Structural basis for quinine-dependent antibody binding to platelet integrin αIIbβ3

Jianghai Zhu, Jieqing Zhu, Daniel W. Bougie, Richard H. Aster and Timothy A. Springer

Article Figures & Data


  • Figure 1

    Alignment of 314.1 and 314.3 V domains and corresponding germline sequences. Residues that differ between 314.1 and 314.3 are in red. Segments derived from the D and J regions are colored in green and cyan, respectively.

  • Figure 2

    Crystal structures of Fabs 314.1 and 314.3 and their complexes with quinine. View of the antigen-binding site of Fab 314.1 and its quinine complex (A,C,E,G) and Fab 314.3 and its quinine complex (B,D,F,H) in identical orientations. Quinine is shown in stick with carbon atoms wheat, oxygens red, and nitrogens blue (C,D) or as a wheat surface (G,H). CDR loops are colored red (H3), blue (L3), yellow (H2 and L2), and green (H1 and L1). The Fab solvent-accessible surface is transparent in panels A-D, revealing CDR loops and framework (white) shown in cartoon. In panels E-H, the solvent-accessible surfaces include quinine when present and are opaque to better show the marked differences in topography of the surfaces that occur because of both quinine binding and CDR H3 and L3 loop backbone reorientation.

  • Figure 3

    Details of quinine binding. Quinine complexes with Fab 314.1 (A) and Fab 314.3 (B). Quinine is shown in thicker stick with wheat carbons; Fab CDR loops and framework are shown in thinner stick with silver carbons. Oxygens are red and nitrogens are blue. Fabs are shown with white transparent surfaces. 2Fo-Fc quinine omit map density is shown as magenta mesh contoured at 3σ. Interactions between quinine and Fab 314.1 (C) and Fab 314.3 (D) shown schematically with Maestro (Schrodinger, New York, NY). Hydrophobic residues are shown in green, hydrophilic residues are cyan, positively charged residues are violet, negatively charged residues are pink, and glycine residues are white. Stripes show backbone contact regions. Arrows show hydrogen bonds. Green line shows π-π stacking. Red-blue line shows a salt bridge. Red curved lines show π-cation interaction. Shadows on atoms in quinine schematize their amount of exposure to solvent. Comparisons for Fab 314.1 (E) and Fab 314.3 (F) with and without quinine. Carbons for the Fabs are shown in silver (no quinine) and in cyan (with quinine).

  • Figure 4

    Quinine-Fab binding. (A-B) Isothermal titration calorimetry. Titrations of quinine into 314.1 (A) and 314.3 (B) IgG in the calorimetry cell are as described in “Methods.” Because the 2 binding sites in IgG bind quinine independently, the results are expressed in terms of the concentration of half of an IgG, ie, the concentration of heavy and light chains. (C) Gel-filtration chromatography of the αIIbβ3 headpiece, with or without Fab 314.1, with and without quinine, and with and without quinine in the running buffer.

  • Figure 5

    Models for DDAb binding to a cell-surface molecule. (A) The sandwich model. (B) The hybrid paratope model. The antigen could represent any cell-surface molecule, including integrin αIIbβ3.


  • Table 1

    Crystallographic statistics

    Fab314.1Fab314.1 + quinineFab314.3Fab314.3 + quinine
    Wavelength (Å)0.979360.979461.006951.03321
    Space groupP312P41212P21P21
    a, b, c (Å)73.8,73.8,190.362.3,62.3,232.967.3,145.2,101.554.2,55.5,72.2
    α, β, γ (°)90.0,90.0,120.090.0, 90.0, 90.090.0, 90.9, 90.090.0, 99.0, 90.0
    Resolution (Å)50 - 2.050 - 2.8550 - 2.750 - 2.5
    Reflections (total/unique)221 231/40 094 (10 840/2,412)*57 965/10 563 (4,865/816)218 092/53 487 (15 958/3,905)53 941/14 747 (3,050/1,025)
    I/σ18.2 (8.7)17.2 (2.6)7.4 (0.9)9.6 (1.5)
    CC1/2 (%)99.8 (97.0)99.9 (91.6)97.5 (36.4)98.9 (58.2)
    Completeness (%)96.6 (80.4)92.6 (100)99.9 (100)99.3 (95.6)
    Rmerge (%)6.4 (16.9)6.4 (84.8)25.5 (174)13.0 (76.3)
     Bond (Å)0.0070.0040.0050.003
     Angle (°)1.1490.8561.0180.882
    MolProbity percentile99th99th97th98th
    • RMSD, root mean square deviation; PDB, Protein Data Bank.

    • * Numbers in parentheses correspond to the last-resolution shell.

    • Rmerge = ∑hi|Ii(h) − < I(h) > |/∑hi|Ii(h)I, where Ii(h) and < I(h) > are the ith and mean measurement of the intensity of reflection h.

    • Rwork = ∑h||Fobs (h)| − |Fcalc (h)||/∑h|Fobs (h)|, where Fobs (h) and Fcalc (h) are the observed and calculated structure factors, respectively. No I/σ(I) cutoff was applied.

    • § Rfree is the R value obtained for a test set of 1000 randomly selected reflections excluded from refinement.

  • Table 2

    Quinine-Fab interface characteristics

    Fab 314.1Fab 314.3
    Solvent-accessible surface area (Å2)*
    Quinine total502509
    Buried on quinine440 (87.6%)452 (88.8%)
    Buried on H chain154202
    Buried on L chain127128
    Buried total on Fab281330
    Shape complementarity index0.7640.809
    • * Calculated by PISA.18

    • Computed by the SC program in CCP4 suite.19