title: Theoretical study of the mechanism of protein arginine deiminase 4 (PAD4) inhibition by F-amidine.
abstract: Protein arginine deiminase 4 (PAD4) catalyzes the hydrolysis of a peptidylarginine residue to form a citrulline residue and ammonia during posttranslational modification. This process plays a pivotal role in rheumatoid arthritis (RA) and gene regulation. F-amidine belongs to a series of haloacetamidine compounds that are the most potent PAD4 inhibitors described to date. F-amidine acts as a mechanism-based inhibitor of PAD4, inactivating PAD4 by the covalent modification of the active site Cys645. In this manuscript, the fundamental mechanism of PAD4 inhibition by F-amidine is investigated using a QM/MM approach. Our simulations show that in the PAD4-F-amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated. This is consistent with the reverse protonation mechanism wherein the active site nucleophile, Cys645, in PAD4 exists as a thiolate in the active form of the enzyme. Inhibition of PAD4 by F-amidine is initiated by the nucleophilic addition of Sγ to the Cζ of F-amidine, leading to the formation of a tetrahedral intermediate. His471 serves as a proton donor, helping F to leave the fluoroacetamidine moiety of F-amidine; meanwhile, Sγ forms a three-membered ring with Cζ and Cη of F-amidine. Subsequently, the three-membered sulfonium ring collapses and rearranges to the final thioether product. His471 acts as a proton donor in the transition state and facilitates the inhibition reaction of PAD4.
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China; Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China. Electronic address: dongmeili@nankai.edu.cn.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Li
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China; Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China.
authors.forename: Cui
authors.initials: C
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China; Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China. Electronic address: jianpinglin@nankai.edu.cn.
authors.forename: Jianping
authors.initials: J
authors.lastname: Lin
mesh_terms: D000578:Amidines / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006845:Hydrocarbons, Fluorinated / Q000737:chemistry / Q000494:pharmacology*; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011522:Protons; D011789:Quantum Theory; D013816:Thermodynamics
chemical_list: D000578:Amidines; C509292:F-amidine; D006845:Hydrocarbons, Fluorinated; D011522:Protons; D006867:Hydrolases; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases
keywords: ONIOM; PAD4; Protonation state; QM/MM; Transition state