Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity
Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity
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DOI：
10.1073/pnas.95.18.10437
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发表时间：
1998-09-01
影响因子：
11.1
通讯作者：
Holt, DA
中科院分区：
文献类型：
--
作者：
Clackson, T;Yang, W;Holt, DA
FKBP配体同二聚体可用于激活细胞和动物内的信号传导事件，所述细胞和动物已被工程化以表达适当的信号传导结构域与FKBP之间的融合。然而，这些二聚体在体内的使用可能受到与内源性FKBP结合的配体的限制。我们已经设计了通过重塑FKBP-配体界面以引入特异性结合口袋而与突变的FKBP特异性结合超过野生型蛋白的配体。带有乙基取代基代替羰基的化合物对具有苯丙氨酸残基的补偿性截短的突变体FKBP表现出亚纳摩尔亲和力和1，000倍选择性。新口袋的结构和功能分析显示，识别令人惊讶地放松，修饰的配体仅部分填充工程化空腔。我们将特异性口袋掺入含有FKBP和Fas受体的胞内结构域的融合蛋白中，表达这种修饰的嵌合蛋白的细胞响应于AP 1903（修饰的配体的同源二聚体）而有效地经历凋亡，无论是在培养中还是植入小鼠体内。重塑的二聚体如AP 1903是用于控制已通过基因治疗程序修饰的细胞活性的理想试剂，而不受内源性FKBP的干扰。
FKBP ligand homadimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP, We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity, We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor, Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.