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

2004; Springer Nature; Volume: 23; Issue: 6 Linguagem: Inglês

10.1038/sj.emboj.7600166

1460-2075

Marc Kvansakul, Josephine C. Adams, Erhard Hohenester,

Protease and Inhibitor Mechanisms

Article11 March 2004free access Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats Marc Kvansakul Marc Kvansakul Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Josephine C Adams Josephine C Adams Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA Search for more papers by this author Erhard Hohenester Corresponding Author Erhard Hohenester Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Marc Kvansakul Marc Kvansakul Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Josephine C Adams Josephine C Adams Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA Search for more papers by this author Erhard Hohenester Corresponding Author Erhard Hohenester Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Author Information Marc Kvansakul1, Josephine C Adams2 and Erhard Hohenester 1 1Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK 2Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA *Corresponding author. Biophysics Group, Blackett Laboratory, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. Tel.: +44 20 7594 7701; Fax: +44 20 7589 0191; E-mail: [email protected] The EMBO Journal (2004)23:1223-1233https://doi.org/10.1038/sj.emboj.7600166 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 β-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. Introduction The thrombospondins (TSPs) are large, secreted, multimeric glycoproteins that modulate extracellular matrix (ECM) structure and cell behaviour. There are five TSPs in vertebrates, each the product of a different gene. TSP-1 and TSP-2 are homotrimers, whereas TSP-3, TSP-4 and TSP-5 (usually referred to as cartilage oligomeric matrix protein, COMP) are homopentamers (for reviews, see Lawler, 2000; Adams, 2001; Armstrong and Bornstein, 2002). There exists a single pentameric TSP in Drosophila (Adams et al, 2003). TSP-1 has been the most intensively studied family member and the prototype for functional analysis of other TSPs. Each subunit contains binding sites for cellular receptors, cytokines and ECM molecules. Its multifunctionality allows TSP-1 to assemble multiprotein complexes at the cell surface and thereby modulate cell behaviour. TSP-1 is involved in platelet aggregation, cell adhesion and migration, the regulation of proliferation, angiogenesis, wound healing and tumour growth (for reviews, see Lawler, 2000; Adams, 2001; Armstrong and Bornstein, 2002). Each protomer of TSP-1 and TSP-2 consists of an N-terminal domain, a coiled-coil oligomerisation domain, a von Willebrand factor-type C or procollagen homology domain, three TSP type 1 or properdin domains of known structure (Tan et al, 2002), three TSP type 2 (T2) or EGF-like domains, seven calcium-binding type 3 (T3) repeats and a C-terminal domain (CTD) of ≈230 residues that shows no sequence similarity to any other protein domain (Figure 1A). The pentameric TSPs have a different N-terminal domain organisation and lack the type 1 repeats, but have in common with TSP-1 and TSP-2 the T2–T3–CTD arrangement. The T3 repeats and CTD are the most highly conserved region between all TSPs (Adams and Lawler, 1993a; Adams et al, 2003). The structure of this region is thus of high interest for understanding the molecular interactions and functions of the TSP family. Figure 1.Cell binding to T35–7–CTD. (A) Domain organisation of TSP-1 and its T35–7–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 T35–7–CTD C974S fragment. Download figure Download PowerPoint The most striking property of the C-terminal region of TSPs is the profound dependence of protein conformation and function on calcium binding. The T3 repeats are a tandem of aspartic acid-rich motifs, which resemble EF hands in the spacing of acidic side chains (Lawler and Hynes, 1986). Unlike EF hands, however, the calcium-binding loops of the T3 repeats are not flanked by secondary structure elements. TSPs have been shown to bind 10–12 calcium ions per subunit with high cooperativity and moderate affinity (average KD≈0.1 mM) (Lawler and Simons, 1983; Misenheimer and Mosher, 1995; Chen et al, 2000). Chelation of calcium by EDTA leads to a large shape change, visualised by rotary shadowing of intact molecules as an elongation of the C-terminal ‘arms’ and shrinking of the C-terminal globule (Lawler et al, 1985). This has been attributed to collapse of the T3 repeats and indeed the entire T2–T3–CTD assembly (Misenheimer et al, 2003). Other functions of the C-terminal region include cell adhesion and migration (for review, see Adams, 2001). Crucially, cell attachment activity depends on the calcium loading