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Host invasion by a number of pathogenic bacteria such as staphylococci and streptococci involves binding to fibronectin, a ubiquitous extracellular matrix protein. On the bacterial side, host extracellular matrix adherence is mediated by MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) which, in some cases, have been identified to be important virulence factors. In this study we used nuclear magnetic resonance spectroscopy to characterize the interaction of B3, a synthetic peptide derived from an adhesin of Streptococcus dysgalactiae, with the N-terminal module pair 1F12F1 of human fibronectin. 1F12F1 chemical shift changes occurring on formation of the 1F12F1/B3 complex indicate that both modules bind to the peptide and that a similar region of each module is involved. A similar surface of the 4F15F1 module pair had previously been identified as the binding site for a fibronectin-binding peptide from Staphylococcus aureus.

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The solution structure of the (6)F1(1)F2(2)F2 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 (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 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'.

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The structure of a pair of modules (6F1(1)F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from 1H-1H NOE and 3J(HalphaHN) data [Bocquier et al. (1999) Structure, 7, 1451-1460] and a weak module-module interface, comprising Leu19 and Leu28, in 6F1, and Tyr68 in 2F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of 15N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and 3J(HalphaHN) data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where 15N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443-449].

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The lone (1)F2(2)F2 modular pair of fibronectin is found in the collagen-binding region. This exclusive localization suggests the (1)F2(2)F2 pair plays an important role in the recognition of collagen. However, no information is currently available about the interaction between the two F2 modules and, thus, the orientation of their putative collagen-binding sites with respect to one another. Comparison of a variety of high-resolution NMR parameters from the F2 modules in isolation and the (1)F2(2)F2 pair was used to establish the extent of interaction between the F2 modules in the pair. Chemical shifts of the F2 modules and the (1)F2(2)F2 pair indicate that the structures of the modules are preserved in the pair and that, with the exception of the covalent linkage, they do not interact. (15)N NMR relaxation data identify significant motion occurring in the linker region of the (1)F2(2)F2 pair, and analyses of the anisotropic diffusion properties of the (1)F2(2)F2 pair are consistent with the modules in the F2 pair tumbling independent of one another.

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Many pathogenic Gram-positive bacteria express cell surface proteins that bind to components of the extracellular matrix. This paper describes studies of the interaction between ligand binding repeats (D3 and D1-D4) of a fibronectin-binding protein from Staphylococcus aureus with a module pair ((4)F1(5)F1) from the N-terminal region of fibronectin. When D3 was added to isotope-labeled (4)F1(5)F1, (1)H, (15)N, and (13)C NMR chemical shift changes indicate that binding is primarily via residues in (4)F1, although a few residues in (5)F1 are also affected. Both hydrophobic and electrostatic interactions appear to be involved. The NMR data indicate that part of the D3 repeat converts from a disordered to a more ordered, extended conformation on binding to (4)F1(5)F1. In further NMR experiments, selective reduction of the intensity of D1-D4 resonances was observed on binding to (4)F1(5)F1, consistent with previous suggestions that in each of D1, D2, and D3 repeats, the main fibronectin binding site is in the C-terminal region of the repeat. In D1-D4, these regions also appear to go from a disordered to a more ordered conformation of fibronectin binding. Although the regions of the two proteins which interact had been previously identified, the findings presented here identify, for the first time, the specific residues in both proteins that are likely to be involved in the interaction.

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Multiple sites within the N-terminal domain (1-5F1) of fibronectin have been implicated previously in fibronectin matrix assembly, heparin binding, and binding to cell surface proteins of pathogenic bacteria. The solution structure of 1F1(2)F1, the N-terminal F1 module pair from human fibronectin, has been determined using NMR spectroscopy. Both modules in the pair conform to the F1 consensus fold. In 4F1(5)F1, the only other F1 module pair structure available, there is a well-defined intermodule interface; in 1F1(2)F1, however, there is no detectable interface between the modules. Comparison of the backbone 15N-{1H} NOE values for both module pairs confirms that the longer intermodule sequence in 1F1(2)F1 is flexible and that the stabilization of the 4F1 C-D loop observed in 4F1(5)F1, as a result of the intermodule interface, is not observed in 1F1(2)F1.

