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

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

Apparent activation energies of protein–protein complex dissociation in the gas–phase determined by electrospray mass spectrometry

We have developed a method to determine apparent activation energies of dissociation for ionized protein–protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein–protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation ( E#A m0gEA m0g# ) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein–protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants