Structural Basis for the Interaction of Fibrin with the Very Low-Density Lipoprotein Receptor Revealed by NMR and Site-Directed Mutagenesis.

Abstract

Interaction of fibrin with the very low-density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation. To establish the structural basis for this interaction, we have previously localized the VLDLR-binding site to fibrin βN-domains including fibrin β chain sequence 15-64 and determined the NMR solution structure of the VLDLR(2-4) fragment containing fibrin-binding CR domains 2-4 of VLDLR. In this study, we identified amino acid residues in VLDLR and the βN-domains that are involved in the interaction using NMR and site-directed mutagenesis. The results obtained revealed that Lys47 and Lys53 of the second and third positively charged clusters of the βN-domain, respectively, interact with Trp20 and Asp25 of the CR2 domain and Trp63 and Glu68 of the CR3 domain, respectively. This finding indicates that Lys residues of the βN-domain interact with the Lys-binding site of the CR domains in a manner proposed earlier for the interaction of other members of the LDL receptor family with their ligands. In addition, Gly15 of the βN-domain and its first positively charged cluster contribute to the high-affinity interaction with VLDLR. Molecular modeling based on the results obtained and analysis of the previously published structures of such domains complexed with RAP and HRV2 allowed us to propose a model of interaction of fibrin βN-domains with the fibrin-binding CR domains of the VLDL receptor.