Selection and Structure of Ion-selective Ligands for Platelet Integrin αIIbβ3

Abstract

Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca 2؉ and Mg 2؉ bind to distinct sites on integrin and can have opposing effects on ligand binding. These effects are presumably brought about by alterations of the shape of the ligand binding pocket. To gain insight into the nature of these structural differences, we probed the integrin ligand binding site with an RGD-based library of unparalleled complexity. A cysteine-constrained phage library containing six random amino acids and the RGD motif present in seven different registers was used to select for ligands that exhibit ion-selective binding to integrin ␣ IIb ␤ 3 . The library was used to select for peptides that bind to the integrin ␣ IIb ␤ 3 preferentially in Ca 2؉ versus Mg 2؉ . Peptides were identified which bound selectively in each ion. The Ca 2؉ -selective peptides had a range of sequences, with the only obvious consensus involving a motif that had four cysteine residues bonded in a 1,4:2,3 arrangement. Interestingly though, the Mg 2؉ -selective peptides exhibited a well defined consensus motif containing Cys-X-aromatic-L/G-R-G-D-hydrophobic-R-R/K-Cys. As a first step toward understanding the structural basis for this selectivity, solution NMR structures were obtained for representatives of both sets of peptides. All peptides formed turns, with the RGD motif at the apex. The Mg 2؉ -selected peptides contained a unique basic patch that protrudes from the base of the turn.

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

“…To decipher the mechanisms underlying the experimental differences we observed using optical force spectroscopy, we turned to computational modeling using the reported crystal structures of the open and closed αIIbβ3 headpiece and NMR structures for cRGDFK and γC-12 that we determined experimentally. It is important to note that the structures we found for cRGDFK and γC-12 peptides were consistent with previously reported NMR and crystallographic structures for cyclic RGD peptides and for γC-12. ,− …”