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BACKGROUND: Fibronectin has a role in vital physiological processes such as cell migration during embryogenesis and wound healing. It mediates the attachment of cells to extracellular matrices that contain fibrous collagens. The affinity of fibronectin for native collagen and denatured collagen (gelatin) is located within a 42 kDa domain that contains four type 1 (F1) and two type 2 (F2) modules. A putative ligand-binding site has been located on an isolated F2 module, but the accessibility of this site in the intact domain is unknown. Thus, structural studies of module pairs and larger fragments are required for a better understanding of the interaction between fibronectin and collagen. RESULTS: The solution structure of the 101-residue 6F1 1F2 module pair, which has a weak affinity for gelatin, has been determined by multidimensional NMR spectroscopy. The tertiary structures determined for each module conform to the F1 and F2 consensus folds established previously. The experimental data suggest that the two modules interact via a small hydrophobic interface but may not be tightly associated. Near-random-coil 1H NMR chemical shifts and fast dynamics for backbone atoms in the linker indicate that this region is unlikely to be involved in the overall stabilisation of the module pair. CONCLUSIONS: The modules in the 6F1 1F2 module pair interact with each other via a flexible linker and a hydrophobic patch, which lies on the opposite side of the 1F2 module to the putative collagen-binding site. The intermodule interaction is relatively weak and transient.

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Fibronectin is an extracellular matrix glycoprotein that plays a role in a number of physiological processes involving cell adhesion and migration. The modules of the fibronectin monomer are organized into proteolytically resistant domains that in isolation retain their affinity for various ligands. The tertiary structure of the glycosylated second type 2 module (2F2) from the gelatin-binding domain of fibronectin was determined by two-dimensional nuclear magnetic resonance spectroscopy and simulated annealing. The structure is well defined with an overall fold typical of F2 modules, showing two double-stranded antiparallel beta-sheets and a partially solvent-exposed hydrophobic cluster. An N-terminal beta-sheet, that was not present in previously determined F2 module structures, may be important for defining the relative orientation of adjacent F2 modules in fibronectin. This is the first three-dimensional structure of a glycosylated module of fibronectin, and provides insight into the possible role of the glycosylation in protein stability, protease resistance and modulation of collagen binding. Based on the structures of the isolated modules, models for the 1F22F2 pair were generated by randomly changing the orientation of the linker peptide between the modules. The models suggest that the two putative collagen binding sites in the pair form discrete binding sites, rather than combining to form a single binding site.

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BACKGROUND: Fibronectin is an extracellular matrix glycoprotein involved in cell adhesion and migration events in a range of important physiological processes. Aberrant adhesion of cells to the matrix may contribute to the breakdown of normal tissue function associated with various diseases. The adhesive properties of fibronectin may be mediated by its interaction with collagen, the most abundant extracellular matrix protein. The collagen-binding activity of fibronectin has been localized to a 42 kDa proteolytic fragment on the basis of this fragment's affinity for denatured collagen (gelatin). This gelatin-binding domain contains the only type 2 (F2) modules found in the protein. The F2 modules of the matrix metalloproteinases MMP2 and MMP9 are responsible for the affinity of these proteins for gelatin. Knowledge of the structure of fibronectin will provide insights into its interactions with other proteins, and will contribute to our understanding of the structure and function of the extracellular matrix, in both normal and disease-altered tissues. RESULTS: We have determined the solution structure of the first F2 (1F2) module from human fibronectin by two-dimensional NMR spectroscopy. The tertiary structure of the 1F2 module is similar to that of a shorter F2 module, PDC-109b, from the bovine seminal plasma protein PDC-109. The 1F2 module has two double-stranded antiparallel beta sheets oriented approximately perpendicular to each other, and enclosing a cluster of highly conserved aromatic residues, five of which form a solvent-exposed hydrophobic surface. The N-terminal extension in 1F2 brings the N and C termini of the module into close proximity. CONCLUSIONS: The close proximity of the N and C termini in 1F2 allows for interactions between non-contiguous modules in the gelatin-binding domain. Thus, instead of forming an extended, linear chain of modules, the domain may have a more compact, globular structure. A pocket in the module's solvent-exposed hydrophobic surface may bind nonpolar residues in the putative fibronectin-binding site of the extracellular matrix component type I collagen.

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Sequence analysis of the region of the mumps virus genome encoding the putative small hydrophobic protein gene confirms that it is a highly variable region. Jeryl Lynn, the mumps vaccine strain used in the U.K., is shown to be a mixture of two closely related viruses, both probably of American origin.

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