Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry
Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry
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DOI：
10.1039/c9sc01032g
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发表时间：
2019-09-14
期刊：
影响因子：
8.4
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
--
作者：
Mehaffey, M. Rachel;Schardon, Christopher L.;Brodbelt, Jennifer S.
GTP酶K - Ras的突变，特别是在密码子G12处的突变，仍然是人类癌症中最常见的基因改变。调控下游信号通路激活的机制以及它们如何与突变的特性相关联仍未完全明确。在此，我们利用天然质谱（MS）结合紫外光解离（UVPD）来研究三种G12X突变（G12C、G12V、G12S）对K - Ras同源二聚化以及与下游效应蛋白Raf异源二聚化的影响。电喷雾电离（ESI）被用于将结合鸟苷5'-二磷酸（GDP）或GppNHp（GTP的不可水解类似物）的野生型（WT）或G12X K - Ras复合物转移到气相中。从ESI - MS光谱估计同源或异源二聚体复合物的相对丰度。用UVPD激活K - Ras + Raf异源复合物，以探究导致每种变体所形成异源复合物数量差异的结构变化。光解离产生的全（配体结合）碎片离子表明G12X突变体沿着预期的效应物结合区域（β界面）结合Raf，但也可能通过另一种α界面与Raf相互作用。利用每种变体在紫外光激活过程中主链裂解效率的变化，将突变特性与可能影响下游信号传导的结构变化相关联。具体而言，氢键氨基酸取代（G12C、G12S）的致癌性上调是通过稳定与Raf的β界面相互作用实现的，而体积更大的疏水性G12V取代会导致该界面不稳定，反而增加了α螺旋束上残基的接近程度。这项研究为不同K - Ras突变如何影响下游信号传导这一复杂谜题提供了新的线索。
Mutations in the GTPase enzyme K-Ras, specifically at codon G12, remain the most common genetic alterations in human cancers. The mechanisms governing activation of downstream signaling pathways and how they relate back to the identity of the mutation have yet to be completely defined. Here we use native mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) to investigate the impact of three G12X mutations (G12C, G12V, G12S) on the homodimerization of K-Ras as well as heterodimerization with a downstream effector protein, Raf. Electrospray ionization (ESI) was used to transfer complexes of WT or G12X K-Ras bound to guanosine 5 '-diphosphate (GDP) or GppNHp (non-hydrolyzable analogue of GTP) into the gas phase. Relative abundances of homo- or hetero-dimer complexes were estimated from ESI-MS spectra. K-Ras + Raf heterocomplexes were activated with UVPD to probe structural changes responsible for observed differences in the amount of heterocomplex formed for each variant. Holo (ligand-bound) fragment ions resulting from photodissociation suggest the G12X mutants bind Raf along the expected effector binding region (beta-interface) but may interact with Raf via an alternative alpha-interface as well. Variations in backbone cleavage efficiencies during UV photoactivation of each variant were used to relate mutation identity to structural changes that might impact downstream signaling. Specifically, oncogenic upregulation for hydrogen-bonding amino acid substitutions (G12C, G12S) is achieved by stabilizing beta-interface interactions with Raf, while a bulkier, hydrophobic G12V substitution leads to destabilization of this interface and instead increases the proximity of residues along the alpha-helical bundles. This study deciphers new pieces of the complex puzzle of how different K-Ras mutations exert influence in downstream signaling.