Analysis of nidogen-1/laminin γ1 interaction by cross-linking, mass spectrometry, and computational modeling reveals multiple binding modes.
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.
Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Philip
P
Lössl
Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Knut
K
Kölbel
Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Dirk
D
Tänzler
Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN, United States of America.
David
D
Nannemann
Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Christian H
CH
Ihling
Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany.
Manuel V
MV
Keller
Research Group Artificial Binding Proteins, Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Marian
M
Schneider
Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany.
Frank
F
Zaucke
Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN, United States of America.
Jens
J
Meiler
Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Andrea
A
Sinz
D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D007797:Laminin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry
D003432:Cross-Linking Reagents; D007797:Laminin; D008562:Membrane Glycoproteins; C089141:laminin gamma 1; C029967:nidogen