The α-subunit regulates stability of the metal ion at the ligand-associated metal ion-binding site in β3 integrins

Abstract

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β3 integrins αIIbβ3 and αVβ3, but a potential role of this interaction on stability of the metal ion at LIMBS in β3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbβ3 versus αVβ3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVβ3. Replacing the αIIb-LIMBS interface residue Phe(191) in αIIb (equivalent to Trp(179) in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbβ3; a Trp(179) to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbβ3 and a W179/F substitution in αVβ3 reduced and increased, respectively, the apparent affinity of Mn(2+) to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVβ3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

Keywords: Cell Adhesion; Cell Biology; Integrin; Molecular Dynamics; Signal Transduction.

FIGURE 1.

α-subunit residues facing LIMBS in β₃ integrins. A ribbon diagram showing the residues from α_IIb and α_V facing LIMBS loop residues Arg²¹⁶-Ala²¹⁸ is presented. The β₃ subunits of unliganded α_IIbβ₃ (green, 3fcs.pdb) and α_Vβ₃ (yellow, 4g1e.pdb) ectodomains were superposed with Chimera. The metal ion (M²⁺) at LIMBS (sphere) has the color of the respective integrin. α_IIb and α_V residues are labeled in green and black, respectively, and the LIMBS residues are labeled in red.

FIGURE 2.

α-subunit/LIMBS interaction energies and LIMBS conformations in β₃ integrins. A, computed energy of interaction between α_IIb and α_V subunits and LIMBS loop residues Arg²¹⁶-Ala²¹⁸. The two trajectories appear to equilibrate after around 8 ns of simulation. The transient peak seen afterward (at ∼16 ns) most likely represents a high energy local minimum state that the system occasionally takes, rather than simply a random fluctuation. B and C, snapshots of molecular dynamics simulations at t = 20 ns, showing structures of LIMBS in α_IIbβ₃ (B) and α_Vβ₃ (C), in the same orientation, with the LIMBS Ca²⁺ shown as a large sphere in each case. In α_IIbβ₃, the LIMBS metal ion contacts six primary oxygens (magnified red spheres) and two secondary oxygens (small red spheres) (B). In α_Vβ₃, the LIMBS metal ion also contacts six primary oxygens but only one secondary oxygen (C). Note that the side chain of Arg²¹⁶ is stretched out in C versus in B and that five primary oxygens in α_Vβ₃ (C) lie in one plane, in contrast to the octahedral arrangement of the primary oxygens in α_IIbβ₃ (B).

FIGURE 3.

α-subunit residues interacting with the LIMBS loop. A, snapshot of MD simulations at 20 ns showing interactions of the LIMBS loop Arg²¹⁶-Ala²¹⁸ residues (shown as ball and stick) with α_V subunit residues (shown as stick). The structures in A and C are shown in the same orientation after superposing LIMBS of each. LIMBS residues are labeled red in A and C (also in Figs. 4B and 5B). In α_V, the LIMBS loop interacts with the Phe¹⁵⁴, Tyr¹⁷⁸, Trp¹⁷⁹, Asp²¹⁹, Glu¹²¹, and Glu¹²³ of α_V. B, MD simulations showing van der Waals energy of interaction between Arg²¹⁶ of the LIMBS loop with Trp¹⁷⁹ and Phe¹⁵⁴ of α_V. C, snapshot of MD simulations at 20 ns showing interactions of the LIMBS loop Arg²¹⁶-Ala²¹⁸ residues with α_IIb subunit residues (shown as stick). In α_IIb, interactions are limited to the corresponding residues Tyr¹⁶⁶, Tyr¹⁹⁰, Phe¹⁹¹, and Asp²³² and a transient interaction with Glu¹²³. D, electrostatic energy of interaction between Arg²¹⁶ of the LIMBS loop and Glu¹²³ of α_IIb. Occasional jumps to higher energy levels represent ionic bonds between Arg²¹⁶ and Glu¹²³. The energy peaks at t = 7 and 16 ns show sharp increases to the same value of about 100 kcal/mol, suggesting that the ionic bond occurs at a local energy minimum that the system continues to take, whereas the Arg²¹⁶-Glu¹²³ bond spends most of the simulation time in a lower electrostatic energy state (i.e. longer bond distance).

