AMPylation is a prevalent posttranslational modification that involves the addition of adenosine monophosphate (AMP) to proteins. Exactly how Huntingtin-associated yeast-interacting protein E (HYPE), as the first human protein, is involved in the transformation of the AMP moiety to its substrate target protein (the endoplasmic reticulum chaperone binding to immunoglobulin protein (BiP)) is still an open question. Additionally, a conserved glutamine plays a vital key role in the AMPylation reaction in most filamentation processes induced by the cAMP (Fic) protein. In the present work, the detailed catalytic AMPylation mechanisms in HYPE were determined based on the density functional theory (DFT) method. Molecular dynamics (MD) simulations were further used to investigate the exact role of the inhibitory glutamate. The metal center, Mg2+, in HYPE has been examined in various coordination configurations, including 4-coordrinated, 5-coordinated and 6-coordinated. DFT calculations revealed that the transformation of the AMP moiety of HYPE with BiP followed a sequential pathway. The model with a 4-coordinated metal center had a barrier of 14.7 kcal/mol, which was consistent with the experimental value and lower than the 38.7 kcal/mol barrier of the model with a 6-coordinated metal center and the 31.1 kcal/mol barrier of the model with a 5-coordinated metal center. Furthermore, DFT results indicated that Thr518 residue oxygen directly attacks the phosphorus, while the His363 residue acts as H-bond acceptor. At the same time, an MD study indicated that Glu234 played an inhibitory role in the α-inhibition helix by regulating the hydrogen bond interaction between Arg374 and the Pγ of the ATP molecule. The revealed sequential pathway and the inhibitory role of Glu234 in HYPE were inspirational for understanding the catalytic and inhibitory mechanisms of Fic-mediated AMP transfer, paving the way for further studies on the physiological role of Fic enzymes.
Keywords: AMPylation; Fic-mediated; HYPE; density functional theory.