Abstract
The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.
Highlights
Integrins are cell adhesion molecules that transmit bidirectional signals across the plasma membrane [1,2].Divalent metal ions regulate integrin ligand binding and signaling
Design of Mutant αIIbβ3 Integrins While studying the variation in crystal structures between the liganded and unliganded αIIbβ3 [16,17] and the unliganded αXβ2 [47], we proposed that the β3 Ala252 might contribute significantly to the difference in ligand binding and adhesion among different integrins [25,31]
While this Ala252 residue does not appear to interact with metal ion-dependent adhesion site (MIDAS) Mg2+ ion, the corresponding residue Asp243 in the αXβ2 integrin [47] does appear to contribute negative charge to the MIDAS that could have an effect on the ligand binding ability
Summary
Integrins are cell adhesion molecules that transmit bidirectional signals across the plasma membrane [1,2].Divalent metal ions regulate integrin ligand binding and signaling. Integrin inside-out activation causes the β3 hybrid domain to swing out, followed by the downward displacement of the β3 I domain α7 helix and breaking the bond between Met335 and the ADMIDAS ion [16,21,26,27,28,29] Following these conformational changes, the metal ion-binding sites change conformation, the Asp251 side chain carboxyl moves toward the ADMIDAS site and coordinates with its Ca2+ ion. The metal ion-binding sites change conformation, the Asp251 side chain carboxyl moves toward the ADMIDAS site and coordinates with its Ca2+ ion Because these residue shifts, the αIIbβ integrin is in the high affinity open headpiece conformation [16], making the MIDAS become more positive and able to bind ligands with higher affinity [17]. The ADMIDAS ion stabilizes the low affinity closed headpiece conformation and stabilizes the high affinity state with the open headpiece conformation, which has been confirmed by mutagenesis studies [17,25]
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