FIGURE 4.

Effect of F191/W change in α_IIbβ₃ on interaction energies and shape of LIMBS. A, computed energy of interaction between LIMBS loop residues Arg²¹⁶-Ala²¹⁸ and Trp¹⁹¹ in α_IIb^F/W. B, snapshot at t = 20 ns of the interactions of LIMBS loop with α_IIb^F/W residues Tyr¹⁶⁶, Tyr¹⁹⁰, Trp¹⁹¹, Asp²³², and Glu¹²³. Mutating Phe¹⁹¹ to Trp in α_IIb^F/W enhanced interactions of the larger Trp¹⁹¹ side chain with the LIMBS loop, especially with Arg²¹⁶, modifying the conformation of the LIMBS pocket (see “Results”).

FIGURE 5.

Effect of W179/F in α_Vβ₃ on interaction energies and shape of LIMBS. A, computed energy of interaction between the LIMBS loop residues Arg²¹⁶-Ala²¹⁸ and Phe¹⁷⁹ in α_V^W/F. Mutating Trp¹⁷⁹ to Phe does not change the energy of interaction with the LIMBS loop significantly. B, snapshot at t = 20 ns of interactions of the LIMBS loop with α_V subunit residues Glu¹²¹, Glu¹²³, Phe¹⁵⁴, Phe¹⁷⁹, and Asp²¹⁹. The W179/F mutation weakens interaction of LIMBS loop with α_V^W/F, reshaping the LIMBS pocket. The structure is shown in the same orientation as in Fig. 4B.

FIGURE 6.

Effect of α_IIb F191/W mutation in α_IIbβ₃ on cell surface expression, activation, and binding to soluble ligand. A, histograms (mean ± S.D.; n = 3) comparing cell surface expression and heterodimer formation of α_IIbβ₃, α_IIb^F/Wβ₃, and constitutively active α_IIb^FF/AAβ₃, α_IIbβ₃^Δ-genu, α_IIb^F/W+FF/AAβ₃, and α_IIb^F/Wβ₃^Δ-genu. Constitutive activation reduced expression of the wild type and F191/W integrin to equivalent degrees. B, histograms (mean ± S.D.; n = 3) showing binding of the LIBS mAb AP5 to α_IIbβ₃ and to constitutively active α_IIb^FF/AAβ₃, α_IIb^F/W+FF/AAβ₃, α_IIbβ₃^Δ-genu, and α_IIb ^F/Wβ₃^Δ-genu. Binding was expressed as a percentage of binding of the heterodimer-specific mAb CD41-P2. C, histograms (mean ± S.D.; n = 3) showing binding of wild-type and constitutively active α_IIb^F/W+FF/AAβ₃ to saturating amounts of soluble Alexa Fluor 488-FB (Alex488-FB) in 1 mm Ca²⁺ plus 1 mm Mg²⁺ (Ca²⁺/Mg²⁺) or 1 mm Mn²⁺. Binding is expressed as a percentage of Alexa Fluor 647-AP3 mAb staining. F191/W did not significantly impair ligand binding to constitutively active α_IIbβ₃ in Mn²⁺. However, ligand binding to constitutively active α_IIbβ₃ in Mn²⁺ was abolished when the ligand contact residue Tyr¹⁸⁹ was simultaneously mutated to Ala.

FIGURE 7.

Effect of α_IIb^F/W, α_V^W/F, and β₃^D/N mutations on integrin-ligand interactions in the presence of varying concentrations of Mn²⁺. A and B, dose-response curves comparing binding of α_IIb^FF/AAβ₃ (A) and α_IIb^FF/AAβ₃^D/N and α_IIb^F/W+FF/AAβ₃ (B) with saturating amounts of soluble Alexa Fluor 488-FB (Alex488-FB) in the presence of increasing concentrations of Mn²⁺. C and D, dose-response curves comparing binding of cellular α_V^FF/AAβ₃ (C) and α_V^W/F+FF/AAβ₃ (D) with saturating amounts of soluble Alexa Fluor 488-FN10 in the presence of increasing concentrations of Mn²⁺. Binding was expressed as a percentage of Alexa Fluor 647-AP3 mAb binding.