Stabilizing the integrin alpha M inserted domain in alternative conformations with a range of engineered disulfide bonds

Abstract

Conformational movement of the C-terminal alpha7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an alpha/beta Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the alpha7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cbeta atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation. We also find more success with substituting partially exposed than buried residues. Disulfides stabilizing the open conformation resulted in constitutively active alphaMbeta2 heterodimers and isolated alphaM inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive alphaMbeta2 that was resistant to activation but became activatable after dithiothreitol treatment.

Fig 1.

Spatial relationships of residues substituted for cysteine in the open and closed conformations of the αM I domain. Superimposed structures of open (PDB ID code , ref. 4) and closed (PDB ID code , ref. 5) αM I domains are shown as ribbon diagrams. Backbone regions where conformational changes are significant are shown in yellow (open) and blue (closed). Other backbone regions are gray. MIDAS metal ions are shown as spheres (open, yellow; closed, blue). Side chains of the residues mutated to cysteines are in orange (open) and dark blue (closed). Cβ–Cβ distances are shown as dashed lines in orange (open) and blue (closed). The structures were superimposed by using residues 132–141, 166–206, 211–241, 246–270, and 287–294.

Fig 2.

Ligand binding and expression of activation epitopes by αMβ2 cysteine-substitution mutants. (A) Binding of 293T transient transfectants to immobilized iC3b. Mock, transfected with empty vector. (B) Binding of K562 stable transfectants to immobilized iC3b. Binding of the transfectants was determined in L15/FBS that contained Mg²⁺ and Ca²⁺ in the presence of 10 μg/ml of the activating mAb CBR LFA-1/2, control nonbinding IgG X63, or 10 mM DTT as indicated. (C) Expression of CBRM1/5 activation epitope by K562 transfectants. Binding of the CBRM1/5 mAb was determined by flow cytometry as specific mean fluorescence intensity and is expressed as a percentage of wild type. All results are mean ± SEM of three independent experiments with duplicate samples.

Fig 3.

Ligand binding and expression of activation epitope by 293T transfectants expressing mutant αMβ2. (A) Binding of transfectants was determined in L15/FBS medium that contains Mg²⁺ and Ca²⁺ in the presence of 10 μg/ml of the activating mAb CBR LFA-1/2 or control nonbinding IgG X63, and in the presence or absence of 10 mM DTT as indicated. (B) Expression of the CBRM1/5 activation epitope. Binding of CBRM1/5 mAb was determined by flow cytometry. Cells were stained with phycoerythrin-labeled CBRM1/5 mAb or phycoerythrin-labeled control IgG at 37°C in the presence of CBR LFA-1/2 mAb (+) or X63 control IgG1 (−), and in the presence or absence of 10 mM DTT as indicated. Values are specific mean fluorescence intensity and are expressed as a percentage of wild type (WT). Results are mean ± SEM of three independent experiments with duplicate samples.

Fig 4.

Ligand binding by the isolated cell surface αM I domains. (A) Effects of inhibitory mAb and disulfide reduction by DTT. Binding of K562 stable transfectants expressing mutant or wild-type isolated I domains was examined in L15/FBS containing Mg²⁺ and Ca²⁺ in the presence of the inhibitory I domain mAb CBRM1/5 (10 μg/ml), the control nonbinding IgG X63 (control), or 10 mM DTT. (B) Effect of divalent cations. Binding was performed in Hepes/NaCl/glucose supplemented with 1 mM MgCl₂, 1 mM MnCl₂, or 2 mM EDTA. All results are mean ± SEM of three independent experiments with duplicate samples.