of TSP-1 (Lawler et al, 1988). The cytoskeletal rearrangements associated with cell spreading are only elicited when C-terminal fragments of TSP-1 or TSP-2 are presented in trimeric form (Anilkumar et al, 2002). Candidate receptors for this region include integrins that bind an RGD motif in T37 of TSP-1 (Lawler et al, 1988), CD47 (Gao et al, 1996) and syndecan-1 (Adams et al, 2001). The C-terminal regions of COMP and TSP-4 bind ECM molecules including collagens I, II and IX, and are thus implicated in ECM assembly (Rosenberg et al, 1998; Narouz-Ott et al, 2000; Adams, 2001; Holden et al, 2001). The C-terminal regions of TSPs are also involved in human disease. Two related autosomal dominant skeletal disorders, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), are linked to mutations in COMP (Briggs et al, 1995; Hecht et al, 1995). Notably, the great majority of mutations affect calcium-binding residues within the T3 repeats (for review, see Briggs and Chapman, 2002). Mutant COMP aggregates in the endoplasmic reticulum of chondrocytes, leading to impaired COMP secretion, reduced cell viability and altered pericellular matrix (Delot et al, 1998; Holden et al, 2001; Dinser et al, 2002; Hashimoto et al, 2003). The T2–T3–CTD region appears to be extraordinarily sensitive to mutation, with single missense or deletion mutations typically resulting in the loss of about half the exchangeable calcium ions (Chen et al, 2000; Maddox et al, 2000; Thur et al, 2001; Kleerekoper et al, 2002). Recently, polymorphism of TSPs (N700S at the N-terminus of the T3 repeats of TSP-1 and A387P in the last T2 repeat of TSP-4) has been associated with familial premature coronary heart disease (Topol et al, 2001). Like the COMP mutations, the TSP-1 polymorphism reduces calcium binding (Hannah et al, 2003), but how the structural change predisposes to coronary heart disease remains to be established. We have determined the crystal structure of a recombinant C-terminal fragment of TSP-1, spanning the last three T3 repeats and the CTD. The T3 repeats are devoid of secondary structure and are held together by a core of 12 calcium ions. The three T3 repeats and the lectin-like β-sandwich of the CTD are assembled into a compact unit. These results demonstrate how calcium binding so dramatically affects the properties of TSPs and indicate how mutations of calcium-binding residues in COMP can cause misfolding and disease. Results Construct design and crystallisation To search for a crystallisable C-terminal fragment of TSP-1, we systematically extended from the N-terminus of the CTD and expressed these constructs in human embryonic kidney cells. The smallest construct that was both highly expressed and soluble spans the last three T3 repeats and the CTD (T35–7–CTD; amino-acid residues 816–1152). To prevent disulphide isomerisation, which is known to occur in TSP-1 (Sun et al, 1992) and would frustrate crystallisation attempts, we mutated the free cysteine at position 974 to serine, the amino acid at this position in most other TSPs. Far-UV circular dichroism, fluorescence spectroscopy and limited trypsin digestion indicated that T35–7–CTD C974S bound calcium and was saturated at ≈2 mM calcium. Calcium binding was associated with a considerable compaction of the protein, as evidenced by a 0.9 ml shift in elution volume from a 24 ml gel filtration column (not shown). To show that the T35–7–CTD C974S mutant retained the biological functions of its parent molecule, we examined its cell-binding properties. C2C12 skeletal myoblasts and rat vascular smooth muscle cells attached to immobilised calcium-replete T35–7–CTD C974S as strongly as to a previously studied recombinant T23–T3–CTD fragment (Figure 1B). Being monomeric, T35–7–CTD C974S only supported cell attachment but not spreading (Anilkumar et al, 2002). Because we failed to obtain crystals of T35–7–CTD C974S, we removed the N-linked glycan by mutagenesis of the putative acceptor site at Asn1049. The N1049K mutation abolished glycosylation, as indicated by a shift to lower molecular mass on SDS–PAGE gels, but did not affect protein solubility or cell binding (not shown). The T35–7–CTD C974S/N1049K double mutant could be crystallised in the presence of 5 mM calcium and its structure was determined to a resolution of 1.9 Å (Table I). Table 1. Crystallographic statistics Crystal Native 2a Native 1 K2PtCl4 Sm(CH3COO)3 Data collection and phasing Resolution range (Å) 20–1.9 20–2.4 20–2.8 20–2.8 Unique reflections 32 099 16 840 10 725 10 637 Multiplicity 5.4 (4.0) 14.9 10.6 7.2 Completeness (%) 95.3 (92.9) 99.8 99.7 99.7 Rmergeb 0.086 (0.37) 0.104 0.079 0.081 Rderivc 0.196 0.141 RCullis (centric/acentric)d 0.82/0.88 0.76/0.84 Phasing power (centric/acentric)e 0.72/0.81 0.82/0.98 Refinement Resolution range (Å) 20–1.9 Å Reflections (working set/test set) 28 902/3197 Protein atoms 2667 Solvent atoms 262 H2O, 16 Ca2+ Rcryst/Rfreef 0.199/0.227 r.m.s.d. bonds (Å) 0.005 r.m.s.d. angles (deg) 1.4 r.m.s.d. B-factors (Å2) 1.6 Ramachandran plot (%)g 80.4/19.3/0/0.4 a Numbers in parentheses refer to the highest resolution shell (2.00–1.90 Å). b Rmerge=∑h∑i∣Ii(h)−〈I(h)〉∣/∑h∑iIi(h), where Ii(h) is the ith measurement of reflection h and 〈I(h)〉 is the weighted mean of all measurements of h. c Rderiv=∑h∣∣FPH∣−∣FP∣∣/∑h∣FP∣, where FP and FPH are the native and derivative structure factors, respectively. The native 1 data were used for phasing. d RCullis=∑h∣∣∣FPH∣−∣FP∣∣−∣FH∣∣/∑h∣∣FPH∣−∣FP∣∣, where FH is the calculated heavy atom structure factor. e The phasing power is defined as (r.m.s. FH/r.m.s. lack-of-closure). f R=∑h∣Fobs−Fcalc∣/∑hFobs, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. Rcryst and Rfree were calculated using the working and test set, respectively. g Residues in most favoured, additionally allowed, generously allowed and disallowed regions (Laskowski et al, 1993). Overall structure We observed clear electron density for almost the entire T35–7–CTD fragment. Only the N-terminal tag and residues 962–966 in the β2–β3 hairpin of the CTD are not defined in our structure. The T35–7–CTD fragment adopts a compact structure of approximate dimensions 70 × 50 × 35 Å (Figure 2). At its heart is the globular CTD, which folds into a curved 15-stranded β-sandwich. T35 makes multiple contacts with loop regions at the top of the concave β-sandwich face (view of Figure 2A), while T36 and T37 form a tight hairpin that projects from the body of the structure. The interface between T35 and the CTD buries 940 Å2 of solvent-accessible surface area, and a further 1240 Å2 is buried in the hairpin interface. The glycosylation site at residue 1049, which was mutated to lysine to obtain crystals, is located below and behind T35. An N-linked glycan could be accommodated at this position without causing steric clashes. There are four disulphide bridges in the T35–7–CTD structure. Three of them link consecutive T3 repeats (see below for a definition of the repeats) and a fourth bridge links T37 to the extreme C-terminus of the CTD. The same sequential disulphide linkage was determined chemically for the T23–T3–CTD region of TSP-2 (Misenheimer et al, 2001). Residue 974, which is a free cysteine in native TSP-1 and serine in our construct (and in most other TSPs), is located between strands β3 and β4, and is buried by the calcium-binding β1–β2 loop. A total of 16 metal ions were identified in the T35–7–CTD structure and assigned as calcium. There are four calcium ions in T35, five ions in T36 and three ions in T37. Four additional ions are bound to the edge of the CTD opposite the T36–7 hairpin. The calcium concentration in our crystals (5 mM) suggests that all observed metal sites are physiologically relevant. Figure 2.Overall structure of T35–7–CTD. (A) Cartoon drawing of the T35–7–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. Download figure Download PowerPoint Structure of the C-terminal domain The globular CTD is a β-sandwich of two curved antiparallel β-sheets. The fold is an elaboration of the jelly roll topology, with strands β3–β7, β11 and β14–β15 forming the eight-stranded jelly roll motif. The smaller seven-stranded sheet presents a convex face to the solvent, whereas the more radically curved eight-stranded sheet creates a deep concave cleft, which is completely filled by a short α-helix and residues contributed by four loops. The long β7–β8 loop is particularly prominent, closing off the pocket and all but burying the α-helix. The CTD contains two large hydrophobic cores, one between the two β-sheets and one within the concave cleft. Despite its unique sequence, the CTD was found to belong to a larger family of lectin-like folds. The DALI server (Holm and Sander, 1997) retrieved two calcium-binding lectin domains, which match the CTD of TSP-1 in its entirety. These structural homologues are p58/ERGIC-53 (Velloso et al, 2002), an animal lectin functioning in the secretory pathway (r.m.s. deviation 2.7 Å for 164 Cα atoms, 7% sequence identity) and a plant lectin from Griffonia simplicifolia (Delbaere et al, 1993) (r.m.s. deviation 2.6 Å for 162 Cα atoms, 12% sequence identity), both members of the L-type lectin family (Dodd and Drickamer, 2001). The CTD of TSP-1 and the lectins are most similar in the β-sheets, but differ markedly in many loop regions, in particular those shaping the concave cleft. There are four calcium ions bound to the lower rim of the CTD sandwich. The first ion is coordinated by three main-chain carbonyl groups in the β1–β2 loop and the side chains of Asp956 and Asp975. The second and third ions are bound in close proximity to each