Structural basis of sequence-specific collagen recognition by SPARC

Abstract

Protein interactions with the collagen triple helix play a critical role in collagen fibril formation, cell adhesion, and signaling. However, structural insight into sequence-specific collagen recognition is limited to an integrin-peptide complex. A GVMGFO motif in fibrillar collagens (O denotes 4-hydroxyproline) binds 3 unrelated proteins: von Willebrand factor (VWF), discoidin domain receptor 2 (DDR2), and the extracellular matrix protein SPARC/osteonectin/BM-40. We report the crystal structure at 3.2 A resolution of human SPARC bound to a triple-helical 33-residue peptide harboring the promiscuous GVMGFO motif. SPARC recognizes the GVMGFO motifs of the middle and trailing collagen chains, burying a total of 720 A(2) of solvent-accessible collagen surface. SPARC binding does not distort the canonical triple helix of the collagen peptide. In contrast, a critical loop in SPARC is substantially remodelled upon collagen binding, creating a deep pocket that accommodates the phenylalanine residue of the trailing collagen chain ("Phe pocket"). This highly restrictive specificity pocket is shared with the collagen-binding integrin I-domains but differs strikingly from the shallow collagen-binding grooves of the platelet receptor glycoprotein VI and microbial adhesins. We speculate that binding of the GVMGFO motif to VWF and DDR2 also results in structural changes and the formation of a Phe pocket.

Fig. 1.

Crystal structure of SPARC FS-EC ΔαC bound to a 33-residue collagen peptide (stereoview). The FS and EC domains of SPARC are in green and cyan, respectively. Disulphide bridges are in pale pink, the glycan attached to N99 is in gray, and a calcium ion is shown as a purple sphere. The collagen peptide is shown as a Cα ribbon (leading chain, yellow; middle chain, orange; trailing chain, red). The chain termini, selected helices and the location of the αC deletion are labeled.

Fig. 2.

Details of the SPARC-collagen interaction. (A) Sequence of the collagen peptide, indicating the 1-residue stagger between the chains. The sequence numbering of the leading strand is indicated at the top, and the SPARC-binding motifs of the middle and trailing chains are colored. (B) Surface representation of the collagen binding site of SPARC. The FS and EC domains of SPARC are in green and cyan, respectively. The collagen peptide is shown as a Cα ribbon (leading chain, yellow; middle chain, orange; trailing chain, red). Selected residues of the middle and trailing chains are shown in atomic detail and are labeled. (C) Interactions of SPARC with the trailing chain. SPARC is shown as a ribbon diagram with semitransparent surface rendering. Selected SPARC residues are shown in atomic detail and are labeled. The collagen leading and middle chains are shown as a Cα ribbon and the trailing chain is shown in atomic detail. Hydrogen bonds are indicated by dashed lines. (D) Interactions of SPARC with the collagen middle chain.

Fig. 3.

Structural changes within SPARC upon collagen binding. (A) Surface representation of the collagen binding region in apo SPARC. The FS and EC domains are in green and cyan, respectively. Residues implicated in collagen binding by mutagenesis (20) are in purple and labeled. W153 is shown in blue and the location of P244 (see Structural Changes within SPARC) is indicated. (B) Surface representation of the collagen-binding site in the SPARC-collagen complex. The color scheme is the same as in A. The side chain of F23 of the collagen trailing chain is shown in atomic detail (red) to facilitate comparison with Figs. 1 and 2. (C) Stereoview of a superposition of the region surrounding the Phe pocket in apo SPARC (light brown) and the SPARC-collagen complex (cyan). Selected residues are shown in atomic detail and are labeled. F23 of the collagen trailing chain is shown as a semitransparent space-filling model (red).

Fig. 4.

Putative SPARC-binding sites in collagen IV. Shown are partial sequences of human collagen III (SwissProt entry P02461) and collagen IV (α1 chain, P02462; α2 chain, P08572). The SPARC-binding site in collagen III is highlighted; residues that are predicted to be strictly required for SPARC binding (see Prediction of SPARC-Binding Sites in Collagens I–IV) are in red, residues that should be apolar are in orange. The same coloring scheme is used to indicate the 4 putative SPARC-binding sites in collagen IV.