair and 2F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of 6F11F22F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'. Introduction The extracellular matrix glycoprotein fibronectin is a large, multifunctional molecule involved in adhesion and migration events in a range of important physiological processes such as embryogenesis, wound healing, haemostasis and thrombosis (Hynes, 1990). As a soluble dimer in plasma, it is involved in blood coagulation through its affinity for fibrin and platelets. As an insoluble network in the extracellular matrix, it interacts with cell surface receptors and with other matrix components such as collagens and proteoglycans, thus assisting cell migration and the maintenance of tissue integrity (Hynes, 1990). The interaction between fibronectin and collagen in the extracellular matrix is well documented (Hynes, 1990) but poorly understood at the molecular level. The two proteins are co-distributed in tissues, as shown by immunofluorescence and immunocytochemical studies. Addition of extraneous fibronectin also promotes the attachment of fibroblastic cells to collagen substrates in vitro. Further more, fibronectin is observed in a regularly distributed array along collagen fibres synthesized in culture. Knowledge of the molecular basis of fibronectin's interaction with collagen would provide a better understanding of the structure and function of the extracellular matrix. The only viable approach to correlating the structure and function of the large, flexible fibronectin monomer at the atomic level is to dissect it into manageable units (Campbell and Downing, 1998). Fortunately, like many other proteins of the extracellular matrix, fibronectin is a mosaic protein consisting of repeating sequence elements or 'modules' that are capable of folding independently (Bork et al., 1996). Its primary sequence is composed almost entirely of three types of module (F1, F2 and F3), which are organized into functional domains (Figure 1A). These domains may be isolated in the form of proteolytic fragments that retain affinity for various ligands. Consequently, many of the ligand-binding sites have been mapped to specific regions of the fibronectin polypeptide. The collagen-binding domain can be isolated as a 42 kDa proteolytic fragment that retains affinity for heat-denatured collagen (gelatin) (Hynes, 1990). This domain has the module composition 6F11F22F27F18F19F1, where nFX represents the nth type X module in the native protein. Further dissection of this gelatin-binding domain by proteolysis produces three non-overlapping module pairs (6F11F2, 2F27F1 and 8F19F1) each of which retains some degree of gelatin-binding activity (Ingham et al., 1989; Litvinovich et al., 1991). This suggests that the gelatin-binding site (or sites) spans multiple modules in the domain. Figure 1.Binding of fibronectin fragments to immobilized collagen α1(I) chains. (A) The mosaic structure of a fibronectin monomer is shown, with the positions of the alternatively spliced regions EDB, EDA and IIICS depicted below. The major binding sites for cells and for other matrix components are labelled. (B) Assaying the binding of fibronectin fragments to immobilized α1 chains by surface plasmon resonance (SPR). A 20 μl aliquot of 300 μM samples of 6F11F22F2 (—), 1F22F2 (– – –), 6F11F2 (........), 1F2 (– - – -) and 2F2 (– - - –) was injected over the same flow cell of immobilized collagen α1(I) chains at a flow rate of 20 μl/min. The surface was regenerated with 50 mM HCl after each injection and the individual sensorgrams overlaid with the BIAevaluation 3.0 software (Biacore). (C) Response (RU) 25 s after the end of the injection of the fibronectin fragments as a comparison of collagen α1(I) chain binding ability. Significant binding was only detected in this assay for 6F11F22F2 and 1F22F2. Values represent the mean of triplicate samples, with standard deviation of replicate samples shown as error bars. Download figure Download PowerPoint Attempts to localize the gelatin-binding site further by recombinant expression in Escherichia coli have yielded conflicting results. Analysis of recombinant fragments produced as β-galactosidase fusion proteins showed that both the 1F2 module and the 1F22F2 module pair could bind immobilized gelatin (Banyai