Structural requirements for activation in alphaIIb beta3 integrin

Abstract

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.

FIGURE 1.

Effect of β-head/β-tail interface stabilization on the αIIbβ3-Fbg interaction. A, crystal structure of the αVβ3 integrin. The entire αV chain is shown as the gray ribbon. The βA and hybrid domains that compose the head region of the β3 chain are shown as red and orange ribbons, respectively. The EGF-3, EGF-4, and βT domains that compose the β-tail of the β3 chain are shown as the green ribbon. The β-head/β-tail interface shown in the rectangle is magnified in the right-hand panel. Amino acid residues Ser-367 and Gly-382 in the hybrid domain and Val-332 in the βA domain are shown as magenta spacefill. These residues are closely located to Ser-551 in the EGF-3 domain, Thr-564 in the EGF-4 domain, and Ser-674 in the βT domain, respectively, which are shown as yellow spacefill. Only the residues in the β-tail are labeled. To constrain αIIbβ3 in its bent conformation, these four residue couples were simultaneously mutated to Cys to facilitate disulfide bridge formation. B, Fbg binding to cells expressing αIIbβ3 constrained in the bent conformation in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ and control antibody (solid column), in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ and PT25-2 (hatched column), and in cells pretreated with DTT in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ (open column) is shown. MFI, mean fluorescent intensity. Error bars indicate S.E.

FIGURE 2.

Role of the βA/CD loop interaction in αIIbβ3 activation. A, the βA and βT domains are shown as red and green ribbons, respectively. The βA/CD loop interface shown in the rectangle is magnified on the right-hand side. Amino acid residues 671–676 in the β3 domain that contain the CD loop in the βT domain are shown in yellow. Val-332 in the βA domain and Ser-674 in the CD loop are shown as magenta and yellow spacefill, respectively. B, the CD loop sequences were either deleted (del-CD) or replaced with an irrelevant FLAG tag sequence (FLAG-CD). MFI, mean fluorescent intensity. C, bulky N-glycan-binding sites were introduced either at Val-332 (V332N) or at Ser-674 (S674N/K676T), or in combination (332N/674N). Fbg binding to the cells in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ and a control antibody and in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ and PT25-2 is shown as the solid and hatched columns, respectively. Fbg binding in the presence of 1 mm Mn²⁺ with control antibody is shown as the gray column. Error bars indicate S.E.

FIGURE 3.

Effect of integrin extension on ligand binding. A, the three-dimensional structure of the αV chain is shown as semitransparent blue spacefill with its backbone as a blue ribbon. The β3 chain is shown as a gray ribbon. Structures around α-genu in the rectangle are magnified below. The positions of the amino acid residues Thr-466, Pro-583, and Gln-589, which are homologous to Thr-478, Asp-589, and Gln-595 in αIIb, are shown as cyan spacefill and labeled as such. Note that Gln-595 is located immediately behind the genu. B, an N-glycosylation site was introduced at Thr-478, Asp-589, and Gln-595 in the αIIb chain or at Ser-674 in the β3 chain. The resulting αIIbβ3 mutants T478N, D589N/H591T, Q595N/R597T, and S674N/K676T were expressed in CHO cells. Fbg binding to cells in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ and control antibody and in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ and PT25-2 is shown as the solid column and hatched columns, respectively. Fbg binding in the presence of 1 mm Mn²⁺ with control antibody is shown as the gray column. MFI, mean fluorescent intensity. Error bars indicate S.E.

FIGURE 4.

Constitutive activation depends on the binding of N-glycan in the Q595N/R597T mutant. Three amino acid residues starting from Gln-595 in the αIIb chain were mutated to various sequences. Fbg binding to the cells in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ with a control antibody (solid column) or with PT25-2 (hatched column) and in the presence of 1 mm Mn²⁺ with a control antibody (gray column) was examined. MFI, mean fluorescent intensity. Error bars indicate S.E.

FIGURE 5.

Swing-out of the hybrid domain is required for integrin activation. A, crystal structure of the αVβ3 head domains derived from the bent conformation complexed with RGD peptide is overlaid on the crystal structure of the αIIbβ3 head domains complexed with the ligand, which is shown as a semitransparent ribbon. In both parts of this graphic, the β-propeller domain in the α-chain is shown as a blue ribbon. The βA and the hybrid domains in the β3 chain are shown as red and orange ribbons, respectively. Note that Asp-319 in αIIb (Asp-306 in αV), shown as cyan spacefill, and Val-359 in β3, shown as magenta spacefill, are close to each other in the closed head (as in αVβ3) but are separated in the open head (as in αIIbβ3). B, to investigate the role of the swing movement of the hybrid domain in integrin activation, Asp-319 in αIIb and/or Val-359 in β3 were mutated to Cys to facilitate disulfide bridge formation between the two residues. Fbg binding to cells in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ is shown. The solid column represents binding in the presence of control antibody, and the hatched column represents binding in the presence of PT25-2. The open column represents binding to cells pretreated with DTT. MFI, mean fluorescent intensity. Error bars indicate S.E.

FIGURE 6.

mAb binding to cells expressing αIIbβ3 constrained in the bent conformation or in the closed head. The binding of the ligand-mimetic mAb OP-G2 (A and B), anti-LIBS mAbs anti-LIBS2 (C and D), and anti-LIBS6 (E and F) to cells expressing αIIbβ3 constrained in the bent conformation (A, C, and E) or in the closed head (B, D, and F) was examined. Values in the y axis represent normalized mAb binding. The open column represents mAb binding in the presence of 1 mm Ca²⁺/1 mm Mg²⁺. The hatched column represents binding in the presence of 1 mm GRGDS peptide under the same cation conditions. Error bars indicate S.E.

FIGURE 7.

Swing-out of the hybrid domain is required for activation induced by integrin extension. The effect of the swing-out of the hybrid domain on extension-induced integrin activation was examined. To constrain αIIbβ3 in the extended conformation with a closed head, the Q595N/R597T and D319C/V359C mutations were combined (595-319/359). Fbg binding to the cells in the presence of 1 mm Ca²⁺/1 mm Mg²⁺ and the control antibody (solid column) or PT25-2 (hatched column) and Fbg binding to cells pretreated with DTT (open column) is shown. MFI, mean fluorescent intensity. Error bars indicate S.E.