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摘要
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.
- 出版日期2014-11-11
