Mapping of mutual binding sites in the hemidesmosomal proteins integrin α6ß4 and BPAG1e

Abstract

Hemidesmosomes (HDs) are junctional complexes that mediate the stable adhesion of epithelial cells to the basement membrane by linking the extracellular matrix to the intermediate filament system of the cytoskeleton. In (pseudo)stratified epithelia HDs contain integrin α6ß4, the bullous pemphigoid antigen 2 (BPAG2), the tetraspanin CD151, plectin, and BPAG1e. The integrin alpha6beta4 is connected to the cytokeratins by plectin and BPAG1e, which belong to the plakin family of cytolinkers. BPAG1e interacts with the third and fourth FnIII domains (FnIII-3,4) of the ß4 cytoplasmic moiety. We have recently elucidated the structure of the FnIII-3,4 of ß4, which revealed a fixed arrangement of the two FnIII domains and an evolutionary conserved surface. In this work, we have mapped in detail the reciprocal binding sites in BPAG1e and beta4. On the one hand, binding to beta4 is mediated by a segment of ~30 residues near the N-terminus of BPAG1e. On the other hand, using structure-based point mutants of the FnIII-3,4, we have found that residues in ß4 that are important for BPAG1e-binding cluster on the conserved surface of the FnIII-3,4 structure. In addition, disruption of the FnIII-3,4 inter-domain arrangement inhibits the binding to BPAG1e, supporting that the BPAG1e-binding site expands both FnIII domains and suggesting that the interaction could be modulated by stretching of the ß4 cytodomain.