Electron microscopy shows that binding of monoclonal antibody PT25-2 primes integrin αIIbβ3 for ligand binding

Abstract

The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the αIIb β propeller, a site distant from the Arg-Gly-Asp-binding pocket. To elucidate the mechanism, we studied the αIIbβ3-PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in αIIbβ3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the β3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the αIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent αIIbβ3 crystal structure showed that binding of PT25-2 creates clashes with the αIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return to its bent conformation. Kinetic studies of the binding of PT25-2 compared with mAbs 10E5 and 7E3 support this hypothesis. We conclude that PT25-2 induces αIIbβ3 ligand binding by binding to extended conformations and by preventing the interactions between the αIIb and β3 leg domains and subsequently the βI and β3 leg domains required for the bent-closed conformation.

Graphical abstract

Figure 1.

Negative-stain EM characterization of the αIIbβ3 ectodomain in complex with PT25-2 Fab. (A) Raw image of negatively stained αIIbβ3 ectodomain. Some integrins in the fully bent-closed conformation are indicated by dashed circles, whereas some integrins in more extended conformations are indicated by ovals. (B) Selected 2D class averages of negatively stained αIIbβ3 ectodomain by itself. Average 1 represents the fully bent-closed conformation. The diagram in panel E illustrates the ∼45° angle between the head and leg domains. Averages 2 to 5 show increasingly extended closed conformations. Average 6 shows a side view, and averages 7 and 8 show face-on views of the fully extended-closed conformation. (C) Raw image of negatively stained αIIbβ3 ectodomain in complex with PT25-2 Fab. Some integrins with a bound Fab are indicated by ovals. (D) Selected 2D class averages of negatively stained αIIbβ3 ectodomain in complex with PT25-2 Fab. Average 1 shows the complex in the most compact conformation. The diagram in panel F illustrates the ∼80° angle between the head and leg domains, and the arrowhead points to the PT25-2 Fab that prevents a closer association of the leg domains with the headpiece. Averages 2 to 7 show increasingly extended closed conformations of the PT25-2 Fab-bound αIIbβ3 ectodomain. Average 8 shows a side view of the fully extended-closed conformation. The scale bars in panels A and C are 50 nm, and the side length of the individual class averages in panels B and D is 43.2 nm.

Figure 2.

Binding of LIBS antibodies to platelets treated with PT25-2. Washed platelets were either untreated or incubated with T6 (10 μM), PT25-2 (15 μg/mL), eptifibatide (10 μM), or EDTA (10 mM) for 10 minutes at 22°C. Then, LIBS mAbs were added: AP5 (A), LIBS1 (B), or LIBS6 (C). After 30 minutes at 22°C, the platelets were washed and analyzed by flow cytometry. To normalize for differences between donor platelets, the geometric mean fluorescence of the sample incubated with eptifibatide was defined as 100%, and all other values were expressed as a percentage of the eptifibatide value. Data are presented as mean ± standard deviation. The data for AP5 and LIBS1 are from 3 separate experiments, and the data for LIBS6 are from 4 separate experiments.

Figure 3.

Cryo-EM structure of the αIIbβ3 headpiece in complex with the PT25-2 Fab. (A) Density map of the αIIbβ3–PT25-2 Fab complex at 3.3-Å resolution, segmented and colored according to the individual polypeptide chains. Asterisk indicates the ligand-binding site. (B) Atomic model of the αIIbβ3–PT25-2 Fab complex colored as in panel A. VH, variable domain of the heavy chain; CH1, first constant domain of the heavy chain; VL, variable domain of the light chain; CL, constant domain of the light chain. (C) Interaction surfaces of αIIb (i) and the PT25-2 Fab (ii). Side chains of residues that are within 4 Å from a side chain of the interacting protein are shown in stick representation.

Figure 4.

PT25-2 Fab binding prevents the αIIbβ3 ectodomain from adopting the fully bent-closed conformation. (A) Two perpendicular views of a hybrid atomic model of the PT25-2 Fab bound to the αIIbβ3 headpiece are shown. The PT25-2 Fab causes steric clashes with the αIIb leg and prevents the interaction of the β3 lower leg with the β3 hybrid domain that stabilizes the fully bent-closed conformation. (B) View of the hybrid atomic model of the PT25-2 Fab-bound αIIbβ3 headpiece showing the arrangement of the domains in the lower legs. Binding of the PT25-2 Fab to the αIIb β propeller displaces the αIIb calf-1 and calf-2 domains, which thus no longer stabilize the position of the β3 lower leg domains and, in turn, no longer support the interaction of the EGF-3 and EGF-4 domains with the hybrid domain, the main interactions stabilizing the fully bent-closed conformation.

Figure 5.

PT25-2 binding does not induce hybrid domain swing-out. (A) Overlay of the αIIbβ3 headpiece in complex with PT25-2 (red) and the headpiece of unliganded αIIbβ3 (PDB: 3FCS) (blue). (B) Overlay of the αIIbβ3 headpiece in complex with PT25-2 (red) and the headpiece of αIIbβ3 in complex with the fibrinogen γ-chain C-terminal dodecapeptide (PDB: 2VDO) (green). (C) Overlay of all 3 αIIbβ3 headpiece structures. ADMIDAS, adjacent to MIDAS; βI, β-inserted domain.

Figure 6.

Kinetics of binding of mAbs 10E5, 7E3, and PT25-2 to washed platelets. Washed platelets (2 × 10⁸/mL) were incubated with the Alexa⁴⁸⁸-labeled mAbs at 10 μg/mL for the indicated times, and then the samples were diluted 10-fold with HBMT to slow the reaction and immediately analyzed by flow cytometry. The data were normalized by expressing the results as the percentage of antibody bound at 1 hour, the time required to achieve near-saturating levels of mAb 7E3, the mAb that binds slowest. The blue asterisks above the 10E5 curve indicate the time points at which the binding of PT25-2 differs from that of 10E5, and the red asterisks below the 7E3 curve indicate the time points at which the PT25-2 binding differs from that of 7E3 as judged by P < .05 by analysis of variance, followed by Dunnett's post hoc t tests.