Liu Jing, Kurella Vinodh B, LeCour Louis, Vanagunas Tomas, Worthylake David K
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-New Orleans , 1901 Perdido Street, New Orleans, Louisiana 70112, United States.
Biochemistry. 2016 Nov 22;55(46):6433-6444. doi: 10.1021/acs.biochem.6b00745. Epub 2016 Nov 10.
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 Ca/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 Ca/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues KK are important for CaM binding, and indeed, the EE double mutation displays a reduced level of binding to Ca/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca/calmodulin binding, and the KK pair, near the interface, is also used for Ca/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.
IQGAP1是一种参与多种细胞过程的多结构域支架蛋白。我们已经确定了一个包含完整钙调蛋白同源结构域的N端片段（以下简称CHDF）跨越第1至191位残基的晶体结构。CHDF的结构与其他3型钙调蛋白同源结构域（如来自钙调蛋白、Vav和酵母IQGAP1直系同源物Rng2的结构域）非常相似。然而，在晶体中，两个CHDF分子通过主要的疏水相互作用形成“头对头”或平行二聚体。结合实验表明，CHDF与F-肌动蛋白和钙/钙调蛋白都结合，但结合是相互排斥的。基于该结构，引入了两个二聚体界面替代物。虽然在凝胶过滤实验中CHDFL157D破坏了二聚体，但氧化的CHDFK161C稳定了二聚体。这些结果表明，CHDF在溶液中形成的二聚体与晶体结构中所见的相同。二硫键稳定的二聚体在沉降试验中显示出F-肌动蛋白结合水平降低，并且在等温滴定量热法（ITC）实验中显示不与钙/钙调蛋白结合，表明界面残基用于这两种结合事件。钙调蛋白靶标数据库预测KK残基对钙调蛋白结合很重要，实际上，EE双突变在ITC实验中显示出与钙/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-肌动蛋白和钙/钙调蛋白结合，并且靠近界面的KK对也用于钙/钙调蛋白结合。这些结果也与全长IQGAP1形成平行同型二聚体一致。