et al., 1990). In an earlier study, however, the 6F11F22F2 module construct only bound to gelatin if the 14 N-terminal residues of 7F1 were present at the C-terminus of the triplet fragment (Owens and Baralle, 1986). A third study identified the smallest recombinant fragment capable of binding to immobilized gelatin as 6F11F22F27F1 (Skorstengaard et al., 1994). The occurrence of F2 modules in other gelatin-binding proteins, such as the matrix metalloproteinases (MMP) 2 and 9, provides evidence for their involvement in gelatin binding by fibronectin (Collier et al., 1988; Wilhelm et al., 1989). Furthermore, recombinant expression of F2 modules from these MMPs has produced fragments with high affinity for gelatin (Banyai and Patthy, 1991; Collier et al., 1992; Banyai et al., 1994), whereas recombinant MMP2 lacking F2 modules was devoid of gelatin-binding activity (Murphy et al., 1994; Allan et al., 1995). Here we describe the gelatin-binding properties and solution structure of the 6F11F22F2 fragment from fibro nectin (Protein Data Bank entry codes 1e88 and 1e8b). Dissection of this fragment into the 6F11F2 module pair and the individual 2F2 module results in a drastic reduction in gelatin affinity, suggesting that module–module interactions are essential for optimal binding activity. A comparison of backbone amide chemical shifts of 6F11F22F2 with 6F11F2 and 1F22F2 revealed long-range interactions between the non-contiguous 6F1 and 2F2 modules. In the solution structure of 6F11F22F2, the 6F1 and 2F2 modules interact via an extensive hydrophobic interface whose buried surface area is the largest intermodule contact yet seen in fibronectin. The central 1F2 module shows no non-covalent interactions with either 6F1 or 2F2, implying that the 6F1–1F2 interface observed previously (Bocquier et al., 1999) is disrupted in the presence of additional modules. The hairpin topology of 6F11F22F2 may facilitate intramolecular contact between the flanking 1F12F13F14F15F1 and 1F3 fragments, an interaction that is believed to modulate fibronectin fibrillogenesis in the extracellular matrix. Its conformation may also account for the previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin at high ionic strength. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. Results and discussion Enhanced gelatin-binding activity in the 6F11F22F2 fragment The isolated 1F2 and 2F2 modules, the 6F11F2 and 1F22F2 module pairs and the 6F11F22F2 module triplet were produced as described in Materials and methods. During their purification, the isolated 1F2 and 2F2 modules and the 6F11F2 module pair bound weakly to the gelatin affinity column and were separated from the non-binding contaminants by isocratic elution; the 1F22F2 module pair and the 6F11F22F2 module triplet bound more tightly and were eluted with a urea gradient. The binding of 1F2, 2F2, 6F11F2, 1F22F2 and 6F11F22F2 to immobilized collagen α1(I) chains was analysed in greater detail using surface plasmon resonance (SPR) (Figure 1B and C). For the 6F11F22F2 binding to the α1(I) polypeptide chains, an acceptable fit (χ2 <2) could be obtained with a model that assumed surface heterogeneity, a common observation of immobilization through amine side chains. This treatment resolved the data into two components, with the major component exhibiting association (kon) and dissociation (koff) rates of 115 ± 12 M−1s−1 and 0.0036 ± 0.001 s−1, respectively, and an equilibrium dissociation constant of 31 ± 6 μM (Kd = koff/kon). No significant changes in the 6F11F22F2 binding were observed under acidic conditions (pH 4.5). The 1F22F2 bound with much lower affinity, with kon and koff values of 94 ± 21 M−1s−1 and 0.012 ± 0.001 s−1, respectively, which is equivalent to an equilibrium dissociation constant of 131 ± 23 μM. The binding of 1F2, 2F2 and 6F11F2 to immobilized α1(I) chains was too weak to quantify the kinetics with this assay. The very low affinity of the 1F2 and 2F2 modules and of the 6F11F2 module pair for α1(I) chains demonstrates that the gelatin-binding site of 6F11F22F2 does not reside entirely within any single module (Figure 1C). The moderate affinity of the 1F22F2 module pair may result from cooperativity between weak, independent binding sites on each module since there is no defined interface between the modules and they tumble independently of each other (Smith et al., 2000). The enhanced binding activity of 6F11F22F2 must therefore involve the formation of a composite binding site involving 1F2 and/or 2F2 and the 6F1 module, or cooperativity between independent, weak gelatin-binding sites on separate modules. The structural basis for this affinity was investigated by nuclear magnetic resonance (NMR) spectroscopy. 6F1 and 2F2 interact in the 6F11F22F2 fragment Preliminary information regarding the nature and site of any intermodule interaction in a mosaic protein can be derived from a comparison of the NMR chemical shifts of overlapping fragments under identical solution conditions. We compared the backbone amide resonances (NH and HN) of the 6F11F22F2 fragment with the overlapping 6F11F2 and 1F22F2 module pairs (Figure 2). Removal of the 2F2 module from 6F11F22F2 results in shift perturbations at the C-terminus of the 1F2 module (Figure 2A), as expected from the change in charge. However, many major shift changes are seen in 6F1, in the region of Val10–Lys20. Similarly, removal of 6F1 from 6F11F22F2 results in shift perturbations at the N-terminus of the 1F2 module, but the largest shift changes are seen in the 2F2 module, in the region of Ser111–His117 (Figure 2B). Therefore, the 6F1 and 2F2 modules must interact in 6F11F22F2 despite being separated in sequence by the 1F2 module. Figure 2.Amide chemical shift perturbation upon dissection of 6F11F22F2. (A and B) Combined chemical shift perturbation (Δδ) of the HN and NH backbone resonances, upon (A) removal of the 2F2 module from 6F11F22F2, and (B) removal of the 6F1 module from 6F11F22F2. In each case, Δδ = {|ΔδHN| + (|ΔδNH|/5)}/2, where ΔδHN and ΔδNH are the amide proton and amide nitrogen chemical shift differences, respectively. Download figure Download PowerPoint Solution structure of 6F11F22F2 The solution structure of the 6F11F22F2 fragment could, in theory, be modelled by combining the NMR restraints derived from the independent studies on the overlapping 6F11F2 and 1F22F2 module pairs (Bocquier et al., 1999; Smith et al., 2000). However, because of the extensive amide chemical shift changes in the 6F1 and 2F2 modules, and thus the possibility of significant structural alterations, the solution structure was determined ab initio using only restraints derived from NMR experiments on 6F11F22F2 itself. Of the 100 structures calculated, 20 were selected on the basis of their good agreement with the experimental restraints and their minimal deviations from ideal covalent geometry (Table I). Table 1. Experimental restraints and structural statistics R.m.s. deviations from experimental dataa all 2024 NOE restraints 0.009 ± 0.001 Å 515 intraresidue NOEs {i = j} 0.008 ± 0.002 Å 598 sequential NOEs {|i−j| = 1} 0.007 ± 0.002 Å 231 short-range NOEs {1 < |i−j| < 5} 0.014 ± 0.003 Å 539 long-range NOEs {|i−j| > 4} 0.009 ± 0.002 Å 41 6F1–2F2 intermodule NOEs 0.006 ± 0.003 Å 100 ambiguous NOEs 0.011 ± 0.004 Å 106 hydrogen bond restraints 0.010 ± 0.002 Å 69 dihedral φ angles 0.066 ± 0.036° R.m.s. deviations from ideal covalent geometry bonds 0.0013 ± 0.0001 Å angles 0.245 ± 0.004° impropers 0.123 ± 0.014° Ramachandran analysisb residues in favoured regions 55.0% residues in additional allowed regions 39.9% residues in generously allowed regions 4.4% residues in disallowed regions 0.7% Coordinate precision: secondary structure backbone, all heavy atomsc 6F1(1F22F2) 0.46 ± 0.17 Å, 1.21 ± 0.82 Å (6F1)1F2(2F2) 0.33 ± 0.09 Å, 0.99 ± 0.88 Å (6F11F2)2F2 0.37 ± 0.06 Å, 0.98 ± 0.69 Å 6F1(1F2)2F2 0.53 ± 0.14 Å, 1.15 ± 0.73 Å (6F1)1F2(2F2) 11.7 ± 3.48 Å, 10.2 ± 3.59 Å a None of the 20 accepted structures showed distance restraint violations of >0.3 Å or dihedral restraint violations of >2°. No distance or dihedral restraints were consistently violated by >0.1 Å or 1°, respectively. b Prolines, glycines and terminal residues are excluded. c Coordinate r.m.s. deviations were calculated following best-fit superposition over the secondary structure elements of the underlined module(s). Residues from modules in parentheses were excluded from the calculation. The tertiary structures of the individual modules in 6F11F22F2 are very similar to those in previous studies of the 6F11F2 module pair (Bocquier et al., 1999) and the isolated 2F2 module (Sticht et al., 1998). The back bone heavy atom (NH, Cα, C) root mean square (r.m.s.) deviations over secondary structure elements are 0.98 Å for 6F1, 0.65 Å for 1F2 and 1.21 Å for 2F2. The 6F1 module (Figure 3A) comprises a short N-terminal double-stranded antiparallel β-sheet (AB) that folds over a triple-stranded β-sheet (CDE). The β-sheets are linked by two, conserved disulfide bridges in a 1–3 and 2–4 pattern connecting strands A–D and D–E, respectively. The module core is composed primarily of two highly conserved aromatic residues Tyr12 and Trp18, and a hydrophobic residue, Val36. Figure 3.Structural definition of the individual modules in 6F11F22F2. (A–C) Individual module overlays for the ensemble of 20 lowest energy structures of 6F11F22F2. Each module has been overlaid onto the lowest energy structure by best-fit superposition over the backbone heavy atoms of its secondary structure elements. The secondary structure is coloured green in 6F1, gold in 1F2 and red in 2F2. The disulfide bridges are shown in yellow, and the side chains of non-polar residues that are invariant or highly conserved between modules are shown in cyan for 6F1, pink for 1F2 and purple for 2F2. (D) Average atomic r.m.s. deviation between the 20 accepted structures for the backbone heavy atoms (solid line) and all heavy atoms (dotted line). The vertical dashed lines mark the exon boundaries between the modules. (E) 15N-{1H}-NOE for the 6F11F22F2 fragment. Lower 15N-{1H}-NOE values indicate regions with increased backbone flexibility. Download figure Download PowerPoint The 1F2 module (Figure 3B) comprises two double-stranded antiparallel β-sheets (AB and CD), oriented approximately perpendicular to each other, with a single α-helical turn located between strands C and D. The cleft between the β-sheets is occupied by side chains of invariant hydrophobic and aromatic residues, which make up the module core. On the opposite face of the second β-sheet, two disulfide bonds link the invariant cysteines, with connectivities 1–3 and 2–4. The topology of 2F2 (Figure 3C) is very similar to that of 1F2, but includes the additional A′A″ β-sheet preceding the first cysteine residue. In general, the individual 6F1, 1F2 and 2F2 modules in 6F11F22F2 are well defined (Figure 3A–C) with low backbone heavy atom r.m.s. deviations over their secondary structure elements (Table I). The low 15N-{1H}-NOE for the C–D loop in 6F1, and the B–C loops in 1F2 and 2F2, each of which have backbone r.m.s. deviations >1.0 Å, shows that the poorer definition of these regions arises from backbone flexibility rather than a lack of experimental data (Figure 3D and E). The list of long-range NOEs used in the final round of 6F11F22F2 structure calculations included an extensive array of intermodule restraints between nine residues in 6F1 and eight in 2F2 (Table I). This resulted in a precise definition of the relative locations and orientations of the 6F1 and 2F2 modules in 6F11F22F2 (Figure 4; Table I). The 6F1–2F2 interface is formed by the docking of the external edges of β-strands C of 6F1 and A′ of 2F2, and by the annealing of two extensive hydrophobic surfaces involving Val10, Tyr12, Met16, Leu19 and Leu28 from 6F1, and Leu103, Ala114, Leu115 and Thr145 from 2F2 (Figure 5A). These non-polar residues account for 53% of the total buried surface area between the two modules. There are no titratable groups involved in the remaining 47% that could disrupt the hairpin topology within the pH range 4.5–7.4. Figure 4.Solution structure of 6F11F22F2. (A) A stereoview of the ensemble of the 20 lowest energy structures of 6F11F22F2 is shown. The structures were superimposed on the backbone heavy atoms of the secondary structure elements of the 6F1 and 2F2 modules of the lowest energy structure. The colour scheme for the protein backbone and side chain residues is the same as in Figure 3A–C. (B) Orthogonal stereoview of the 6F11F22F2 ensemble generated by a 90° rotation about the horizontal. For clarity, the disordered 1F2 module has been omitted. Download figure Download PowerPoint Figure 5.Module reorganization upon dissection of 6F11F22F2. Ribbon diagrams of the minimized average structures of (A) 6F11F22F2 and (B) 6F11F2. The colour scheme for the secondary structure elements