Mutations designed to destabilize the receptor-bound conformation increase MICA-NKG2D association rate and affinity
Mutations designed to destabilize the receptor-bound conformation increase MICA-NKG2D association rate and affinity
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DOI：
10.1074/jbc.m704513200
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发表时间：
2007-10-19
影响因子：
4.8
通讯作者：
McFarland, Benjamin J.
中科院分区：
文献类型：
--
作者：
Lengyel, Candice S. E.;Willis, Lindsey J.;McFarland, Benjamin J.
云母是一种主要组织相容性复合物样蛋白，在结合其免疫受体NKG 2D后经历从无序到有序的结构转变。我们用RosettaDesign重新设计了云母的无序区域以增加NKG 2D结合。预期稳定该区域的突变增加缔合动力学而不改变解离动力学，增加相互作用的亲和力，并减少结合时的熵损失。云母突变体在溶液中是稳定的，并且它们适合于NKG 2D结合动力学和热力学的表面等离子体共振评价。几种云母突变体以增强的亲和力结合NKG 2D，在缔合过程中主要观察到动力学变化，熵的热力学变化与预期一致。然而，预测稳定受体结合云母构象的15种突变组合均未增强NKG 2D亲和力，而预测不稳定的所有10种突变体均以增加的结合率结合NKG 2D。其中5个通过1 - 3个非接触取代使亲和力比野生型提高0.9-1.8 kcal/ mol。因此，在这种情况下，设计为轻度破坏蛋白质稳定性的突变增强了缔合和亲和力。
MICA is a major histocompatibility complex-like protein that undergoes a structural transition from disorder to order upon binding its immunoreceptor, NKG2D. We redesigned the disordered region of MICA with RosettaDesign to increase NKG2D binding. Mutations that stabilize this region were expected to increase association kinetics without changing dissociation kinetics, increase affinity of interaction, and reduce entropy loss upon binding. MICA mutants were stable in solution, and they were amenable to surface plasmon resonance evaluation of NKG2D binding kinetics and thermodynamics. Several MICA mutants bound NKG2D with enhanced affinity, kinetic changes were primarily observed during association, and thermodynamic changes in entropy were as expected. However, none of the 15 combinations of mutations predicted to stabilize the receptor-bound MICA conformation enhanced NKG2D affinity, whereas all 10 mutants predicted to be destabilized bound NKG2D with increased on-rates. Five of these had affinities enhanced by 0.9-1.8 kcal/ mol over wild type by one to three non-contacting substitutions. Therefore, in this case, mutations designed to mildly destabilize a protein enhanced association and affinity.