Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex

Abstract

Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High affinity VWF-binding sequences have been identified in the homotrimeric collagen types II and III, however, it is unclear how VWF recognizes the heterotrimeric collagen type I, the superstructure of which is unknown. Here we present the crystal structure of VWF domain A3 bound to a collagen type III-derived homotrimeric peptide. Our structure reveals that VWF-A3 interacts with all three collagen chains and binds through conformational selection to a sequence that is one triplet longer than was previously appreciated from platelet and VWF binding studies. The VWF-binding site overlaps those of SPARC (also known as osteonectin) and discodin domain receptor 2, but is more extended and shifted toward the collagen amino terminus. The observed collagen-binding mode of VWF-A3 provides direct structural constraints on collagen I chain registry. A VWF-binding site can be generated from the sequences RGQAGVMF, present in the two α1(I) chains, and RGEOGNIGF, in the unique α2(I) chain, provided that α2(I) is in the middle or trailing position. Combining these data with previous structural data on integrin binding to collagen yields strong support for the trailing position of the α2(I) chain, shedding light on the fundamental and long-standing question of the collagen I chain registry.

Fig. 1.

Structure of the A3/collagen-peptide complex. (A) Overview of the complex showing the orientation of the peptide with respect to the A3 domain. In all three peptide chains the Arg and Phe residues are shown in stick representation for reference. (B) The collagen-A3 interface seen along the long axis of the collagen peptide. Residues of the peptide that provide the major interactions with A3 (L:Val, L:Phe, M:Arg, T:Arg, and T:Hyp15) are shown in sticks as is A3 E1764. Peptide numbering is as used in the text. (C) Detailed view of the interaction interface. Residues that are involved in the interaction are shown in sticks. Stereo representations of this view can be found in Fig. S3. (D) Surface representation of the A3 domain showing the collagen peptide as a ribbon with side chains of key interacting residues. A3-residues that interact with the peptide are labeled and their surfaces are shown in red, yellow, orange, or green. In A–C, the A3 protein is shown in yellow and in all panels, the collagen leading, middle, and trailing chains in light blue, blue, and dark blue, respectively. Hydrogen bonds are indicated by dashed lines.

Fig. 2.

Conformational flexibility of the α3β4 loop. Stereoview of the different conformations of the α3β4 loop region. Shown in pale blue is the collagen leading chain of the A3/peptide complex with side chains of L:Met and L:Phe in stick representation. Shown in orange is the α3β4 loop region as observed in A3 crystal structure 1AO3 and the A3/peptide complex; the Phe-binding pocket is fully formed with M1785 projecting outward and H1786 hydrogen bonding to S1783 and, in the complex, to the backbone of L:Met. Superposed in yellow is the α3β4 loop region in A3 structure 1ATZ showing H1786 projecting outward into the solvent and M1785 projecting inward into the space which is occupied by L:Phe in the peptide complex.

Fig. 3.

Analysis of the VWF-binding site. (A) Surface rendering of the triple-helical peptide with atoms that are within 4.5 Å of the A3 domain indicated in red. Residues that form the core binding site are indicated in yellow, additional binding site residues are indicated in white. (B) Schematic drawing of collagen III showing core residues that would contact A3 if it binds to L:Phe, M:Phe, and T:Phe, respectively. (C) Schematic representations of the collagen I surfaces formed with the α2(I) chain in the leading, middle, and trailing position. In both B and C residues that correspond to the binding site found in the crystal structure are depicted in red. The collagen main chain is colored as in Fig. 1.

Fig. 4.

Analysis of the integrin α₂β₁ binding site in collagen I. (A) Surface rendering of the integrin-binding peptide from PDB ID code 1DZI. Residues within 4.5 Å of integrin α₂β₁ are shown in red. (B) Sequences of the collagen I α1 and α2 chain and of the triple-helical peptide that was used in the crystal structure. (C–E) Schematic representations of the collagen surface encountered by integrin α₂β₁ with the α2(I) chain in the leading (C), middle (D), and trailing (E) position and with the leading, middle, or trailing chain glutamate coordinating the α2β1 metal-ion dependent adhesion site (MIDAS). Residues that correspond to the binding site found in the crystal structure are depicted in red. The collagen main chain is colored as in Fig. 1.

Fig. 5.

Collagen binding by A3, DDR2, and SPARC. (A) A3, DDR2, and SPARC bind to a different surface of the collagen III helix. Crystal structures were oriented by superposition of the triple-helical peptides. (B) Surface drawing of a collagen III peptide showing the binding surfaces of A3, DDR2, and SPARC, respectively. Atoms within 4.5 Å of these proteins in their respective complexes are shown in red. (C–E) Close-up of the Phe-binding pocket of A3 (C), DDR2 (D), and SPARC (E). Key residues from the binding pockets are shown in sticks and hydrogen bonds are indicated by black dashes. Only the two collagen strands that interact with the protein are shown.