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| - | <h1>Decorin, Dimeric bovine tissue-extracted decorin, crystal form 1 </h1> | + | <h1>CRE RECOMBINASE/DNA COMPLEX REACTION INTERMEDIATE I </h1> |
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| - | Decorin is a small extracellular matrix proteoglycan present in a variety of connective tissues, typically in association with or “decorating” collagen fibrils (1, 2, 3). It is involved in several fundamental biological functions, including the formation and/or organization of collagen fibrils (4, 5) and the modulation of cell adhesion mediated by fibronectin and thrombospondin (6). Decorin also modulates the activity of growth factors, such as transforming growth factor-β (7), and has other, transforming growth factor-β-independent effects on cell proliferation and behavior (8, 9).
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| - | Mammalian decorin contains a protein core and a single chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to a serine residue near the N terminus (10). Decorin is the best characterized member of the growing family of small leucine-rich repeat proteoglycans and proteins (SLRPs) (3, 11), all having a domain of tandem leucine-rich repeats (LRRs), flanked on either side by clusters of conserved Cys residues. Most SLRPs have been grouped into three different classes on the basis of gene organization, amino acid sequence similarity, number of LRRs, and the spacing of Cys residues in the N-terminal segment. Thus, class I includes decorin, biglycan, and asporin; class II includes fibromodulin, osteoadherin, lumican, proline arginine-rich end LRR protein (PRELP), and keratocan; and class III includes opticin, osteoglycin/mimecan, and epiphycan/PGLb (3, 12). Three further proteins, extracellular matrix 2 (ECM2), chondroadherin, and nyctalopin, have LRR domains with significant homology to the SLRP family (12). The structural and functional similarities between different SLRPs suggest that they share biological functions. For instance, several SLRPs are known to regulate collagen fibrillogenesis, and there is evidence that they are able to compensate for each other in studies on knockout mice (11). Conversely, the wide variation in their expression patterns would indicate that their functions are regulated in a cell- or tissue-specific manner.
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| - | The LRR motif is very widely distributed and has been found in >100 intracellular, cell surface, and extracellular proteins (the LRR superfamily) (13, 14). Several crystal structures of LRR domains have been determined (15, 16, 17, 18, 19). All of them adopt a curved solenoid fold, with a parallel β-sheet forming the inner concave face and a variety of secondary structure topologies forming the outer convex face. To date, crystal structures of complexes of LRR domains with their protein ligands have shown that the concave surface contains the ligand-binding sites (16, 20, 21). It has been assumed that decorin and SLRPs interact with their ligands in the same way (3, 22). However, biophysical analyses have demonstrated that decorin is dimeric in solution, and low-angle x-ray scattering data have suggested that the concave surfaces are involved in dimerization, potentially making them unavailable for ligand-binding (23). A recent article suggested that decorin is in fact a monomer and that dimerization is artifactual (24). However, the crystal structure of the decorin dimer presented here confirms that the concave surfaces mediate dimerization in a highly specific and conserved manner, almost certainly precluding artifact. The highly specific self-recognition by an LRR domain suggests that current models of decorin-ligand interactions need to be reevaluated.
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| - | Materials and Methods.
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| - | Sample Preparation, Characterization, and Crystallization. Two different decorin samples have been used in this study: a recombinant decorin (DcnR), expressed in HEK 293A cells and purified without chaotropic agents, and a tissue-derived decorin (DcnT), extracted from calfskin and refolded from solutions containing urea. Both forms have been shown to be biologically active as they interact with collagen, inhibit collagen fibrillogenesis, and inhibit fibroblast proliferation (9, 25) (Fig. 5, which is published as supporting information on the PNAS web site). The core proteins were prepared by removal of the GAG chain as described in ref. 23. Further details of the biochemical characterization of both samples are given in Supporting Methods, which is published as supporting information on the PNAS web site. Both light-scattering experiments (23) and sedimentation equilibrium (Fig. 6, which is published as supporting information on the PNAS web site) indicate that decorin is dimeric in solution.
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| - | Crystals of both DcnT and DcnR were grown at 20°C by vapor-diffusion methods. Hanging drops (10 μl) were prepared by mixing equal volumes of protein solution (2-3 mg/ml) and 25% (vol/vol) polyethylene glycol 400, both in 0.06 M Tris (pH 7.75)/0.01% β-octyl d-glucoside/0.02% sodium azide. The drops were allowed to equilibrate against 1 ml of the same polyethylene glycol solution. Orthorhombic plate-like crystals appeared within 2 or 3 days and grew to 0.2-0.3 mm in the longest dimension in ≈2 weeks. Crystals suitable for x-ray diffraction were mounted in cryoloops (Hampton Research, Aliso Viejo, CA), flash-cooled, and stored in liquid nitrogen until used for data collection. Derivatives were prepared by soaking crystals overnight in 0.5 mM mercury(II) acetate dissolved in precipitant and then soaking in precipitant solution for several minutes immediately before flash-cooling and x-ray diffraction.
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| - | Structure Determination and Refinement. Both decorin forms gave crystals in two different space groups, C2221 and P212121, which were indistinguishable by visual inspection. Several complete sets of data from native and derivatized DcnR and DcnT crystals were collected at different in-house and synchrotron sources, and structures were determined for DcnR and DcnT in each space group. Tables Tables11 and and22 show the statistics for the two most representative forms, and additional data are summarized in Table 3, which is published as supporting information on the PNAS web site. See Supporting Methods for details of data collection and processing for all crystal forms. The native data for C2221 DcnR were collected with very high redundancy (Table 1), and anomalous data were measured.
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