Sequence Alignment between vWF and Peptides Inhibiting the vWF-collagen Interaction Does not Result in the Identification of a Collagen-binding Site in vWF (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 2012
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.
von Willebrand factor binds to native collagen VI primarily via its A1 domain
The Biochemical journal, 1997
Collagen VI is abundant in the arterial subendothelium. To investigate its mechanism of interaction with von Willebrand factor (vWF), collagen VI was isolated from human placenta and from the extracellular matrix of the human lung fibroblast cell line MRC-5. Purified vWF bound to non-digested collagen VI with moderately high affinity (EC50 approximately 5 nM) and could be inhibited by the Hirudo medicinalis collagen inhibitor calin. The anti-(human vWF A1 domain) monoclonal antibody (AJvW-2), as well as aurin tricarboxylic acid (ATA), at concentrations that saturate the vWF A1 domain, also inhibited this binding. In contrast, the monoclonal anti-(human vWF A3 domain) antibody (82D6A3) inhibited vWF binding to collagens I, III and IV, but had no effect on vWF binding to collagen VI. Likewise, vWF binding to collagen VI was not inhibited by the recombinant vWF domain D4. Polyclonal anti-(collagen VI) antibodies, specifically neutralizing the binding of vWF to collagen VI, confirmed th...
Identification of binding partners interacting with the α1-N-propeptide of type V collagen
Biochemical Journal, 2011
The predominant form of type V collagen is the [α1(V)] 2 α2(V) heterotrimer. Mutations in COL5A1 or COL5A2, encoding respectively the α1(V)-and α2(V)-collagen chain, cause classic Ehlers-Danlos syndrome (EDS), a heritable connective tissue disorder, characterized by fragile hyperextensible skin and joint hypermobility. Approximately half of the classic EDS cases remains unexplained. Type V collagen controls collagen fibrillogenesis through its conserved α1(V)-amino(N)propeptide domain. To gain insight into the role of this domain, a yeast-two-hybrid screen among proteins expressed in human dermal fibroblasts was performed utilizing the amino(N)-propeptide as a bait. We identified 12 interacting proteins including extracellular matrix proteins and proteins involved in collagen biosynthesis. Eleven interactions were confirmed by surface plasmon resonance and/or coimmunoprecipitation: α1(I)-and α2(I)-collagen chains, α1(VI)-, α2(VI)-and α3(VI)-collagen chains, tenascin-C, fibronectin, procollagen C-proteinase enhancer-1 (PCPE-1), tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-2 and transforming growth factor-β1.
Biochemical Society Transactions, 2008
Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. We have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin α2β1 binds to several GXX GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where O is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.
Journal of Biological Chemistry, 2003
To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg 963 , Pro 981 , Asp 1009 , Arg 1016 , Ser 1020 , Met 1022 , and His 1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser 1020 , Arg 1016 , Pro 981 , and Trp 982 in the threedimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser 1020 and Arg 1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp 982 . In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.
[Isolation and characterization of collagen-binding domains from human von Willebrand factor]
Molekuliarnaia genetika, mikrobiologiia i virusologiia, 2009
DNA fragments encoding for two collagen binding decapeptides from the human von Willebrand factor (vWF-H1 and vWF-H2) were cloned in the Escherichia coil culture. Overproducing strains of the chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD consisting of the corresponding decapeptide, Gly-Ser spacer and a cellulose binding domain (CBD) from Anaerocellum thermophilum were constructed. Using one-stage purification on cellulose, the highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained and the ability of these proteins to bind collagen was studied. These constructions are planned to be used for development of the recombinant collagen binding proteins with different biological activities, which, in its turn, will be used for development of the new generation products and materials for medicine, such as different kinds of implants, the coats, etc.
Journal of Biological Chemistry, 2006
Here we describe a novel collagen belonging to the class of von Willebrand factor A (VWA) domain-containing proteins. This novel protein was identified by screening the EST data base and was subsequently recombinantly expressed and characterized as an authentic tissue component. The COL28A1 gene on human chromosome 7p21.3 and on mouse chromosome 6A1 encodes a novel protein that structurally resembles the beaded filament-forming collagens. The collagenous domain contains several very short interruptions arranged in a repeat pattern. As shown for other novel minor collagens, the expression of collagen XXVIII protein in mouse is very restricted. In addition to small amounts in skin and calvaria, the major signals were in dorsal root ganglia and peripheral nerves. By immunoelectron microscopy, collagen XXVIII was detected in the sciatic nerve, at the basement membrane of certain Schwann cells surrounding the nerve fibers. Even though the protein is present in the adult sciatic nerve, collagen XXVIII mRNA was only detected in sciatic nerve of newborn mice, indicating that the protein persists for an extended period after synthesis.
Journal of Thrombosis and Haemostasis, 2006
Summary. Background: It is established that the A3 domain in von Willebrand factor (VWF) contains the major collagen-binding site. However, there are conflicting reports describing the capacity of the A1 domain to interact with collagen types I and III. Methods: In this study, we have used recombinant VWF-A1 polypeptides, as well as conformation-specific monoclonal antibodies (mAb), to analyze the A1–collagen interaction. Results: The A1 domain bound to collagen with Kd ∼ 8.0 nm and this binding was blocked by the mAb 6G1, which blocks the interaction between ristocetin and VWF. In addition, collagen-bound A1 protein was able to support flow-dependent adhesion of platelets, demonstrating that the binding sites for collagen and glycoprotein (GP)Ib are different. Analysis with two conformation-specific mAb demonstrated that the structure of the A1 domain changed as a result of the binding to collagen. In contrast, the antibodies failed to detect conformational change in the G1324S mutant (type 2M von Willebrand disease). Thus, direct binding to collagen induces a change in the structural conformation within the VWF-A1 domain, and the G1324S substitution prevents this conformational change. Conclusion: This study has shown that the isolated A1 domain can simultaneously bind to collagen and platelet GPIb, supporting platelet adhesion under high-flow conditions. In addition, this study has used mAb to demonstrate that the binding of the isolated A1 domain or full-length VWF to collagen is accompanied by a conformational change in A1 domain.
Identification of the Collagen-binding Site of the von Willebrand Factor A3-domain
Journal of Biological Chemistry, 2000
Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of residues Arg 963 , Glu 987 , His 990 , Arg 1016 , and His 1023 , located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His 1023 abolished collagen binding, whereas mutation of Arg 963 and Arg 1016 reduced collagen binding by 25-35%. These residues are part of loops ␣34 and ␣12 and ␣-helix 3, respectively, and lie near the bottom face of the domain. His 1023 and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.
Identification and Characterization of Novel Collagen Chains
2010
Collagen VI and collagen XXVIII are two extracellular matrix proteins that belong to the superfamily of von Willebrand Factor A (VWA) domain containing molecules. Earlier studies on collagen VI indicated that this widely distributed protein is composed of α1, α2 and α3 polypeptide chains, which form a microfibrillar network in close association with basement membranes in muscle and other tissues. In contrast, an initial study on collagen XXVIII reported that it forms a homotrimer and has a very restricted localization at specific basement membranes of peripheral nerves. In this dissertation, the identification and characterization of three novel collagen VI chains, α4, α5 and α6, that show similarity to the collagen VI α3 chain is described. The genes coding for the new chains are arranged in tandem on mouse chromosome 9. The proteins contain seven N-terminal VWA domains followed by a collagenous domain, two C-terminal VWA domains and a unique domain. In addition the collagen VI α4 ...