Engineered allosteric mutants of the integrin αMβ2 I domain:: structural and functional studies
Engineered allosteric mutants of the integrin αMβ2 I domain:: structural and functional studies
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DOI：
10.1042/bj20021273
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发表时间：
2003-05-15
影响因子：
4.1
通讯作者：
Liddington, RC
中科院分区：
文献类型：
--
作者：
McCleverty, CJ;Liddington, RC
在白细胞整联蛋白如α M β 2和α L β 2的α-亚基中发现的α-I结构域在开放和闭合三级构象之间切换，反映了调节细胞粘附和迁移所需的整联蛋白的高亲和力和低亲和力配体结合状态。在本研究中，我们表明，通过使用点突变和工程二硫键，配体亲和力可以减少或增加变构通过改变封闭和开放状态之间的平衡。我们确定了两个配体，纤维蛋白原和细胞间细胞粘附分子1，通过表面等离子体共振的α M-I结构域的结合的平衡常数，并确定了低亲和力突变体的晶体结构。将结构域锁定在开放构象中使亲和力增加不大于10倍，这与在不存在配体的情况下两种构象之间的紧密平衡的平衡一致。这种行为与unliganded alphaL-1结构域的行为形成对比，后者的平衡强烈地有利于闭合构象。这些结果表明显着差异的方式的父母整合素调节I结构域的构象，因此配体亲和力。
The alpha-I domain, found in the a-subunit of the leucocyte integrins such as alphaMbeta2 and alphaLbeta2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the alphaM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded alphaL-1 domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity.