Structure of the Nidogen Binding LE Module of the Laminin γ1 Chain in Solution

Max-Planck-Institut für Biochemie, Mastinsreid bei München, FRG.
Journal of Molecular Biology (Impact Factor: 4.33). 05/1996; 257(3):658-68. DOI: 10.1006/jmbi.1996.0192
Source: PubMed


The structure of the single LE module between residues 791 and 848 of the laminin gamma1 chain, which contains the high affinity binding site for nidogen, has been probed using NMR methods. The module folds into an autonomous domain which has a stable and unique three-dimensional (3D) structure in solution. The 3D structure was determined on the basis of 362 interproton distance constraints derived from nuclear Overhauser enhancement measurements and 39 phi angles, supplemented by 5 psi and 22 chi1 angles. The main features of the NMR structures are two-stranded antiparallel beta-sheets which are separated by loops and cross-connected by four disulfide bridges. The N-terminal segment which contains the first three disulfide bridges is similar to epidermal growth factor. The C-terminal segment has an S-like backbone profile with a crossover at the last disulfide bridge and comprises two three-residue long beta-strands that form an antiparallel beta-sheet. The LE module possesses an exposed nidogen binding loop that projects away from the main body of the protein. The side-chains of three amino acids which are crucial for binding (Asp, Asn, Val) are all exposed at the domain surface. An inactivating Asn-Ser mutation in this region showed the same 3D structure indicating that these three residues, and possibly an additional Tyr in an adjacent loop, provide direct contacts in the interaction with nidogen.

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    • "In Alport syndrome, mutations in a single collagen gene result in the absence of three collagen proteins in the basement membrane because of improper protomer assembly (Kalluri et al., 1993; Gunwar et al., 1998). In laminins, synonymous LEdomain loop structures have been shown to be essential for nidogen binding and stability (Baumgartner et al., 1996; Dreyer et al., 2000). A knock-in mouse was recently described where the nidogen binding domain in the laminin γ1 chain was specifically ablated. "
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    ABSTRACT: Usherin is a basement membrane protein encoded by the gene associated with Usher syndrome type IIa, the most common deaf/blind disorder. This report demonstrates a specific interaction between type IV collagen and usherin in the basement membrane, with a 1:1 stoichiometry for binding. Genetic and biochemical approaches were used to explore the role of type IV collagen binding in usherin function. We demonstrate binding occurs between the LE domain of usherin and the 7S domain of type IV collagen. A purified fusion peptide comprising the first four LE modules was shown to compete with full-length recombinant usherin for type IV collagen binding. However, synonymous fusion peptides with single amino acid substitutions resulting from missense mutations that were known to cause Usher syndrome type IIa in humans, failed to compete. Only mutations in loop b of the LE domain abolished binding activity. Co-immunoprecipitation and western blot analysis of testicular basement membranes from the Alport mouse model show a 70% reduction in type IV collagen is associated with a similar reduction in usherin, suggesting the usherin/collagen (IV) interaction stabilizes usherin in the basement membrane. Thus, the domain-specific interaction between usherin and type IV collagen appears essential to usherin stability in vivo, and loss of this interaction may result in Usher pathology in humans.
    Full-text · Article · Feb 2004 · Journal of Cell Science
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    • "Nidogen-1, a sulphated 150 kDa glycoprotein, co-purifies with laminin-1 upon EDTA extraction (Paulsson et al., 1987). The nidogen-binding site on laminin-1 has been localized to laminin EGF-like (LE) module 4, which has subsequently been determined at atomic resolution (Stetefeld et al., 1996; Baumgartner et al., 1996). It was shown that this interaction might be significant for proper basement membrane formation in organ cultures, as the presence of antibodies raised against the nidogen-binding site on laminin-1 disrupted the basement membrane and reduced branching epithelial morphogenesis in a number of different organs (Ekblom et al., 1994; Kadoya et al., 1997). "
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    ABSTRACT: Basement membranes contain two major molecular networks consisting of laminin and collagen IV. Previous antibody perturbation experiments suggest that the interaction between laminin and nidogen-1 is necessary for proper basement membrane formation and epithelial development, whereas results from gene ablation experiments in mice show that both basement membranes and general development are grossly normal in the absence of nidogen-1. To refine the perturbation approach, we produced F9-teratocarcinoma-cell-derived embryoid bodies in the presence of recombinantly expressed nidogen-binding sites localized within the gamma1III3-5 laminin fragment. We found basement membranes were disrupted in gamma1III3-5-expressing embryoid bodies. As a measurement of basement membrane function, we tested permeability and detected drastically increased diffusion rates in correlation with basement membrane disruption. Furthermore, TROMA-1 localization in embryoid bodies expressing the nidogen-binding site was altered, suggesting separation of epithelium-specific gene expression from the formation of the actual epithelium when occurring in the absence of an organized basement membrane.
    Preview · Article · Apr 2003 · Journal of Cell Science
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    • "Nidogen, also called entactin (Durkin et al., 1988; Mann et al., 1989), is a basement membrane protein that binds with high affinity to laminin γ1 chain (Mayer et al., 1993a). The few crucial residues important for nidogen binding on a single epidermal growth factor-like module of the γ1 chain (III4) have been identified (Timpl and Brown, 1996) and the structure of the loops where these reside has been clarified in detail (Baumgartner et al., 1996; Stetefeld et al., 1996). These and many other studies suggest that nidogen forms a crucial link between basement membrane components (Timpl and Brown, 1996). "
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    ABSTRACT: Epithelial-mesenchymal interactions are major driving forces for the development of most solid organs. The importance of these interactions was first shown for the embryonic submandibular gland more than 40 years ago. We here present evidence that interactions between two basement membrane components, nidogen (entactin) and laminin gamma1 chain, could be important for epithelial-mesenchymal interactions in this gland. Nidogen mRNA was detected by in situ hybridization in the mesenchyme, and yet the protein was detected in epithelial and endothelial basement membranes. The role of nidogen-laminin interactions for epithelial morphogenesis was studied by applying antibodies to submandibular gland organ cultures. Antibodies reacting strongly with the nidogen-binding site of laminin gamma1 chain drastically perturbed branching epithelial morphogenesis. Electron microscopy of the epithelial-mesenchymal interface showed that blocking antibodies disrupted the formation of the basement membrane. Epidermal growth factor was shown to increase the expression of nidogen in mesenchyme, and could counteract the effect of the blocking antibodies. We suggest that nidogen could be an important mesenchymal factor for submandibular gland development.
    Full-text · Article · Mar 1997 · Development
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