Walter Göhring

Binding of the G domains of laminin α1 and α2 chains and perlecan to heparin, sulfatides, α‐dystroglycan and several extracellular matrix proteins

The C‐terminal G domain of the mouse laminin α2 chain consists of five lamin‐type G domain (LG) modules (α2LG1 to α2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays α2LG1‐3 and α2LG4‐5. These fragments were compared with similar modules from the laminin α1 chain and from the C–terminal region of perlecan (PGV) in several binding studies. Major heparin‐binding sites were located on the two tandem fragments and the individual α2LG1, α2LG3 and α2LG5 modules. The binding epitope on α2LG5 could be localized to a cluster of lysines by site‐directed mutagenesis. In the α1 chain, however, strong heparin binding was found on α1LG4 and not on α1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments α2LG1–3 and α2LG4‐5 also bound to fibulin‐1, fibulin‐2 and nidogen‐2 with Kd = 13–150 nM. Both tandem fragments, but not the individual modules, bound strongly to α‐dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to α‐dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell–matrix interactions and supramolecular assembly.

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C‐terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non‐covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25–32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N‐terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin‐1 and fibulin‐2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin‐1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22–38 kDa) and in serum (120–300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N‐terminal association region (∼50 residues), a central protease‐sensitive hinge region (∼70 residues) and a C‐terminal stable endostatin domain (∼180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix‐associated to a more soluble endocrine form.

Fibrillin-1 and Fibulin-2 Interact and Are Colocalized in Some Tissues

Microfibrils 10-12 nm in diameter are found in elastic and non-elastic tissues with fibrillin as a major component. Little is known about the supramolecular structure of these microfibrils and the protein interactions it is based on. To identify protein binding ligands of fibrillin-1, we tested binding of recombinant fibrillin-1 peptides to different extracellular matrix proteins in solid phase assays. Among the proteins tested, only fibulin-2 showed significant binding to rF11, the N-terminal half of fibrillin-1, in a calcium-dependent manner. Surface plasmon resonance demonstrated high affinity binding with a Kd = 56 nM. With overlapping recombinant fibrillin-1 peptides, the binding site for fibulin-2 was narrowed down to the N terminus of fibrillin-1 (amino acid positions 45-450). Immunofluorescence in tissues demonstrated colocalization of fibrillin and fibulin-2 in skin, perichondrium, elastic intima of blood vessels, and kidney glomerulus. Fibulin-2 was not present in ocular ciliary zonules, tendon, and the connective tissue around kidney tubules and lung alveoli, which all contain fibrillin. Immunogold labeling of fibulin-2 on microfibrils in skin was found preferentially at the interface between microfibrils and the amorphous elastin core, suggesting that in vivo the interaction between fibrillin-1 and fibulin-2 is regulated by cellular expression and deposition as well as by protein-protein interactions.

Structural basis for Gas6–Axl signalling

Receptor tyrosine kinases of the Axl family are activated by the vitamin K‐dependent protein Gas6. Axl signalling plays important roles in cancer, spermatogenesis, immunity, and platelet function. The crystal structure at 3.3 Å resolution of a minimal human Gas6/Axl complex reveals an assembly of 2:2 stoichiometry, in which the two immunoglobulin‐like domains of the Axl ectodomain are crosslinked by the first laminin G‐like domain of Gas6, with no direct Axl/Axl or Gas6/Gas6 contacts. There are two distinct Gas6/Axl contacts of very different size, both featuring interactions between edge β‐strands. Structure‐based mutagenesis, protein binding assays and receptor activation experiments demonstrate that both the major and minor Gas6 binding sites are required for productive transmembrane signalling. Gas6‐mediated Axl dimerisation is likely to occur in two steps, with a high‐affinity 1:1 Gas6/Axl complex forming first. Only the minor Gas6 binding site is highly conserved in the other Axl family receptors, Sky/Tyro3 and Mer. Specificity at the major contact is suggested to result from the segregation of charged and apolar residues to opposite faces of the newly formed β‐sheet.

Crystal structure and mapping by site‐directed mutagenesis of the collagen‐binding epitope of an activated form of BM‐40/SPARC/osteonectin

The extracellular calcium‐binding domain (positions 138–286) of the matrix protein BM‐40 possesses a binding epitope of moderate affinity for several collagen types. This epitope was predicted to reside in helix αA and to be partially masked by helix αC. Here we show that deletion of helix αC produces a 10‐fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix. The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 Å resolution. This constitutively activated mutant was used to map the collagen‐binding site following alanine mutagenesis at 13 positions. Five residues were crucial for binding, R149 and N156 in helix αA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM‐40. These residues are spatially close and form a flat ring of 15 Å diameter which matches the diameter of a triple‐helical collagen domain. The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens. Selected mutations in the non‐activated mutant ΔI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM‐40.

Analysis of Neurocan Structures Interacting with the Neural Cell Adhesion Molecule N-CAM

Neurocan is a brain-specific chondroitin sulfate proteoglycan, which has been shown to bind to the neural cell adhesion molecule N-CAM and to inhibit its homophilic interaction. To study in more detail the structures of neurocan responsible for this interaction, various recombinant neurocan fragments were generated. The ability of these fragments to interact with N-CAM was investigated in several different in vitro assay systems, enzyme-linked immunosorbent assay-type binding assays, Covasphere-aggregation assays, and assays based on an optical biosensor (BIAcore™) system. The analysis of the homophilic N-CAM interaction in the BIAcore system revealed a KD of 64 nM. This homophilic interaction could be reduced by preincubation of soluble N-CAM with neurocan. Direct binding of N-CAM to immobilized neurocan core protein and recombinant neurocan fragments could also be demonstrated, and KD values between 25 and 100 nM were obtained. In addition, direct binding of N-CAM to chondroitin sulfate could be demonstrated. Binding of N-CAM to the immobilized neurocan core protein could be inhibited with all recombinant fragments containing chondroitin sulfate or major parts of the mucin-like central region of neurocan. For the inhibition of homophilic N-CAM interactions, however, a combination of globular and extended structures was required.

The N-terminal globular domain of the laminin α1 chain binds to α1β1 and α2β1 integrins and to the heparan sulfate-containing domains of perlecan

The N‐terminal domains VI plus V (62 kDa) and V alone (43 kDa) of the laminin α1 chain were obtained as recombinant products and shown to be folded into a native form by electron microscopy and immunological assays. Domain VI alone, which corresponds to an LN module, did not represent an autonomously folding unit in mammalian cells, however. Fragment α1VI/V, but not fragment α1V, bound to purified α1β1 and α2β1 integrins, to heparin, and to heparan sulfate‐substituted domains I and V of perlecan. This localized the binding activities to the LN module, which contains two basic sequences suitable for heparin interactions.

Mapping of a Defined Neurocan Binding Site to Distinct Domains of Tenascin-C

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded aK D value of 17 nm for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins

Elastic fibers in vessel walls and other tissues consist of cross‐linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K d about 1 nM) of tropoelastin for microfibrillar fibulin‐2 and the recently described nidogen‐2 isoform. More moderate affinities were observed for fibulin‐1, laminin‐1 and perlecan, while several other ligands such as collagens, nidogen‐1, fibronectin and BM‐40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin‐2 and nidogen‐2, while the reaction with fibulin‐1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.

Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded β-barrel with a central helix. The β-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the β-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.