Structural Analysis of the Heparin-Binding Site of the NC1 Domain of Collagen XIV by CD and NMR †,‡ Roland Montserret, § Elisabeth Aubert-Foucher, § Michael J. McLeish, |,⊥ Joanna M. Hill, | Damien Ficheux, § Michel Jaquinod, # Michel van der Rest, # Gilbert Dele ´age, § and Franc ¸ ois Penin* ,§ Institut de Biologie et de Chimie des Prote ´ ines, CNRS UPR 412, 7 passage du Vercors, 69367 Lyon Cedex 07, France, Victorian College of Pharmacy, Monash UniVersity, 381 Royal Pde, ParkVille 3052, Australia, and Institut de Biologie Structurale Jean Pierre Ebel, CEA-CNRS, 43 aVenue des Martyrs, 38027 Grenoble Cedex 1, France ReceiVed January 5, 1999; ReVised Manuscript ReceiVed March 11, 1999 ABSTRACT: Type XIV collagen, a fibril-associated collagen with interrupted triple helices (FACIT), interacts with the surrounding extracellular matrix and/or with cells via its binding to glycosaminoglycans (GAGs). To further characterize such interactions in the NC1 domain of chicken collagen XIV, we identified amino acids essential for heparin binding by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116). The 3D structure of this peptide was then obtained using circular dichroism and NMR. The NC1(84-116) peptide appeared poorly structured in water, but the stabilization of its conformation by the interaction with hydrophobic surfaces or by using cosolvents (TFE, SDS) revealed a high propensity to adopt an R-helical folding. A 3D structure model of NC1(84-116), calculated from NMR data recorded in a TFE/water mixture, showed that the NC1-heparin binding site forms a amphipathic R-helix exhibiting a twisted basic groove. It is structurally similar to the consensus spatial R-helix model of heparin-binding [Margalit et al. (1993) J. Biol. Chem. 268, 19228-19231], except that the GAG binding domain of NC1 may be extended over 18 residues, that is, the NC1(94-111) segment. In addition, the formation of a hydrophobic groove upon helix formation suggests the contribution of additional sequences to ensure the stability of the GAG-binding domain. Overall the NC1(84-116) model exhibits a nativelike conformation which presents suitably oriented residues for the interaction with a specific GAG. Collagens are the most abundant proteins present in the extracellular matrix. They are characterized by a triple helical structure consisting of the repetitive sequence Gly-Xaa-Yaa (where Xaa is often a proline residue and Yaa is often a hydroxyproline residue). They have been classified according to their structural organization in the extracellular matrix (for reviews, see refs 1-3). Among the nonfibrillar collagens, a class called FACITs 1 for fibril-associated collagens with interrupted triple helix (4, 5) has been defined since their discovery in the neighborhood of quarter-staggered fibrils. In the FACIT’s family, collagen IX is clearly associated at the surface of the fibers, and covalent links between collagen IX and the fibrils forming collagen II have been demonstrated (6, 7). For other members of the family, like collagens XII and XIV, the mode of association has yet to be identified. However, it has been proposed that proteoglycans, such as decorin, could provide a link between collagen XIV and the fibrils through their glycosaminoglycan (GAG) chains (8). Collagen XIV is a homotrimeric protein which contains two short triple helical domains (COL1 and COL2) and three non-triple helical domains (NC1, NC2, and NC3). Intensive study has been undertaken to understand the trimeric assembling of collagen XIV (9), its overall function, and the function of its different domains (8, 10, 11). As is the case for numerous proteins of the extracellular matrix, the interaction of collagen XIV with glycosaminoglycans (GAGs) is well-documented. Apart from its binding with the dermatan sulfate chain of decorin (8), it has also been found that collagen XIV interacts with the heparan sulfate chain of perlecan, with heparin (10), and with a proteoglycan form of the cellular receptor CD44 (12). Two sites involved in GAG binding have been described for collagen XIV and † This work was supported by the Centre National de la Recherche Scientifique, by a Program Emergence grant from the Re ´gion Rho ˆne- Alpes (France), and by the Monash Research Fund (Australia). ‡ The coordinates have been deposited with the Brookhaven Protein Data Bank under the accession codes 1B9P (average structure), 1B9Q (set of 19 structures), and R1B9QMR (NMR restraints). * To whom correspondence should be addressed. E-mail: f.penin@ ibcp.fr. Fax: 334 72 76 90 50. § Institut de Biologie et de Chimie des Prote ´ines. | Monash University. ⊥ Present address: College of Pharmacy, University of Michigan, Ann Arbor MI 48109. # Institut de Biologie Structurale. 1 Abbreviations: 2D and 3D, two and three-dimensional; CD, circular dichroism; cmc, critical micellar concentration; DQF-COSY, double quantum filtered correlation spectroscopy; DTNB, 5,5′dithiobis-(2- nitrobenzoic acid); DTT, dithiothreitol; FACIT, fibril-associated col- lagen with interrupted triple helices; FID, free induction decay; GAG, glycosaminoglycan; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; NOE, nuclear Over- hauser enhancement; NOESY, nuclear Overhauser enhancement spec- troscopy; NMR, nuclear magnetic resonance; ppm, parts per million; PMSF, phenylmethylsulphonyl fluoride; rmsd, root-mean-squared deviation; ROESY, rotating frame Overhauser effect spectroscopy; RP- HPLC, reversed-phase high performance liquid chromatography; SCUBA, stimulated cross-peaks under bleached alphas; TFE, 2,2,2- trifluoroethanol; TOCSY, total correlation spectroscopy; TPPI, time proportional phase increment; TSP-d4, 3-(trimethysilyl)propionic- 2,2,3,3-d 4 acid; WATERGATE, water suppression by gradient-tailored excitation. 6479 Biochemistry 1999, 38, 6479-6488 10.1021/bi9900222 CCC: $18.00 © 1999 American Chemical Society Published on Web 04/29/1999