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首页> 外文期刊>Journal of Molecular Biology >The Disintegrin Echistatin Stabilizes Integrin alphaIIbbeta3's Open Conformation and Promotes Its Oligomerization.
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The Disintegrin Echistatin Stabilizes Integrin alphaIIbbeta3's Open Conformation and Promotes Its Oligomerization.

机译：Disintegrin Echistatin可稳定整联蛋白alphaIIbbeta3的开放构型并促进其低聚。

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We have employed echistatin, a 5.4kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting alphaIIbbeta3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human alphaIIbbeta3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward beta3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of alphaIIbbeta3 function. Electron microscopy of rotary shadowed specimens yielded a variety of alphaIIbbeta3 conformers ranging from compact, spherical particles (maximum dimension 22nm) to the classical "head with two tails" forms (32nm). The population of larger particles (42-56nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed alphaIIbbeta3's solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected alphaIIbbeta3 from thermal aggregation, raising its transition mid-point from 46 degrees C to 69 degrees C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial alphaIIbbeta3 dimerization. van't Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to alphaIIbbeta3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate alphaIIbbeta3's structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.
机译：我们已使用echistatin（一种5.4kDa蛇毒双整合素）作为模型蛋白来研究悖论，即小的配体模拟物可以与静止的alphaIIbbeta3整合素结合，而粘附的大分子则不能。我们表征了纯化的人alphaIIbbeta3和两个重组echistatin变体之间的相互作用：rEch（1-49）M28L，其针对beta3整合素的选择性而选择，以及rEch（1-40）M28L，羧基末端截短突变体。虽然两者都包含RGD整合素靶向序列，但只有rEch（1-49）M28L是alphaIIbbeta3功能的有效抑制剂。旋转阴影标本的电子显微镜检查产生了各种alphaIIbbeta3构象异构体，从紧凑的球形颗粒（最大尺寸为22nm）到经典的“有两个尾巴的头”形式（32nm）不等。在存在rEch（1-49）M28L的情况下，较大颗粒（42-56nm）的数量从17％增加到28％，表明配体诱导了寡聚。沉积速度测量表明全长和截短的echistatin都扰乱了alphaIIbbeta3的溶液结构，产生了沉降较慢的开放构象异构体。动态光散射表明，rEch（1-49）M28L保护了alphaIIbbeta3免于热聚集，将其转变中点从46摄氏度提高至69摄氏度; rEch（1-40）M28L产生的位移较小。沉积平衡表明，两个echistatin配体诱导实质性alphaIIbbeta3二聚化。范霍夫（van't Hoff）分析揭示了一种类似于替罗非班的熵/焓补偿模式，替罗非班是一种小型RGD配体模拟物，与alphaIIbbeta3紧密结合，但产生的构象扰动比棘皮抑素小。我们建议echistatin可以作为理解多域粘附大分子的范式，因为它调节αIIbbeta3结构的能力在于RGD环上，而完整的整联蛋白活性则需要一个包括羧基末端的9个氨基酸残基的辅助位点。

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