title: 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.
authors.affiliation: Center for Blood Research, Departments of Pathology, Pediatrics, and Anesthesia, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Motomu
authors.initials: M
authors.lastname: Shimaoka
authors.forename: Chafen
authors.initials: C
authors.lastname: Lu
authors.forename: Azucena
authors.initials: A
authors.lastname: Salas
authors.forename: Tsan
authors.initials: T
authors.lastname: Xiao
authors.forename: Junichi
authors.initials: J
authors.lastname: Takagi
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Springer
mesh_terms: D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D016177:Macrophage-1 Antigen / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D004220:Disulfides; D016177:Macrophage-1 Antigen; D004229:Dithiothreitol