Structure of the calcium-rich signature domain of human thrombospondin-2

Abstract

Thrombospondins (THBSs) are secreted glycoproteins that have key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6-A-resolution crystal structure of the glycosylated signature domain of human THBS2, which includes three epidermal growth factor-like modules, 13 aspartate-rich repeats and a lectin-like module. These elements interact extensively to form three structural regions termed the stalk, wire and globe. The THBS2 signature domain is stabilized by these interactions and by a network of 30 bound Ca(2+) ions and 18 disulfide bonds. The structure suggests how genetic alterations of THBSs result in disease.

Figure 1

Structure of human TSP-2. (a) Schematic diagram of TSP-2. Each TSP-2 monomer is composed of a N-terminal module (N), an oligomerization domain (O), a procollagen module (C), and type-1 or properdin modules (P123) followed by the signature domain common to all TSPs. In human TSP-2, this domain consists of tandem EGF-like modules (EGF1 (residues 551–589, yellow), EGF2 (590–647, orange), and EGF3 (648–692, wheat)), aspartate-rich repeats (693–956, blue), and a lectin-like module (957–1172, magenta). Glycosylation sites are shown as green ovals. The arrowhead above the EGF-like repeats indicates the site of extra EGF-like module in TSP-3, -4 and -5. (b) Stereo diagram of the glycosylated residue Asn1069. The 2F_o-F_c electron-density map for the region is shown contoured at 1.3 σ (c) Four views of the ribbon diagram (top) and corresponding surface representation (bottom) of the crystal structure of the signature domain of human TSP-2. Modules are colored as in Fig. 1a, with Ca²⁺ ions (red), carbohydrates (green), and disulfide bonds (stick representation) shown.

Figure 2

Ca²⁺ coordination in the stalk and wire elements of TSP-2. (a) Ca²⁺ coordination at the interface between EGF1 and EGF2, colored as in Figure 1. Ca²⁺-coordinating residues are shown and labeled. Reside labelled with (mc) ligand via their main chain carbonyl groups. (b) Overlay of the five N-type Ca²⁺-binding motifs. Ca²⁺-binding residues are labeled. “Xxx” represents any residue. Ca²⁺ ions are shown as red spheres. Residues labeled “(mc)” coordinate Ca²⁺ via main chain carbonyl groups. (c) Overlay of the eight C-type Ca²⁺-binding motifs. The insert sequence from repeat 1C has been removed for the alignment. (d) The glycosylated insertion element in repeat 1C and coordination of an additional Ca²⁺ between repeats 2N and 3C are highlighted by showing 1C, 2N, 3C, and 4C in stereo. (e) Alignment of Ca²⁺-binding repeats in the wire. The Ca²⁺ coordination scheme for each residue is colored-coded as follows: side chain binding to two Ca²⁺ ions (red), side chain binding to one Ca²⁺ ion (purple), main chain carbonyl binding to one Ca²⁺ ion (green or underlined), and water-mediated binding (blue or italicized). Disulfide bonds are shown in red lines. The symbols + or - indicate N-type repeats that bind three or one Ca²⁺, respectively, rather than two. The arrowhead above repeat 1C indicates the site of the 13-residue insertion, which is shown. The arrowhead above repeat 11C indicates the location of the 4-residue insertion that is present in TSP-3 and -4. Residue numbers are listed to the left of the sequence and coordination numbering is shown above.

Figure 3

Disease-associated mutations or polymorphisms of TSPs mapped onto the signature domain of human TSP-2. Residues homologous to positions in TSP-5 that are sites where missense mutations are linked to PSACH or EDM1, or to the polymorphism in TSP-1 that is linked to coronary artery disease are colored yellow and shown in stick form. The wire module is shown in cartoon form with the remainder the signature domain shown as a surface representation to highlight the 62 positions of the missense mutations. Fifty-two map to the wire region and 10 map to surfaces on the lectin-module. Twenty-two additional non-missense mutations are not highlighted but also map to the wire module (Supplementary Fig. 2 online).

Supplementary Figure 1

Stereo ribbon diagram of the crystal structure of the signature domain of human TSP-2. Structure is color-coded to indicate the positions of the tandem EGF-like modules (EGF1 (residues 551–589, yellow), EGF2 (590–647, orange), and EGF3 (648–692, wheat)), aspartate-rich repeats (693–956, blue), and a lectin-like module (957–1171, magenta). Ca²⁺ ions (red), carbohydrates (green), and disulfide bonds (stick representation) are shown.

Supplementary Figure 2

Sequence and secondary structure of the signature domain of human TSP-2. Helices (boxes) and β-strands (arrows) are numbered sequentially above the sequence and are color-coded as in Fig. 1. Cysteines connected by lines form disulfide bonds in the structure. Residues colored green are glycosylated. Underlined or bold-face residues are sites that are mutated or deleted, respectively, in either EDM-1 or PSACH. Two insertion mutations map to the position labeled with a “Y”.

Supplementary Figure 3

Comparison of the crystal structures of the TSP-2 signature domain (A) and the TSP-1 signature domain fragment (B). Both structures are colored and shown in the same orientations as for the TSP-2 signature domain in Figure 1 (main text).