Active Site Cysteine Is Protonated in the PAD4 Michaelis Complex: Evidence from Born−Oppenheimer Ab Initio QM/MM Molecular Dynamics Simulations

Abstract

Protein Arginine Deiminase 4 (PAD4) catalyzes the citrullination of the peptidylarginine, and plays a critical role in rheumatoid arthritis (RA) and gene regulation. Understanding its catalytic mechanism is not only of fundamental importance, but also of significant medical interest for the rational design of new inhibitors. By employing on-the-fly Born-Oppenheimer ab initio QM/MM molecular dynamics simulations, we have demonstrated that it is unlikely for the active site cysteine and histidine to exist as a thiolate-imidazolium ion-pair in the PAD4 Michaelis reactant complex. Instead, a substrate-assisted proton transfer mechanism for the deimination reaction step has been characterized: both Cys645 and His471 in the PAD4 active site are neutral prior to the reaction; the deprotonation of Cys645 by the substrate arginine occurs in concert with the nucleophilic addition of the Cys thiolate to C ζ of the substrate, and leads to a covalent tetrahedral intermediate; then the C ζ -N η1 bond cleaves and the resulted ammonia is displaced by a solvent water molecule. The initial deprotonation and nucleophilic attack step is found to be rate-determining. The computed free energy barrier with B3LYP(6-31G*) QM/MM MD simulations and umbrella sampling is 20.9 kcal·mol −1 , consistent with the experimental kinetic data. During the deimination, His471 plays an important role in stabilizing transition state through the formation of the hydrogen bond with the guanidinium group. Our current studies further demonstrated the viability and strength of the ab initio QM/MM molecular dynamics approach in simulating enzyme reactions.

Citrullination of autoantigens implicates NETosis in the induction of autoimmunity

“…During the reaction, the guanidino group of arginine is attacked by a Cys residue of PAD4, resulting in the formation of a tetrahedral adduct 45. A nucleophilic attack by a water molecule then cleaves the adduct to form the keto group of citrulline, while releasing ammonia and regenerating the Cys residue of the enzyme 44 46. The net result is the conversion of arginine to citrulline with a mass shift of +1 Da.…”

Mechanistic similarity and diversity among the guanidine-modifying members of the pentein superfamily

“…In this model, deprotonation of the core Cys occurs in concert with nucleophilic attack on the guanidine carbon. Computational studies with PAD4 have suggested that a protonated Cys is energetically more favorable than a Cys thiolate in the Michaelis complex [38,39]. In addition, the core Cys residue in AGAT is also proposed to be predominantly protonated in the resting state, possibly activated by Asp305 for attack [33].…”

Born−Oppenheimer ab Initio QM/MM Molecular Dynamics Simulations of the Hydrolysis Reaction Catalyzed by Protein Arginine Deiminase 4

“…It plays a pivotal role in human gene regulation [1–5], and the dysregulation of PAD4 has been linked to the onset and progression of the autoimmune disorder relevant to rheumatoid arthritis [6–9]. Thus, there is significant interest in elucidating its catalytic mechanism and in developing new mechanism-based inhibitors [10–15]. …”

“…Very recently, by employing Born-Oppenheimer ab initio QM/MM molecular dynamics simulations and umbrella sampling, we have characterized the Michaelis reactant complex and determined a substrate-assisted proton transfer mechanism for the initial deimination reaction catalyzed by PAD4 [15], as shown in Fig. 1.…”