(α/β+α)-Peptide antagonists of BH3 Domain/Bcl-xL recognition:: Toward general strategies for foldamer-based inhibition of protein-protein interactions
(α/β+α)-Peptide antagonists of BH3 Domain/Bcl-xL recognition:: Toward general strategies for foldamer-based inhibition of protein-protein interactions
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DOI：
10.1021/ja0662523
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发表时间：
2007-01-10
影响因子：
15
通讯作者：
Gellman, Samuel H.
中科院分区：
文献类型：
--
作者：
Sadowsky, Jack D.;Douglas Fairlie, W.;Gellman, Samuel H.
结合到特定蛋白质表面位点并抑制蛋白质-蛋白质相互作用的分子的开发是分子识别中的基本挑战。应对这一挑战的新策略可能会对生物学和医学产生重要的长期影响。我们正在探索的概念，具有明确定义的构象的非天然低聚物（“折叠体”）可以模拟蛋白质二级结构元件，从而阻止特定的蛋白质-蛋白质相互作用。在这里，我们描述了识别和分析的螺旋肽为基础的折叠体，结合到一个特定的裂缝上的抗凋亡蛋白Bcl-x（L）通过模拟α-螺旋BH 3结构域。采用荧光偏振（FP）竞争测定的初步研究揭示，在几种α/β-和β-肽折叠体骨架中，仅旨在采用14/15-螺旋二级结构的α/β-肽显示与Bcl-x（L）的显著结合。最紧密结合的Bcl-x（L）配体是嵌合寡聚体，其中N-末端α/β-肽区段融合至C-末端α-肽区段（（α/β + α）-肽））。通过标准和非标准技术（分别为丙氨酸扫描和丙氨酸扫描）探测序列亲和力关系，结果使我们能够构建配体/Bcl-x（L）复合物的计算模型。在FP测定条件下，用高亲和力（α/β + α）-肽的分析超离心建立了1：1配体：Bcl-x（L）化学计量。通过表面等离子体共振测量进行的最有效的（α/β + α）-肽的结合选择性研究揭示，该配体与Bcl-w以及Bcl-x（L）紧密结合，而与Bcl-2的结合稍微较弱。未检测到与Mcl-1的结合。我们表明，我们最有效的（α/β + α）-肽可以诱导细胞裂解物中线粒体释放细胞色素C，这是细胞凋亡的早期步骤，并且这种活性依赖于对涉及Bcl-x（L）的蛋白质-蛋白质相互作用的抑制。
The development of molecules that bind to specific protein surface sites and inhibit protein-protein interactions is a fundamental challenge in molecular recognition. New strategies for approaching this challenge could have important long-term ramifications in biology and medicine. We are exploring the concept that unnatural oligomers with well-defined conformations ("foldamers") can mimic protein secondary structural elements and thereby block specific protein-protein interactions. Here, we describe the identification and analysis of helical peptide-based foldamers that bind to a specific cleft on the anti-apoptotic protein Bcl-x(L) by mimicking an alpha-helical BH3 domain. Initial studies, employing a fluorescence polarization (FP) competition assay, revealed that among several alpha/beta- and beta-peptide foldamer backbones only alpha/beta-peptides intended to adopt 14/15-helical secondary structure display significant binding to Bcl-x(L). The most tightly binding Bcl-x(L) ligands are chimeric oligomers in which an N-terminal alpha/beta-peptide segment is fused to a C-terminal alpha-peptide segment ((alpha/beta+alpha)-peptides)). Sequence-affinity relationships were probed via standard and nonstandard techniques (alanine scanning and hydrophile scanning, respectively), and the results allowed us to construct a computational model of the ligand/Bcl-x(L) complex. Analytical ultracentrifugation with a high-affinity (alpha/beta+alpha)-peptide established 1:1 ligand: Bcl-x(L) stoichiometry under FP assay conditions. Binding selectivity studies with the most potent (alpha/beta+alpha)-peptide, conducted via surface plasmon resonance measurements, revealed that this ligand binds tightly to Bcl-w as well as to Bcl-x(L), while binding to Bcl-2 is somewhat weaker. No binding could be detected with Mcl-1. We show that our most potent (alpha/beta+alpha)-peptide can induce cytochrome C release from mitochondria, an early step in apoptosis, in cell lysates, and that this activity is dependent upon inhibition of protein-protein interactions involving Bcl-x(L).