The crystal structure of the signature domain of cartilage oligomeric matrix protein: implications for collagen, glycosaminoglycan and integrin binding

Abstract

Cartilage oligomeric matrix protein (COMP), or thrombospondin-5 (TSP-5), is a secreted glycoprotein that is important for growth plate organization and function. Mutations in COMP cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). In this study, we determined the structure of a recombinant protein that contains the last epidermal growth factor repeat, the type 3 repeats and the C-terminal domain (CTD) of COMP to 3.15-A resolution limit by X-ray crystallography. The CTD is a beta-sandwich that is composed of 15 antiparallel beta-strands, and the type 3 repeats are a contiguous series of calcium binding sites that associate with the CTD at multiple points. The crystal packing reveals an exposed potential metal-ion-dependent adhesion site (MIDAS) on one edge of the beta-sandwich that is common to all TSPs and may serve as a binding site for collagens and other ligands. Disease-causing mutations in COMP disrupt calcium binding, disulfide bond formation, intramolecular interactions, or sites for potential ligand binding. The structure presented here and its unique molecular packing in the crystal identify potential interactive sites for glycosaminoglycans, integrins, and collagens, which are key to cartilage structure and function.

Figure 1.

Overall crystal structure of E4T3C5. A) Ribbon drawing of three COMP monomers in a crystallographic asymmetric unit. They are in a pseudo-3-fold symmetry, colored yellow (monomer A), green (monomer B), and blue (monomer C). The glycans attached to N742 and the SO₄ groups found in the structure are drawn in stick format, and Ca²⁺ ions are drawn as red spheres. The EGF4 domains that form the contact between the monomers are labeled. B) Stereo view ribbon drawing of monomer A. All of the secondary structures of the CTD, the 13 type 3 repeats, and the EGF4 domain are labeled. The three Ca²⁺ ions that are associated with the CTD are labeled as CTD_Ca1, CTD_Ca2, and CTD_Ca3. C) Surface representation of E4T3C5. EGF4 is blue, C-type 3 repeats are dark green or light green, N-type 3 repeats are cyan, CTD is orange, glycans are pink, and Ca ions are red.

Figure 2.

Comparison of the signature domains of COMP and TSP-2. A) Structural superposition of COMP (yellow) and TSP-2 (cyan). The entire CDT domains can be superimposed with the exception of the β1_β2 loops and the extra C-terminal helix (labeled as α2) of COMP, which is unique to the molecule. The major variation of the structural alignment is in the type 3 repeats from repeat 4C to 8N. Other type 3 repeats and the C-terminal region of the EGF repeats (EGF4 of COMP and EGF3 of TSP-2) can be readily aligned. B) Interactions between type 3 repeat 8N and the CTD of TSP-2. All residues involved in the interactions are drawn in stick format. A water molecule that mediates a hydrogen bond network involving N851, Q963, and M964 is drawn as a small magenta ball. Other interactions are described in the text. The two Ca²⁺ ions associated with the 8N repeat are presented as small dark yellow balls. C) The unique C-terminal helix of COMP and its interactions with other parts of the CTD. All primary residues involved in the interactions, as well as the residue N742 before the helix and its attached N-lined glycan, are drawn in stick format.

Figure 3.

Ca²⁺-binding sites and potential MIDAS. A) The two Ca²⁺-binding sites in a C-type 3 repeat, as exemplified by repeat 1C. The first Ca²⁺ ion is completely coordinated by protein atoms, while the second Ca²⁺ ion has one coordinating water molecule that is buried. B) The 2N repeat is shown as an example of an N-type 3 repeat, which contains two Ca²⁺-binding sites. The first Ca²⁺ ion has one coordinating water molecule that is buried. The second Ca²⁺ ion has a similar coordinating water molecule, but the water molecule is opened to the solvent. This Ca²⁺ is lost in some N-type 3 repeats due to unfavorable amino acid substitutions in the coordinating residues. The water molecules shown in A and B were not resolved or assigned in the COMP structure due to its low-resolution limit. They were assigned in the higher-resolution structure of TSP-2 . C) The three Ca²⁺-binding sites in the CTD and their involvement in molecular packing. CTD_Ca1 is coordinated by N565, D593 (two bonds), S728 (two bonds including one to its carbonyl group), and the E341 (two bonds) from a neighboring molecule. CTD_Ca2 is coordinated by D593, D594, D595 (two bonds), Q619, and the E341 (weak bond) from a neighboring molecule. There is a water molecule below CTD_Ca3, completing its coordination . The water molecule was not resolved in the E4T3C5 structure and is not shown.

Figure 4.

RGD motif and other prominent residues. A) The R367GD and other prominent residues in the type 3 repeats are drawn in stick format. Starting from R285 in the insert of the 1C repeat to R381 in the 6C repeat, 10 arginine residues are on the same side of the structure, and all of them are solvent exposed. Another 4 arginine residues in the type 3 repeats that follow 6C, as well R281 on the insertion of 1C, either project out from the other side of the structure or are involved in the interaction between repeats such as R445. E341, which may mimic ligand binding, also points to the other side of the coil. The 9 disulfide bonds associated with type 3 repeats are orange. The β14 strand is labeled to indicate the location of the unpaired C726. B) Structural details of R367GD motif in the 5N repeat. D369 chelates the bound calcium ion via a bridging water molecule in type 3 repeat 5N. The water molecule W1 was not resolved or assigned in the COMP structure due to its low-resolution limit. It was assigned in the higher-resolution structure of TSP2 . The hydrogen bond between the side chain of D369 and the main-chain amide group of T362 is not shown for clarity. C) Location of unpaired C726 on the β14 strand. The residue is completed buried under the β3_β4 and β14_ β15 loops. A residue in the β14_β15 loop, S728, binds to CTD_Ca1.

Figure 5.

Surface potentials and mapping of potential heparin-binding sites. Electrostatic potential surface representation. A) Molecule is shown in the same orientation as that in Fig. 4A. Two SO₄²⁻ groups located in the structure are drawn in the stick format. The figure was prepared based on the structure of the monomer B shown in Fig. 1A to display the binding of R352 and R367 to a SO₄²⁻ group. The SO₄²⁻ group bound to R558 and R738 is found in all three monomers in the COMP crystal structure. Some prominent positively charged residues, which may form heparin-binding sites, are mapped to the molecular surface. B) Molecule is shown as a 90° rotation along the vertical axis relative to panel A.