Mapping the contact surfaces in the Lamin A:AIMP3 complex by hydrogen/deuterium exchange FT-ICR mass spectrometry

Aminoacyl-tRNA synthetases-interacting multifunctional protein3 (AIMP3/p18) is involved in the macromolecular tRNA synthetase complex via its interaction with several aminoacyl-tRNA synthetases. Recent reports reveal a novel function of AIMP3 as a tumor suppressor by accelerating cellular senescence and causing defects in nuclear morphology. AIMP3 specifically mediates degradation of mature Lamin A (LmnA), a major component of the nuclear envelope matrix; however, the mechanism of how AIMP3 interacts with LmnA is unclear. Here we report solution-phase hydrogen/deuterium exchange (HDX) for AIMP3, LmnA, and AIMP3 in association with the LmnA C-terminus. Reversed-phase LC coupled with LTQ 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) results in high mass accuracy and resolving power for comparing the D-uptake profiles for AIMP3, LmnA, and their complex. The results show that the AIMP3-LmnA interaction involves one of the two putative binding sites and an adjacent novel interface on AIMP3. LmnA binds AIMP3 via its extreme C-terminus. Together these findings provide a structural insight for understanding the interaction between AIMP3 and LmnA in AIMP3 degradation.氨酰-tRNA合成酶相互作用多功能蛋白3（AIMP3/p18）可通过与多种氨酰-tRNA合成酶的相互作用，参与大分子tRNA合成酶复合物的组成。近期研究揭示了AIMP3作为肿瘤抑制因子的全新功能：其可加速细胞衰老，并引发细胞核形态异常。AIMP3可特异性介导成熟核纤层蛋白A（LmnA）的降解——后者是核被膜基质的核心组分；但目前AIMP3与LmnA的相互作用机制仍不明晰。本研究针对AIMP3、LmnA以及与LmnA C端结合的AIMP3复合物开展了溶液相氢氘交换（HDX）实验。采用反相液相色谱结合LTQ 14.5 T傅里叶变换离子回旋共振质谱（Fourier transform ion cyclotron resonance mass spectrometry，FT-ICR MS）的分析策略，可实现高精度质量测定与高分辨能力，以便对比AIMP3、LmnA及其复合物的氘摄取谱图。分析结果显示，AIMP3与LmnA的相互作用涉及AIMP3上两个预测结合位点之一，以及其邻近的全新互作界面；LmnA则通过其最末端的C端区域与AIMP3结合。综上，本研究的发现为解析AIMP3降解过程中AIMP3与LmnA的相互作用机制提供了结构层面的新认知。