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, $K_d$, 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 是一种小型 GTP 酶和膜结合信号蛋白。新合成的 KRas 经过膜锚定异戊二烯基团的翻译后修饰。 KRas 伴侣分子由于参与将致癌 KRas 运输到细胞膜而成为癌症的治疗靶点。 SmgGDS 剪接变体是小 GTP 酶的伴侣，其高变域 (HVR) 中带有碱性残基，包括 KRas。 SmgGDS-607 护送预异戊烯化小 GTP 酶，而 SmgGDS-558 护送异戊烯化小 GTP 酶。我们提供了法尼基化和完全加工的 KRas (KRas-FMe) 与 SmgGDS-558 复合物的结构描述，并定义了这种相互作用的生物物理特性。仿生模型膜上的表面等离子体共振测量量化了 SmgGDS 与 KRas 相互作用的热力学，并使用小角 X 射线散射来表征 SmgGDS-558 和 KRas-FMe 复合物的结构。使用蒙特卡罗和分子动力学模拟对结构模型进行了改进。我们的结果表明，SmgGDS-558 与 HVR 和 KRas-FMe 的法呢基化 C 末端相互作用，但不与其 G 结构域相互作用。因此，SmgGDS-558 与异戊二烯化 KRas 的相互作用与异戊二烯化 RhoA 的相互作用不同，在最近的晶体结构中发现 RhoA 的 G 结构域与 SmgGDS-558 紧密接触。使用免疫沉淀测定，我们表明 SmgGDS-558 结合细胞中法尼基化和完全加工的 KRas 的 GTP 结合、GDP 结合和无核苷酸形式，这与 SmgGDS-558 不结合 KRas 的 G 结构域一致。 我们发现解离常数，${\mathrm{<span\; data-immersive-translate-walked="40cdd1a7-c74c-45c9-bd29-5fc5afefb0cc">K</span>}}_{\mathrm{<span\; data-immersive-translate-walked="40cdd1a7-c74c-45c9-bd29-5fc5afefb0cc">d</span>}}$ ，因为 KRas-FMe 与 SmgGDS-558 的结合与 KRas 与 PDEδ 的复合物相当，PDEδ 是一种充分表征的 KRas 分子伴侣，也不与 KRas G 结构域相互作用。这些结果表明 KRas 以类似的方式与两个伴侣 SmgGDS-558 和 PDEδ 相互作用。在 KRas 驱动的癌症中，SmgGDS-558/KRas 复合物的治疗靶向可能与靶向 PDEδ/KRas 复合物一样有用。