Michalak Dennis J, Unger Bethany, Lorimer Ellen, Grishaev Alexander, Williams Carol L, Heinrich Frank, Lösche Mathias
Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania.
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin.
Biophys J. 2022 Oct 4;121(19):3684-3697. doi: 10.1016/j.bpj.2022.05.028. Epub 2022 May 25.
KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, K, for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers.
KRas是一种小GTP酶和膜结合信号蛋白。新合成的KRas在翻译后会被一个膜锚定的异戊二烯基团修饰。KRas伴侣蛋白因其参与将致癌性KRas转运至细胞膜而成为癌症治疗靶点。SmgGDS剪接变体是小GTP酶的伴侣蛋白，其高变区（HVR）含有碱性残基，包括KRas。SmgGDS - 607护送前法尼基化的小GTP酶，而SmgGDS - 558护送法尼基化的小GTP酶。我们提供了与SmgGDS - 558结合的法尼基化且完全加工的KRas（KRas - FMe）的结构描述，并定义了这种相互作用的生物物理特性。在仿生模型膜上进行表面等离子体共振测量，量化了SmgGDS与KRas相互作用的热力学，并用小角X射线散射对SmgGDS - 558和KRas - FMe的复合物进行结构表征。使用蒙特卡罗和分子动力学模拟对结构模型进行优化。我们的结果表明，SmgGDS - 558与KRas - FMe的HVR和法尼基化的C末端相互作用，但不与它的G结构域相互作用。因此，SmgGDS - 558与法尼基化的KRas的相互作用方式不同于法尼基化的RhoA，在最近的晶体结构中发现RhoA的G结构域与SmgGDS - 558紧密接触。通过免疫沉淀试验，我们表明SmgGDS - 558在细胞中能结合法尼基化且完全加工的KRas的GTP结合形式、GDP结合形式和无核苷酸形式，这与SmgGDS - 558不与KRas的G结构域结合一致。我们发现KRas - FMe与SmgGDS - 558结合的解离常数K与KRas和PDEδ复合物的解离常数相当，PDEδ是一种已被充分表征的KRas伴侣蛋白，它也不与KRas的G结构域相互作用。这些结果表明，KRas与两种伴侣蛋白SmgGDS - 558和PDEδ的相互作用方式相似。在KRas驱动的癌症中，针对SmgGDS - 558/KRas复合物进行治疗靶向可能与针对PDEδ/KRas复合物进行治疗靶向一样有用。
