From the Departments of Chemical and Biological Engineering and.
the Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, NCI-Frederick, Frederick, Maryland 21702.
J Biol Chem. 2018 Mar 9;293(10):3685-3699. doi: 10.1074/jbc.RA117.001596. Epub 2018 Jan 22.
IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.
IQ 基序包含的 GTP 酶激活蛋白（IQGAPs）是细胞黏附、极性和运动的支架蛋白，发挥着核心作用。Rho GTP 酶 Cdc42 和 Rac1 以其结合 GTP 的活性形式与所有三种人类 IQGAP 相互作用。IQGAP-Cdc42 相互作用通过增强肌动蛋白聚合促进转移。然而，尽管它们具有很高的序列同一性，但 Cdc42 和 Rac1 在与 IQGAP 的相互作用上存在差异。两个 Cdc42 分子可以结合到 IQGAP 的 GTP 酶激活蛋白（GAP）相关域（GRD）的 Ex 结构域和 RasGAP 位点，并促进 IQGAP 二聚化。只有一个 Rac1 分子可能结合到 GRD 的 RasGAP 位点，并且可能不会促进二聚化，并且 Cdc42 和 Rac1 与 IQGAP 结合的确切机制尚不清楚。使用全原子分子动力学模拟、定点突变和 Western blot，我们揭示了 Cdc42 和 Rac1 与 IQGAP2 相互作用的详细机制。我们观察到 Cdc42 结合到 IQGAP2 的 GRD 的 Ex 结构域（GRD2）释放了 GRD2 的 C 端区域的 Ex 结构域，促进了 IQGAP2 的二聚化。Cdc42 结合到 Ex 结构域促进了 RasGAP 位点的变构变化，为 RasGAP 位点的第二个 Cdc42 提供了结合位点。值得注意的是，Cdc42 的“插入环”对于第一个 Cdc42 与 Ex 结构域的相互作用很重要。相比之下，Rac1 插入环序列和结构的差异使其无法与 Ex 结构域相互作用。Rac1 只能结合到 apo-GRD2 的 RasGAP 位点，并且不能促进 IQGAP2 二聚化。我们详细的机制见解有助于解释 Cdc42 如何刺激转移中的肌动蛋白聚合。
