Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA; Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA; Yale Cancer Center, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Structure. 2018 Nov 6;26(11):1451-1461.e4. doi: 10.1016/j.str.2018.07.015. Epub 2018 Aug 30.
The pseudoGTPases are a rapidly growing and important group of pseudoenzymes. p190RhoGAP proteins are critical regulators of Rho signaling and contain two previously identified pseudoGTPase domains. Here we report that p190RhoGAP proteins contain a third pseudoGTPase domain, termed N-GTPase. We find that GTP constitutively purifies with the N-GTPase domain, and a 2.8-Å crystal structure of p190RhoGAP-A co-purified with GTP reveals an unusual GTP-Mg binding pocket. Six inserts in N-GTPase indicate perturbed catalytic activity and inability to bind to canonical GTPase activating proteins, guanine nucleotide exchange factors, and effector proteins. Biochemical analysis shows that N-GTPase does not detectably hydrolyze GTP, and exchanges nucleotide only under harsh Mg chelation. Furthermore, mutational analysis shows that GTP and Mg binding stabilizes the domain. Therefore, our results support that N-GTPase is a nucleotide binding, non-hydrolyzing, pseudoGTPase domain that may act as a protein-protein interaction domain. Thus, unique among known proteins, p190RhoGAPs contain three pseudoGTPase domains.
假 GTP 酶是一类快速增长且重要的假酶。p190RhoGAP 蛋白是 Rho 信号的关键调节因子，包含两个先前鉴定的假 GTP 酶结构域。在这里，我们报告 p190RhoGAP 蛋白包含第三个假 GTP 酶结构域，称为 N-GTPase。我们发现 GTP 与 N-GTPase 结构域持续共纯化，并且与 GTP 共纯化的 p190RhoGAP-A 的 2.8Å 晶体结构揭示了一个不寻常的 GTP-Mg 结合口袋。N-GTPase 中的六个插入片段表明其催化活性受到干扰，无法与典型的 GTP 酶激活蛋白、鸟嘌呤核苷酸交换因子和效应蛋白结合。生化分析表明，N-GTPase 不能明显水解 GTP，并且仅在苛刻的 Mg 螯合条件下才能进行核苷酸交换。此外，突变分析表明，GTP 和 Mg 结合稳定了该结构域。因此，我们的结果支持 N-GTPase 是一个具有核苷酸结合和非水解功能的假 GTP 酶结构域，可能充当蛋白-蛋白相互作用结构域。因此，在已知的蛋白中，p190RhoGAP 是唯一包含三个假 GTP 酶结构域的蛋白。
