期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2002
卷号：99
期号：26
页码：16737-16741
DOI：10.1073/pnas.252633099
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Conformational movement of the C-terminal 7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an /{beta} Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the 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 M{beta}2 heterodimers and isolated M inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive M{beta}2 that was resistant to activation but became activatable after dithiothreitol treatment.