is as in Figure 3A–C. Side chains for which 6F1–2F2 intermodule NOEs were observed (V10, Y12, S13, M16, L19 and L28 of 6F1, and L103, Q105, S111, N112, A114, L115, T145 and K153 for 2F2) are shown in cyan for 6F1 and purple for 2F2. Removal of the 2F2 module allows the side chain of Y68 (pink) in 1F2 to interact with L19 and L28 in 6F1. Download figure Download PowerPoint No long- or short-range NOEs were observed, either between the 6F1 and 1F2 modules, or between the 1F2 and 2F2 modules. The 6F1–1F2 and 1F2–2F2 linker sequences are flexible, as indicated by their low 15N-{1H}-NOE values (Figure 3E). Hence, the relative location and orientation of 1F2 are somewhat ill defined (Figure 4A; Table I) since the only positional constraints on this central module are the covalent tethering of its N- and C-termini, and steric hindrance from 6F1 and 2F2. The gelatin-binding site(s) of 6F11F22F2 In each F2 module, five of the core aromatic residues (Tyr62, Trp81, Tyr88, Tyr94 and Phe96 in 1F2; Tyr122, Trp141, Tyr148, Phe154 and Phe156 in 2F2) form an extensive, solvent-exposed hydrophobic surface (Figure 3B and C). A pocket in each of these surfaces is thought to provide a binding site for non-polar residues in type I collagen (Pickford et al., 1997). This is supported by the binding of the collagen-like peptide (Pro-Pro-Gly)6 to the 2F2 module from MMP2, which produced backbone HN and NH chemical shift perturbations in the equivalent residues (Briknarová et al., 1999). Furthermore, in the crystal structure of pro-MMP2, a phenylalanine side chain in the inhibitory propeptide occupies the equivalent hydrophobic pocket in the 3F2 module, preventing it from binding its gelatin substrate (Morgunova et al., 1999). In all structures of the 6F11F22F2 ensemble, solvent access to the putative binding sites on the F2 modules is unhindered by the 6F1–2F2 interface. In the average 6F11F22F2 structure (Figure 5A), the binding sites are oriented in opposite directions. This configuration of F2 modules observed here is reminiscent of the pro-MMP2 crystal structure where the three F2 modules are also oriented with their binding sites facing outwards (Morgunova et al., 1999). Recombinant 1F22F23F2 from MMP2 has been shown to be capable of binding multiple collagen triple helices (Steffensen et al., 1995), suggesting that F2 modules form separate binding surfaces that intercalate between molecules in a collagen fibril. The flexibility of the 1F2 module with respect to 6F1 and 2F2 may permit a variety of collagen-binding conformations. It has also been proposed that the observed pliability of the 9F310F3 module pair may allow it to accommodate some variation in the integrin structure to which it binds (Copié et al., 1998). The structural basis for the 4-fold increase in affinity for α(I) chains from 1F22F2 to 6F11F22F2 is not known, but may arise from an extension of the 2F2 binding site onto 6F1. The 6F1 module docks onto 2F2 alongside its gelatin-binding site in an orientation that may allow solvent-exposed residues in the AB sheet of 6F1 to contribute to binding (Figure 4B). Alternatively, the binding enhancement may arise from stabilization of the 2F2 module due to the 6F1–2F2 interface. The backbone HN protons of residues within the AB sheet of 6F1 and the A′A″ sheet of 2F2 undergo much slower solvent exchange in 6F11F22F2 than observed previously for 6F11F2 (Bocquier et al., 1999) and 2F2 (Sticht et al., 1998). Stabilization of the 2F2 module was observed previously in differential scanning calorimetric studies on the 6F11F22F27F1 proteolytic fragment, although this was originally attributed to an interaction between 1F2 and 2F2 (Litvinovich et al., 1991). The contributions of 7F1, 8F1 and 9F1 to collagen binding have yet to be determined. The only structural information available for these domains is the isolated 7F1 (Baron et al., 1990), but there is no information on its interactions with adjacent modules. Given the hairpin structure observed in 6F11F22F2, we believe that the extension of the 6F11F22F2 nucleus with these modules will provide a better examination of the collagen-binding domain function. Module reorganization upon dissection The two techniques capable of providing high-resolution structural information on binding surfaces, namely X-ray