Analysis of nidogen-1/laminin γ1 interaction by cross-linking, mass spectrometry, and computational modeling reveals multiple binding modes.

Lössl P, Kölbel K, Tänzler D, Nannemann D, Ihling CH, Keller MV, Schneider M, Zaucke F, Meiler J, Sinz A
PLoS One. 2014 9 (11): e112886

PMID: 25387007 · PMCID: PMC4227867 · DOI:10.1371/journal.pone.0112886

We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2-4, γ1 LEb2-4 N836D, γ1 short arm, and γ1 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 γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 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 γ1 LEb2-4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 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 γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces.

MeSH Terms (12)

Animals Cross-Linking Reagents HEK293 Cells Humans Kinetics Laminin Membrane Glycoproteins Mice Models, Molecular Protein Structure, Tertiary Surface Plasmon Resonance Tandem Mass Spectrometry

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