Integrin αIIbβ3:ligand interactions are linked to binding-site remodeling
Integrin αIIbβ3:ligand interactions are linked to binding-site remodeling
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DOI：
10.1110/ps.052049506
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发表时间：
2006-08-01
期刊：
影响因子：
8
通讯作者：
Medved, Leonid
中科院分区：
文献类型：
--
作者：
Hantgan, Roy R.;Stahle, Mary C.;Medved, Leonid
本研究测试了大分子配体与α IIb β 3整联蛋白的高亲和力结合与结合位点重塑紧密耦合的假设，这是一种诱导拟合过程，将构象平衡从静止向开放受体转移。aIIbb 3和两种模型配体--刺血菌素（一种具有RGD整合素靶向序列的6-kDa重组蛋白）和纤维蛋白原的γ模块（一种具有KQAGDV整合素结合位点的30-kDa重组蛋白）之间的相互作用通过沉降速度、荧光各向异性和固相测量结合测定，并通过分子图形建模。研究echistatin变体（R24 A，R24 K，D26 A，D26 E，D27 W，D27 F），我们发现在α IIb/β 3界面与带电残基的静电接触，而不是非极性接触，扰乱了静息整合素的构象。天冬氨酸26与附近的MIDAS阳离子相互作用，是结合所必需的，因为D26 A和D26 E是无活性的。相反，R24 K是完全活性的，R24 A是部分活性的，表明带正电荷的精氨酸24有助于整合素识别，但不是整合素识别所需的。此外，我们证明了引发，即，胞外域构象变化和通过在35 ° C下与配体模拟肽cHarGD-一起孵育诱导的寡聚化促进与纤维蛋白原的γ-模块的复合物形成。我们还观察到，γ-模块的灵活的羧基末端是不需要的α IIb β 3整合素结合。我们的研究区分启动配体，结合到静息受体和扰动其构象，从调节配体，结合位点重塑必须首先发生。Echistatin的结合能足以重新排列亚基界面，但受调节的配体如纤维蛋白原必须依赖于引发以克服构象障碍。
This study tested the hypothesis that high-affinity binding of macromolecular ligands to the alpha IIb beta 3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between aIIbb3 and two model ligands-echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's gamma-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site-were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the alpha IIb/beta 3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming-i.e., ectodomain conformational changes and oligomerization induced by incubation at 35 degrees C with the ligand-mimetic peptide cHarGD-promotes complex formation with fibrinogen's gamma-module. We also observed that the gamma-module's flexible carboxy terminus was not required for alpha IIb beta 3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.