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

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

SUBMITTER: Lossl P 

PROVIDER: S-EPMC4227867 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature