Analysis of Nidogen-1/Laminin [gamma]1 Interaction by Cross-Linking, Mass Spectrometry, and Computational Modeling Reveals Multiple Binding Modes

• Published in: PloS one Vol. 9; no. 11
• Main Authors: Lössl, Philip, Kölbel, Knut, Tänzler, Dirk, Nannemann, David, Ihling, Christian H, Keller, Manuel V, Schneider, Marian, Zaucke, Frank, Meiler, Jens, Sinz, Andrea
• Format: Journal Article
• Language: English
• Published: Public Library of Science 11-11-2014
• Subjects: Amino acids; Chemical properties; Computer simulation; Laminin; Spectroscopy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0112886

We describe the detailed structural investigation of nidogen-1/laminin [gamma]1 complexes using full-length nidogen-1 and a number of laminin [gamma]1 variants. The interactions of nidogen-1 with laminin variants [gamma]1 LEb2-4, [gamma]1 LEb2-4 N836D, [gamma]1 short arm, and [gamma]1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin [gamma]1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin [gamma]1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin [gamma]1 LEb2-4, laminin [gamma]1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin [gamma]1 LEb3 domain is not essential for complex formation. Cross-links between laminin [gamma]1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin [gamma]1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces.