Analysis of Nidogen-1/Laminin γ1 Interaction by Cross-Linking, Mass Spectrometry, and Computational Modeling Reveals Multiple Binding Modes

Abstract

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.

Modeling Protein Excited-state Structures from “Over-length” Chemical Cross-links

“…Quite often it has been found that a subset of the highly reliable cross-links cannot be explained by the known structure of the protein or protein complex, or these cross-links are inconsistent with other cross-links or other experimental data (12,13,17,18). In those cases, the over-length cross-links were often discarded or the corresponding distance restraints were relaxed.…”

“…CXMS-based structural modeling has been explored using various software tools, yet a lot of these studies aimed for a single structure that had the lowest energy and best satisfied the experimental data (11,13,24,25). To fully account for the intramolecular cross-links, we developed the DynaXL method.…”

“…The discrepancy between experimental cross-links and protein structures may be due to false identifications of the cross-links. Yet, even after eliminating erroneous assignments with stringent criteria (12), the discrepancy persists (13)(14)(15)(16). As a common practice in protein structure modeling, the distance restraints derived from the over-length cross-links were often relaxed (17,18) or even discarded (19).…”

Laminin L4 domain structure resembles adhesion modules in ephrin receptor and other transmembrane glycoproteins

“…In addition, three L4 domains are found in perlecan, a heparan sulfate proteoglycan of the ECM with an origin as ancient as that of laminin. No experimentally determined structure for an L4 domain has been reported, though a fold prediction based on threading has been made . We produced recombinant versions of the human laminin α1 and α2 L4b domains to investigate the L4 structure at high resolution and to provide a framework for exploring the functions and structural diversification of laminin internal domains.…”

Integrative modelling of cellular assemblies

“…The reliability of NMR restraints is considered by their relative contributions in the scoring function. Distance constraints can be enforced (e.g., [47]) as part of the scoring function involving statistical interaction potentials and evaluation of standard geometry (atoms and secondary structures) and clashes. In a recent structure of S. flexneri type III secretion system (T3SS) needle several solid-state NMR (ssNMR) distance restraints were used to build models with a 7.7 Å cryo EM density map [48].…”