Binding of a fibrinogen mimetic stabilizes integrin alphaIIbbeta3's open conformation.
The platelet integrin alphaIIbbeta3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of alphaIIbbeta3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for alphaIIbbeta3. Eptifibatide binding promptly and reversibly perturbed the conformation of the alphaIIbbeta3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted alphaIIbbeta3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected alphaIIbbeta3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to alphaIIbbeta3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects alphaIIbbeta3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.
Hantgan
R R
RR
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. rhantgan@wfubmc.edu
Rocco
M
M
Nagaswami
C
C
Weisel
J W
JW
D001665:Binding Sites; D019281:Dimerization; D000077542:Eptifibatide; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008027:Light; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D011487:Protein Conformation; D011489:Protein Denaturation; D021122:Protein Subunits; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D008024:Ligands; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D021122:Protein Subunits; D005340:Fibrinogen; D000077542:Eptifibatide