diffraction and NMR, are both limited in the size of mosaic proteins that can be studied (Campbell and Downing, 1998). In the case of X-ray diffraction, the limitation arises from the difficulty in crystallizing a protein that is inherently flexible, whilst for NMR the molecular weight is the limiting factor. Hence, for both techniques, large mosaic proteins must frequently be dissected into fragments that are amenable to analysis. However, a comparison of the solution structures of 6F11F2 and 6F11F22F2 illustrates a potential complication associated with this dissection strategy (Figure 5). The F1 and F2 modules in 6F11F2 were found to interact via a small hydrophobic interface of ∼340 Å2, involving the side chains of Leu19 and Leu28 from 6F1, and Tyr68 from 1F2 (Bocquier et al., 1999). This interaction resulted in a significant upfield shift in the NH resonance of Ser69 relative to the isolated 1F2 module (Hashimoto et al., 2000). However, this 6F1–1F2 interface is inconsistent with the solution structure of 6F11F22F2 presented here. The upfield shift of the Ser69 NH resonance is reversed in 6F11F22F2, consistent with a break up of the weak 6F1–1F2 interface (Figure 2A). None of the 18 weak intermodule NOEs previously observed between 6F1 and 1F2 is apparent in the spectra of 6F11F22F2; manual incorporation of these restraints into the structure calculations of 6F11F22F2 also resulted in structures with 20–25% higher potential energy. Therefore, this contact between the modules must have arisen from a module reorganization; removal of the 2F2 module relieves the covalent and steric constraints on the 1F2 module, allowing a hydrophobic collapse of non-polar residues that are distant and/or buried in the intact protein (Figure 5). Such rearrangements are a well-known phenomenon in intracellular proteins, for example, the rearrangement of SH2 and SH3 domains in Src family tyrosine kinases (Sicheri et al., 1997). A globular domain in a fibrillar protein The 6F1–2F2 interface gives the 6F11F22F2 fragment a compact, hairpin topology (with average dimensions 15 × 19 × 32 Å). This is in sharp contrast to the extended, near-linear arrangement of F3 modules in the crystal structures of the cell-binding 7F38F39F310F3 (Leahy et al., 1996) and heparin-binding 12F313F314F3 (Sharma et al., 1999) fragments of fibronectin (Figure 6). The global topology of 6F11F22F2 is in agreement with previous calorimetric studies, which suggested that the gelatin-binding domain has a relatively compact structure (Litvinovich et al., 1991). Figure 6.Global topologies of multimodule fibronectin fragments. Solvent-accessible surfaces have been superimposed over ribbon diagrams for the minimized average structure of 6F11F22F2, and the crystal structures of 7F38F39F310F3 (Leahy et al., 1996) and 12F313F314F3 (Sharma et al., 1999). The fragment structures are mapped onto the mosaic illustration of fibronectin, which has been folded to account for the hairpin structure of 6F11F22F2. Download figure Download PowerPoint Numerous biophysical and biochemical studies have shown that intact fibronectin undergoes a substantial change in structure from a compact conformation at low ionic strength to a more extended conformation in high salt (Engel et al., 1981; Erickson et al., 1981; Erickson and Carrell, 1983; Rocco et al., 1983; Lai et al., 1993). However, even at high ionic strength, numerous kinks are seen in electron micrographs of the molecule (Engel et al., 1981; Erickson et al., 1981; Erickson and Carrell, 1983; Rocco et al., 1983; Lai et al., 1993; Johnson et al., 1999). The resistance of these bends in the protein to high salt concentrations suggests that the interactions involved are predominantly non-polar in nature. Thus, the solution structure of 6F11F22F2 presented here, with its hairpin conformation and extensive hydrophobic interface between the 6F1 and 2F2 modules, is one possible explanation for the kinks observed towards the N-terminal ends of the fibronectin dimer (Figure 6). Many of the models for the compact structure of fibronectin at low ionic strength involve a folding over of the 1F12F13F14F15F1 fragment allowing it to form interdomain interactions with the F3 modules in the protein (Williams et al., 1982; Homandberg and Erickson, 1986; Rocco et al., 1987; Ing