Search

Conformational movement of the C-terminal [alpha]7 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 [alpha]7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with C[beta] 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 [alpha]M[beta]2 heterodimers and isolated [alpha]M inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive [alpha[M[beta]2 that was resistant to activation but became activatable after dithiothreitol treatment. cell adhesion | protein design | CD11b | Mac-1 | VWA domain

We have used immunoprecipitation with mAbs to probe folding during biosynthesis of the [[Beta].sub.2] integrin subunit of lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) before and after association with the [[Alpha].sub.L] sub-unit. An evolutionarily conserved region is present in the [[Beta].sub.2] subunit between amino acid residues 102 and 344.mAbs to one subregion before the conserved region, and two subregions after the conserved domain, immunoprecipitated both the unassociated [[Beta][prime].sub.2] precursor and mature [[Alpha].sub.L]/[[Beta].sub.2] complex, suggesting portions of these subregions are folded before association with [[Alpha].sub.L]. An activating mAb to the C-terminal cysteine-rich region, KIM127, preferentially bound to the unassociated [Beta] subunit, suggesting that it may bind to an epitope that is in an [Alpha][Beta] interface in unactivated LFA-1. By contrast, mAbs to five different epitopes in the conserved region did not react with unassociated [[Beta][prime].sub.2] precursor, suggesting that this region folds after [[Alpha].sub.L] association and is intimately associated with the [[Alpha].sub.L] subunit in the [[Alpha].sub.L]/[[Beta].sub.2] complex. mAbs to two different epitopes that involve the border between the conserved region and the C-terminal segment, were fully or partially reactive with the [[Beta][prime].sub.2] precursor, suggesting that this region is partially folded before association with [[Alpha].sub.L]. The findings suggest that the conserved region is a distinct folding and hence structural unit, and is intimately associated with the [Alpha] subunit.

The integrin [Alpha]L[Beta]32 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the [Alpha]M and [Alpha]2 subunits has been crystallized in both open and closed conformations; however, the [Alpha]L I domain has been crystallized in only the closed conformation. We hypothesized that the [Alpha]L domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the [Alpha]L I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on [Mg.sup.2+]. Orders of affinity were ICAM-1 [is greater than] ICAM-2 [is greater than] ICAM-3. The [k.sub.on], [k.sub.off], and [K.sub.D ] values for the locked open I domain were within 1.5-fold of values previously determined for the [Alpha]L[Beta]2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized [Alpha]L[Beta]32-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

We used integrin [Alpha]L[Beta]2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins, mAbs to the [Alpha]L I domain and [Beta]2 I-like domain inhibit adhesion of wild-type [Alpha]L[Beta]2 to intercellular adhesion molecule-1. However, with [Alpha]L[Beta]2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, [Alpha]L[Beta]2 containing a locked open I domain was completely resistant to inhibition by mAbs to the [Beta]2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type [Alpha]L[Beta]2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by [Mn.sup.2+], as measured with mAb m24, which we map here to the [Beta]2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or [Mn.sup.2+]-induced exposure of the KIM127 epitope in the [Beta]2 stalk region. Furthermore, locked open I domains, in [Alpha]L[Beta]2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in [Mg.sup.2+] and [Mn.sup.2+]. These results suggest that [Mn.sup.2+] activates [Alpha]L[Beta]2 by binding to a site other than the I domain, most likely the I-like domain of [Beta]2.

We introduced disulfide bonds to lock the integrin [Alpha]L[Beta]2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing [Alpha]L[Beta]2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated [Alpha]L[Beta]2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal [Alpha]-helix of the I domain, inhibited binding to ICAM-1 by [Alpha]L[Beta]2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the [Alpha]L I domain for adhesive function and show that this domain is sufficient for full adhesive activity.