Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats

Abstract

Thrombospondins (TSPs) are extracellular regulators of cell-matrix interactions and cell phenotype. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The T3 repeats lack secondary structure and are organised around a core of calcium ions; two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The CTD forms a lectin-like beta-sandwich and contains four strictly conserved calcium-binding sites. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3-CTD assembly.

Figure 1

Cell binding to T3_5–7–CTD. (A) Domain organisation of TSP-1 and its T3_5–7–CTD fragment. NTD, N-terminal domain; cc, coiled-coil domain; PC, procollagen homology domain; T1, type 1 repeats; T2, type 2 repeats; T3, type 3 repeats; CTD, C-terminal domain. (B) Binding of C2C12 myoblasts (left) and rat vascular smooth muscle cells (right) to immobilised TSP-1, a recombinant T2₃–T3–CTD fragment (Anilkumar et al, 2002) and the T3_5–7–CTD C974S fragment.

Figure 2

Overall structure of T3_5–7–CTD. (A) Cartoon drawing of the T3_5–7–CTD structure. T3₅, T3₆ and T3₇ are in red, yellow and green, respectively. The CTD is in cyan and its β-strands are sequentially labelled 1–15. Disulphide bridges and residue 974 are shown in ball-and-stick representation and are in yellow. Calcium ions are represented by pink spheres. (B) Sequence alignment of human TSPs. The sequence numbering and secondary structure elements of TSP-1 are included above the alignment. Strictly conserved residues are in bold. Cysteines and calcium-binding residues are highlighted in yellow and pink, respectively. An RGD sequence in T3₇ (Lawler and Hynes, 1986) and two putative cell-adhesive sequences in the CTD of TSP-1 (Kosfeld and Frazier, 1993) are in green. TSP-5/COMP residues affected by missense mutations (see text) are indicated by red asterisks.

Figure 3

Double-calcium site in the CTD. Selected loop regions are shown as Cα traces. Calcium-binding residues and Trp1030 are shown in ball-and-stick representation and are labelled. Calcium ions are represented as pink spheres. Calcium-ligand bonds are shown as thin black lines. Water molecules have been omitted for clarity.

Figure 4

Calcium coordination by the type 3 repeats. (A) Sequence alignment of T3 repeats in TSP-1. The calcium-binding aspartates of the D₁xD₃xD₅GxxD₉xxD₁₂ motifs are highlighted in pink. A putative calcium-binding motif in the linker between T2₃ and T3₁ is also marked. Cysteines are highlighted in yellow and the disulphide linkages are indicated by yellow lines. (B) Structural superposition of T3₅ (red), T3₆ (yellow) and T3₇ (green). The repeats were superimposed by fitting their C-type motifs. Calcium ions are represented as small spheres. (C) Stereoviews showing the calcium coordination in the C-type motif (upper panel) and N-type motif (lower panel) of T3₆. Calcium-binding residues are shown in ball-and-stick representation and are labelled. Calcium ions are represented as pink spheres. Calcium-ligand bonds are shown as thin black lines. Water molecules have been omitted for clarity. (D) Schematic drawing of the calcium coordination in the C-type motif (see text). Metal ligands are numbered according to their position in the consensus sequence for comparison with (A). Some ligands are also labelled with the corresponding sequence numbers in T3₆ to facilitate comparison with (C).

Figure 5

T3_6–7 hairpin and RGD site. (A) Cartoon drawing. T3₆ and T3₇ are in yellow and green, respectively. Selected residues are shown in ball-and-stick representation and are labelled. The RGD motif in T3₇ is in grey. Calcium ions are represented as pink spheres. (B) Gel filtration chromatograms of T3_5–7–CTD C974S (blue) and T3₇–CTD C974S (red), showing extensive aggregation of the shorter construct. Both proteins were injected at a concentration of 1 mg/ml. The running buffer was 25 mM Na-HEPES pH 7.5, 140 mM NaCl and 1 mM CaAc₂. The elution volumes of globular molecular mass standards are indicated by labelled arrows. (C) Cell attachment of C2C12 or HISM cells at different calcium concentrations. Solid lines indicate the percentage of cell attachment relative to attachment at 2 mM calcium (UT, untreated). Dotted lines indicate the percentage of cell attachment remaining in the presence of 1 mM GRGDSP peptide for each calcium ion concentration and cell type (non-RGD). GRGESP peptide at 1 mM was non-inhibitory. Each point is the mean from three experiments, bars indicate s.e.m. (D) Effect of calcium ion conditions and reduction on C2C12 cell morphology. (i–iv) Confocal XY images of C2C12 cells plated for 1 h on T3_5–7–CTD C974S/N1049K coated under the indicated conditions, after fixation and processing for phalloidin staining of F-actin. Inset panels in (ii) show the unchanged morphology of cells in the presence of 1 mM GRGESP peptide and the loss of spreading by cells attached in the presence of 1 mM GRGDSP peptide. Bar=10 μm.

Figure 6

Mapping of COMP mutations. Cartoon drawing of the T3_5–7–CTD structure with sites of COMP missense mutations (see text) shown as large Cα atom spheres. The mutations are labelled using COMP sequence numbering (compare Figure 2B). Red spheres and labels indicate mutations of calcium-binding residues; yellow spheres and labels indicate all other mutations. Calcium ions have been omitted for clarity.