Abstract
Collagen IX, located on the surface of collagen fibrils, is crucial for cartilage integrity and stability. The N-terminal NC4 domain of the alpha1(IX) chain is probably important in this because it interacts with various macromolecules such as proteoglycans and cartilage oligomeric matrix protein. At least 17 distinct collagen polypeptides carry an NC4-like unit near their N terminus, but this report, describing the crystal structure of NC4 at 1.8-A resolution, represents the first atomic level structure for these domains. The structure is similar to previously characterized laminin-neurexin-sex hormone binding globulin (LNS) structures, dominated by an antiparallel beta-sheet sandwich. In addition, a zinc ion was found in a position similar to that of the metal binding site of other LNS domains. A partial backbone NMR assignment of NC4 was obtained and utilized in NMR titration studies to investigate the zinc binding in solution state and to quantitate the affinity of metal binding. The K(d) of 11.5 mM suggests a regulatory rather than a structural role for zinc in solution. NMR titration with a heparin tetrasaccharide revealed the presence of a secondary binding site for heparin on NC4, showing structural and functional conservation with thrombospondin-1, but a markedly reduced affinity for the ligand. Also the overall arrangement of the N and C termini of NC4 resembles most closely the N-terminal domain of thrombospondin-1, distinguishing the two from the majority of the published LNS structures.
Highlights
The fibril-associated collagen IX and other FACIT collagens are proposed to mediate the interaction of a collagen fibril with neighboring fibrils and with other extracellular matrix components
The Zn2ϩ binding might become significant only under conditions of stress or injury where elevated Zn2ϩ levels can occur or at specific sites within tissue presenting elevated Zn2ϩ levels. Another possibility is that the affinity of the NC4 domain for Zn2ϩ is higher in intact collagen IX
We have shown previously that NC4 interacts with heparin with a Kd of 0.6 M and mapped the major binding site to the extreme N terminus of the domain [7]
Summary
Production of Recombinant NC4 Domain—The recombinant NC4 domain of human collagen IX without the 23-residue signal peptide was produced and purified essentially as described previously [7]. Additional washing steps with 20 mM BisTris buffer, pH 6.5, were carried out until the calculated concentration of EDTA was 20 M or less This procedure gave a two-dimensional 15N HSQC spectrum identical to that from the size exclusion-based method that we adapted from the procedure used for removal of Ca2ϩ from calerythrin [39]. Average chemical shift changes were calculated with equation ⌬␦ ϭ [(␦HN) ϩ 0.17 ϫ (␦NH)2]1/2 for five residues that could be traced with the highest confidence in the Zn2ϩ titration spectra These values were plotted as a function of Zn2ϩ concentration, and curve fitting with nonlinear regression [40] was performed to obtain dissociation constants for the individual binding curves. Concentrated stock solution of retinoid prepared in deuterated dimethyl sulfoxide was added in excess to a [15N]NC4 NMR sample, keeping the amount of Me2SO below 1%, and a twodimensional 15N HSQC spectrum was measured [42]
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