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Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib

Juliet Morgan, Muhammad Saleem, Ruiqi Ng, Caroline Armstrong, Szu S. Wong, Simon G. Caulton, Alice Fickling, Huw E. L. Williams, Adam D. Munday, José A. López, Mark S. Searle and Jonas Emsley

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Mapping the Mac-1 I-domain binding site with GP1bαN by NMR. (A) Selected peaks showing CSP effects between bound and unbound (the first panel for G228 is shown as a control, in which no perturbation is observed). (B) Representation of CSP effects, with dashed lines showing statistically significant perturbation (lower line, mean CSP +1 SD; upper line, mean CSP +2 SD) residues above the latter. (C) CSP effects mapped to the Mac-1 surface with residues in darker pink corresponding to mean +2 SD, top-down view onto the MIDAS (Mg2+ shown in yellow). (D) Side view showing distal CSP effects removed from the MIDAS site (top). (E) Surface representation showing the binding patch around the MIDAS with key residues in red. (F) Differentiation between the MIDAS binding patch, shown in red, and those distal residues (in green) affected through allosteric changes from binding GP1bαN at the MIDAS site (a few key residues are highlighted).

  • Figure 2.

    Crystal structure of the mouse Mac-1 I-domain. (A) A cartoon diagram is shown for the crystal structure of the mouse Mac-1 I-domain (purple) with the crystal contact stabilizing the active conformation derived from the C-terminal helix α7 in orange. The MIDAS bound Mg2+ ion is shown as a sphere (green) and key residues are shown as sticks and electrostatic interactions as dashed magenta lines. (B) A close-up view of the MIDAS site with residues from helix α7 labeled. (C) A second view of the Mac-1 I-domain MIDAS site with the crystal contact residue E314 shown coordinating the Mg2+ ion and the sidechain hydroxyl of T209. (D) Superposition of the human (gray) and mouse (purple) crystal structures in the region of the MIDAS face. The stabilization glutamic acid is shown as sticks in cyan (human) and orange (mouse).

  • Figure 3.

    Crystal structure of the mouse GP1bαN. (A) A cartoon diagram is shown for the crystal structure of the mouse GPIbαN with the C-terminal LRR capping α-helix colored orange and R-loop colored yellow. GPIbα residues H218 and E222 involved in the interaction with Mac-1 are shown as sticks. The elongated pocket formed in the GPIbα LRR capping region is indicated by a dashed line and represented as a transparent charged surface with key residues lining the pocket shown as sticks. (B) Electron density (2Fo-Fc) from the crystal structure of the mouse GPIbαN C-terminal capping α-helix residues H218 and E222 (gray mesh). (C) Amino acid sequence alignment of GPIbα residues 215 through 223 (human sequence numbering without the signal sequence) from the GPIbα LRR capping α-helix for human, mouse, dog, and cow. Key charged residues 218 and 222 are colored blue and red, respectively.

  • Figure 4.

    The Mac-1 I:GP1bαN complex. (A) A cartoon diagram of the docked complex of the crystal structures of human GPIbαN and the Mac-1 I-domain with the Mac-1 secondary structures colored blue/green and GPIbαN colored red/orange. Boxed is a close-up view of the interface where the Mg2+ ion bound to the Mac-1 MIDAS site is shown as a sphere and electrostatic interactions are shown as dashed purple lines. The GPIbαN C-terminal LRR capping region is colored orange and residue F192 from the LRRs is in gray. (B) A second view related by a 90° rotation. GPIbαN residue D222 coordinates the Mac-1 MIDAS Mg2+ ion and surrounding residues on the MIDAS surface (light blue). (C) Charged surface representation of the GPIbαN LRR capping region showing an elongated pocket formed by GPIbα residues Y215, W219, and F192 is flanked by a region of positive charge (GPIbα R218) and negative charge (GPIbα D222) that form complementary interactions with Mac-1 residues shown as sticks (light blue).

  • Figure 5.

    SPR analysis of Mac-1 I-domain interactions. Plots of SPR sensorgrams measured in RU on the y-axis illustrating wild-type Mac-1 (top) or Mac-1 T209A (bottom) binding to fibrinogen (Fbg; A) and GP1bαN (B), and shown in wild-type Mac-1 I-domain binding to the GP1bαN mutants H218A (C, top) and E222A (C, bottom). RU, response unit.

Tables

  • Table 1.

    Crystallographic data collection and refinement statistics

    SampleMouse GP1bαNMouse Mac-1 I
    Data collection
     Space groupP212121P41212
     Cell dimensions
      a, b, c (Å)61.5, 72.8, 164.062.9, 62.9, 336.2
      α, β, γ (°)90.0, 90.0, 90.090.0, 90.0, 90.0
     Resolution, Å29.9-2.045.9-2.5
     Rmerge*11.112.5
     I/σI; CC(1/2)7.0 (1.9); 0.997 (0.78)8.2 (1.5); 0.903 (0.68)
     Completeness (%)99.0 (90.0)86.7 (67.2)
     Redundancy4.3 (1.3)6.2 (2.5)
    Refinement
     Number of reflections4809521236
     Rwork/Rfree0.192/0.2370.222/0.271
     B factors, Å2
      Protein29.848.2
     RMS deviations
      Bond lengths, Å0.0180.021
      Bond angles, °1.961.85
    • RMS, root mean square.

    • * Rmerge = Sum(h) [Sum(j) [I(hj) − <Ih>]/Sum(hj) <Ih>, where I is the observed intensity and <Ih> is the average intensity of multiple observations from symmetry-related reflections calculated with SCALA.

    • Values in parentheses are for the highest resolution shell.

    • Rwork = Sum(h) ||Fo|h − |Fc|h| / Sum(h)|Fo|h, where Fo and Fc are the observed and calculated structure factors, respectively. Rfree computed as in Rwork, but only for 5% randomly selected reflections, which were omitted in refinement, calculated using REFMAC.