other by the Asp1001–Asp1002–Asp1003 sequence in the β5–β6 loop (Figure 3). Each ion receives five ligands from the CTD. In the crystal lattice, the side chain of Asp825 from a neighbouring molecule completes the metal coordination by bridging the two calcium ions. Finally, a fourth ion is bound loosely by two main-chain carbonyl groups in β5–β6 loop and the side chains of Gln1024 and Asp1086. The location of the double-calcium site (Ca2 and Ca3) is intriguing as it broadly coincides with the calcium-dependent carbohydrate-binding site of the distantly related lectins. The long β7–β8 loop positions the solvent-exposed side chain of Trp1030 just above the double-calcium site, highlighting this region as a prime candidate for an interaction site. Crucially, residues involved in calcium binding to the CTD are strictly conserved across the human TSP family, indicating that they are required for the structure or function of all TSPs. There are no other striking surface features of the CTD offering clues about the location of receptor-binding sites. 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. Download figure Download PowerPoint Calcium coordination by the type 3 repeats The most remarkable feature of the T35–7–CTD structure is the intricate folding of the T3 repeats. The 120 residues preceding the CTD in our construct contain ≈30% aspartic acid and almost no aliphatic or aromatic residues, yet they are fully ordered in the crystal. The polypeptide chain, which apart from β-turns and two turns of 310 helix is devoid of secondary structure, wraps around a series of calcium ions, creating a highly unusual protein structure organised around a metal core. The lack of secondary structure in the T3 repeat region is in reasonable agreement with circular dichroism spectra of TSP-1 and COMP T3 constructs (Thur et al, 2001; Misenheimer et al, 2003). To describe the calcium coordination by the T3 repeats, we must first define the repeating unit. There are 12 D1xD3xD5GxxD9xxD12 motifs in every TSP sequence, which commonly have been grouped into seven repeats (Lawler and Hynes, 1986). In this grouping, T32 and T34 are short and contain only one motif, whereas the other five repeats contain two motifs, which we will refer to as N-type and C-type motifs. In our structure-based definition of the T3 repeats, disulphide bridges link adjacent repeats, and the single motifs in T32 and T34 are identified as C-type motifs (Figure 4). 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). Download figure Download PowerPoint The structurally most conserved part of the T3 repeats is the C-type motif, and T35, T36 and T37 superimpose almost perfectly in this region. Each C-type motif encapsulates two calcium ions, which are likely to be bound with high affinity. The first (upper) calcium ion is coordinated by the side chains at positions 1, 3, 5 and 12 of the D1xD3xD5GxxD9xxD12 motif (Figure 4). The six-fold coordination is completed by the main-chain carbonyl group at position 7 and a water molecule bridging to the aspartate at position 9. The residue at position 6 adopts a main-chain conformation that is favourable only for glycine. The second (lower) calcium ion is seven-fold coordinated by the side chains at positions 3 and 5 (monodentate and bridging to the first calcium), the aspartate at position 12 (bidentate) and three carbonyl groups. The first and second carbonyl group always is donated by main-chain peptide bonds. In T37 the last ligand is also a peptide carbonyl group, but in T35 and T36 this position is taken over by the side-chain oxygen atom of a conserved asparagine (position 19 of the C-type motif). The polypeptide chain wraps around the double-calcium core, with a turn just before the crucial aspartate at position 12 providing the connection between the upper and lower loops. This arrangement is further stabilised by main-chain interactions and conserved residues not engaged in metal binding. The upper loop is stabilised by the conserved aspartate at position 9, and the lower loop is stabilised by a glutamine or glutamic acid at position 22 of the C-type motif. The N-type motif resembles the C-type motif, but seems to allow for more variation. A first calcium ion is coordinated as in the C-type motif. In T37, this is the only ion bound to the N-type motif, because a lysine replaces the crucial aspartic acid at position 12. In T35 and T36, a second calcium ion is bound by the aspartates at positions 3, 5 and 12, with the latter interaction being bidentate as in the C-type motif. However, unlike in the C-type motif, the polypeptide chain does not fully encircle the second calcium ion, leaving the metal coordination sphere incomplete and probably resulting in a lower affinity for calcium. In T35 and T36, residues at positions 14 and 