Liu Meili, Li Li, Wang Zhiqin, Wang Shuang, Tang Xiaowen
Department of Medical Chemistry, School of Pharmacy, Qingdao University, Qingdao, China.
Department of Civil and Architectural Engineering, University of Miami, Coral Gables, FL, United States.
Front Chem. 2023 Jan 25;11:1077188. doi: 10.3389/fchem.2023.1077188. eCollection 2023.
DeAMPylation, as a reversible reaction of AMPylation and mediated by the endoplasmic reticulum-localized enzyme FICD (filamentation induced by cAMP domain protein, also known as HYPE), is an important process in protein posttranslational modifications (PTMs). Elucidating the function and catalytic details of FICD is of vital importance to provide a comprehensive understanding of protein folding homeostasis. However, the detailed deAMPylation mechanism is still unclear. Furthermore, the role of a conserved glutamine (Glu234), that plays an inhibitory role in the AMPylation response, is still an open question in the deAMPylation process. In the present work, the elaborated deAMPylation mechanisms with AMPylation-inhibitory/assistant forms of FICD (wild type and Glu234Ala mutant) were investigated based on the QM(DFT)/MM MD approach. The results revealed that deAMPylation was triggered by proton transfer from protonated histidine (His363) to AMPylated threonine, instead of a nucleophilic attack of water molecules adding to the phosphorus of AMP. The free energy barrier of deAMPylation in the wild type (∼17.3 kcal/mol) is consistent with that in the Glu234Ala mutant of FICD (∼17.1 kcal/mol), suggesting that the alteration of the Glu234 residue does not affect the deAMPylation reaction and indirectly verifying the inducement of deAMPylation in FICD. In the wild type, the proton in the nucleophilic water molecule is transferred to Glu234, whereas it is delivered to Asp367 through the hydrogen-bond network of coordinated water molecules in the Glu234Ala mutant. The present findings were inspirational for understanding the catalytic and inhibitory mechanisms of FICD-mediated AMP transfer, paving the way for further studies on the physiological role of FICD protein.
去腺苷酸化是腺苷酸化的可逆反应，由内质网定位的酶FICD（cAMP结构域诱导丝状化蛋白，也称为HYPE）介导，是蛋白质翻译后修饰（PTM）中的一个重要过程。阐明FICD的功能和催化细节对于全面理解蛋白质折叠稳态至关重要。然而，详细的去腺苷酸化机制仍不清楚。此外，在腺苷酸化反应中起抑制作用的保守谷氨酰胺（Glu234）在去腺苷酸化过程中的作用仍是一个悬而未决的问题。在本工作中，基于QM(DFT)/MM MD方法研究了具有腺苷酸化抑制/辅助形式的FICD（野生型和Glu234Ala突变体）的详细去腺苷酸化机制。结果表明，去腺苷酸化是由质子化组氨酸（His363）向腺苷酸化苏氨酸的质子转移引发的，而不是水分子对腺苷磷的亲核攻击。野生型去腺苷酸化的自由能垒（约17.3 kcal/mol）与FICD的Glu234Ala突变体中的自由能垒（约17.1 kcal/mol）一致，这表明Glu234残基的改变不影响去腺苷酸化反应，并间接验证了FICD中去腺苷酸化的诱导。在野生型中，亲核水分子中的质子转移到Glu234，而在Glu234Ala突变体中，质子通过配位水分子的氢键网络传递到Asp367。本研究结果对于理解FICD介导的AMP转移的催化和抑制机制具有启发性，为进一步研究FICD蛋白的生理作用铺平了道路。
