Lengyel Candice S E, Willis Lindsey J, Mann Patrick, Baker David, Kortemme Tanja, Strong Roland K, McFarland Benjamin J
Department of Chemistry and Biochemistry, Seattle Pacific University, Seattle, Washington 98119, USA.
J Biol Chem. 2007 Oct 19;282(42):30658-66. doi: 10.1074/jbc.M704513200. Epub 2007 Aug 8.
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.
MICA是一种主要组织相容性复合体样蛋白，在与免疫受体NKG2D结合时会经历从无序到有序的结构转变。我们使用RosettaDesign重新设计了MICA的无序区域，以增强与NKG2D的结合。预期稳定该区域的突变会增加结合动力学而不改变解离动力学，增加相互作用亲和力，并减少结合时的熵损失。MICA突变体在溶液中稳定，并且适合通过表面等离子体共振评估NKG2D的结合动力学和热力学。几个MICA突变体以增强的亲和力结合NKG2D，主要在结合过程中观察到动力学变化，并且熵的热力学变化符合预期。然而，预测可稳定受体结合的MICA构象的15种突变组合中，没有一种增强了NKG2D亲和力，而预测为不稳定的所有10种突变体均以增加的结合速率结合NKG2D。其中5种通过一到三个非接触性取代，其亲和力比野生型提高了0.9 - 1.8千卡/摩尔。因此，在这种情况下，设计用于轻微破坏蛋白质稳定性的突变增强了结合和亲和力。