15, respectively, provide bonds to the second calcium ion. As in the C-type motif, position 6 of the N-type motif usually is a glycine. Additionally, there is an invariant glycine at position 8, which allows the close approach of the preceding T3 repeat to form the inter-repeat disulphide bridge. T36 is distinct from T35 and T37 in that a third calcium ion is bound to the outside of the N-type motif by the side chains of Asp880 (bidentate) and Asp887 (monodentate) and two main-chain carbonyl groups. The D1xD3xD5GxxD9xxD12 motifs of TSP T3 repeats resemble the classic EF-hand calcium-binding motif D1xD3xD5GxxxxxE12 (Lawler and Hynes, 1986; Nakayama and Kretsinger, 1994). Structural superposition reveals that the calcium coordination is indeed similar up to position 7. However, there are profound differences in the remainder of the metal coordination, the most obvious distinction being the different number of calcium ions bound (two by the TSP motifs, one by EF hands). EF hands also usually occur as interlocked pairs embedded in an α-helical fold, which sharply contrasts with the dearth of secondary structure in the T3 repeats. The superficial similarity between the T3 and EF-hand motifs is probably due to the constraints of creating a calcium-binding pocket from a contiguous polypeptide segment, which seem to favour regularly spaced aspartic acids. Tertiary structure of the type 3 repeat region The relative orientation of N- and C-type motifs within repeats is highly variable and seems to depend upon the tertiary structure context. We have retained the original definition of a total of seven repeats in TSPs (Lawler and Hynes, 1986), but note that the T3 repeat portion of T35–7–CTD could equally well be described as a chain of alternating high-affinity (C-type) and low-affinity (N-type) calcium-binding motifs. The disulphide bridges between consecutive repeats are clearly important for the tertiary structure of the T3 repeat portion of T35–7–CTD, but they appear to be insufficient to dictate the unique arrangement of repeats. T35 and T36 are joined in a relatively extended conformation, whereas T36 and T37 form a hairpin structure (Figure 5A). Remarkably, this hairpin is stabilised by a well-formed hydrophobic core consisting of residues (Leu894 and Val895 from T36 and Phe916 from T37) that are strictly conserved in all human TSPs and likely to be important for the structure. There is no interaction between T35 and T36, apart from the Cys856–Cys876 disulphide bridge. Thus, the position of T35 in the T35–7–CTD structure appears to depend largely on its contacts with the CTD. The majority of these contacts involve polar residues that are not conserved in all TSPs, and the disposition of T35 relative to the CTD could be different in other TSPs. Figure 5.T36–7 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 T35–7–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 T35–7–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. Download figure Download PowerPoint To determine the contribution of the T36–7 hairpin to TSP-1 folding and stability, we produced an N-terminally truncated construct lacking all of T35 and the N-type motif of T36 (T37–CTD C974S). The shorter construct was produced less efficiently by the 293 cells than T35–7–CTD C974S, indicating impaired folding and/or secretion. Gel filtration chromatography of T37–CTD C974S revealed extensive protein aggregation, in sharp contrast to T35–7–CTD C974S, which is 100% monomeric (Figure 5B). We conclude that disruption of the native T35–7 tertiary structure exposes hydrophobic residues, leading to poor protein solubility. In agreement with this interpretation, an NMR study of a COMP T35–7 construct has shown this region to be a stable subdomain with calcium-dependent tertiary structure (Kleerekoper et al, 2002). Location of the RGD motif in type 3 repeat 7 The single RGD motif in T37 of human TSP-1 has been identified as a binding site for αvβ3 and αIIbβ3 integrins (Lawler et al, 1988; Lawler and Hynes, 1989); however, not all cell types undergo RGD-dependent attachment to TSP-1 (e.g., Long and Dixit, 1990; Adams and Lawler, 1993b). In the structure, the RGD sequence is located near the tip of the T36–7 hairpin and embedded in the N-type calcium-binding site of T37 (Figure 5A). The main-chain carbonyl group of Arg908 coordinates the calcium ion, while its guanidinium group interacts with Asp923 in the C-type motif, thus stabilising the extended conformation of T37. Gly909 is largely buried and Asp910 makes two interactions with Ser903. Both Arg908 and Gly909 adopt an extended main-chain conformation. Because integrin binding requires the RGD motif to be presented in a β-hairpin context (Xiong et al, 2002), native calcium-saturated TSP-1 is unlikely to be cell adhesive directly