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首页> 外文期刊>Biochemistry >The IQGAP1 N-Terminus Forms Dimers, and the Dimer Interface Is Required for Binding F-Actin and Calcium-Bound Calmodulin
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The IQGAP1 N-Terminus Forms Dimers, and the Dimer Interface Is Required for Binding F-Actin and Calcium-Bound Calmodulin

机译：IQGAP1 N 末端形成二聚体，二聚体界面是结合 F-肌动蛋白和钙结合钙调蛋白所必需的

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摘要

IQGAP1 is a multidomain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) that contains the entire calponin homology,domain. The structure of the CHDF is very similar to those of other type 3 calponin homology domains like those from calponin, Vav, and the yeast IQGAP1 ortholog Rng2. However, in the crystal, two CHDF molecules form a "head-to-head" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca2+/calmodulin, but binding is mutually exclusive. On the basis of the structure, two dimer interface substitutions were introduced. While CHDFL157D disrupts the dimer in gel filtration experiments, oxidized CHDFK161C stabilizes the dimer. These results imply that the CHDF forms the same dimer in solution that is seen in the crystal structure. The disulfide-stabilized dimer displays a reduced level of F-actin binding in sedimentation assays and shows no binding to Ca2+/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues (KK94)-K-93 are important for CaM binding, and indeed, the (EE94)-E-93 double mutation displays a reduced level of binding to Ca2+/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca2+/calmodulin binding, and the (KK94)-K-93 pair, near the interface, is also used for Ca2+/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.
机译：IQGAP1 是一种参与许多细胞过程的多结构域支架蛋白。我们已经确定了跨越残基 1-191 的 N 端片段（以下简称 CHDF）的晶体结构，该片段包含整个钙调蛋白同源结构域。CHDF 的结构与其他 3 型钙调蛋白同源结构域的结构非常相似，例如来自钙调蛋白、Vav 和酵母 IQGAP1 直系同源物 Rng2 的结构。然而，在晶体中，两个CHDF分子通过疏水相互作用形成“头对头”或平行二聚体。结合实验表明，CHDF与F-肌动蛋白和Ca2+/钙调蛋白结合，但结合是相互排斥的。在结构的基础上，引入了两种二聚体界面取代。虽然CHDFL157D在凝胶过滤实验中会破坏二聚体，但氧化CHDFK161C稳定二聚体。这些结果表明，CHDF在溶液中形成的二聚体与晶体结构中的二聚体相同。二硫键稳定的二聚体在沉降测定中显示出 F-肌动蛋白结合水平降低，并且在等温滴定量热法 （ITC） 实验中未显示与 Ca2+/钙调蛋白的结合，表明界面残基用于两种结合事件。钙调蛋白靶标数据库预测残基 （KK94）-K-93 对 CaM 结合很重要，事实上，在 ITC 实验中，（EE94）-E-93 双突变显示出与 Ca2+/钙调蛋白结合水平降低。我们的结果表明，CHDF二聚体界面用于F-肌动蛋白和Ca2+/钙调蛋白结合，界面附近的（KK94）-K-93对也用于Ca2+/钙调蛋白结合。这些结果也与形成平行同源二聚体的全长 IQGAP1 一致。

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