Structures of N-terminally processed KRAS provide insight into the role of N-acetylation
Structures of N-terminally processed KRAS provide insight into the role of N-acetylation
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DOI：
10.1038/s41598-019-46846-w
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发表时间：
2019-07-19
影响因子：
4.6
通讯作者：
Simanshu, Dhirendra K.
中科院分区：
文献类型：
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作者：
Dharmaiah, Srisathiyanarayanan;Tran, Timothy H.;Simanshu, Dhirendra K.
尽管 RAS C 端的翻译后修饰已被广泛研究，但对 N 端加工知之甚少。哺乳动物细胞中表达的 KRAS 的质谱表征显示起始子蛋氨酸 (iMet) 的裂解和新生 N 末端的 N-乙酰化。有趣的是，对GDP和GMPPNP结合的KRAS缺乏iMet和N-乙酰化的结构研究导致了具有柔性N末端的无Mg2+结构的KRAS。在无 Mg2+ 的 KRAS-GDP 结构中，柔性 N 末端引起开关间区域的构象变化，导致开关 I 的构象完全开放。在无 Mg2+ 的 KRAS-GMPPNP 结构中，柔性 N 末端引起残基 A59 周围的构象变化，导致 Mg2+ 和开关 I 在非活性状态 1 构象中丢失。对缺乏 iMet 的 N-乙酰化 KRAS-GDP 的结构研究揭示了 Mg2+ 的存在，并且在带有 iMet 的 GDP 结合的未加工 KRAS 的结构中也观察到了开关区域的构象。在没有 iMet 的情况下，N-乙酰基与中央 β-折叠相互作用并稳定 N 末端和开关区域。这些结果表明 N 末端和 Mg2+ 结合位点之间存在串扰，并且 N-乙酰化在 iMet 切除后通过稳定 RAS 的 N 末端发挥着重要作用。
Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP-and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.