title: Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain.
abstract: Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.
authors.affiliation: Departments of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
authors.forename: R A
authors.initials: RA
authors.lastname: Kammerer
authors.forename: T
authors.initials: T
authors.lastname: Schulthess
authors.forename: R
authors.initials: R
authors.lastname: Landwehr
authors.forename: B
authors.initials: B
authors.lastname: Schumacher
authors.forename: A
authors.initials: A
authors.lastname: Lustig
authors.forename: P D
authors.initials: PD
authors.lastname: Yurchenco
authors.forename: M A
authors.initials: MA
authors.lastname: Ruegg
authors.forename: J
authors.initials: J
authors.lastname: Engel
authors.forename: A J
authors.initials: AJ
authors.lastname: Denzer
mesh_terms: D018171:Agrin / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D007797:Laminin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014162:Transfection; D014461:Ultracentrifugation
chemical_list: D018171:Agrin; D018076:DNA, Complementary; D007797:Laminin; D011994:Recombinant Proteins; C121346:laminin 1