Structural Basis for PECAM-1 Homophilic Binding

PECAM-1 is a 130 kDa member of the immunoglobulin gene (Ig) superfamily that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1-mediated homophilic interactions, known to be mediated by its two amino-terminal Ig homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to sense flow, regulate leukocyte transendothelial migration, maintain vascular integrity, and transmit survival signals into the cell. Mutagenesis studies have implicated amino acids on either side of the face of IgD1 in homophilic binding, however the spatial orientation of this domain, the role that IgD2 plays in positioning IgD1 to participate in PECAM-1 homophilic interactions, and the contribution of its sialylated glycans to homophilic PECAM-1/PECAM-1 interactions remain to be critically explored. Complicating things further, PECAM-1 forms dimers and oligomers within the plane of the plasma membrane. The structural features of PECAM-1 responsible for maintaining the PECAM-1/PECAM-1 homophilic contacts necessary for carrying out these activities, however, have heretofore remained unresolved. Given the importance of PECAM-1-mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1-PECAM-1 homophilic binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic binding domain. The structure of IgD1-D2 was determined by multi-crystal anomalous diffraction and refined at 2.8 Šresolution. Interestingly, PECAM-1 crystallized as a strand-swapped dimer, a feature that has been seen in the crystal structures of many other members of the Ig superfamily, including cadherins, NCAM2, CTLA-4, and CD47. The homophilic interface between adjacent IgD1 domains was large, with a total buried interface of 1608 Ų. In addition to the large IgD1/IgD1 interface, interdomain contacts were found between IgD1/IgD2, and IgD2/IgD2 that likely reinforce the PECAM-1/PECAM-1 homophilic interactions that form as a consequence of the large number of PECAM-1 molecules that become concentrated at endothelial cell-cell borders as a result of diffusion trapping. Taken together, these results provide atomic-level detail to the unique architecture of the interface that forms the most abundant component of the endothelial cell intercellular junction.