Molecular Mechanism of Protein Arginine Deiminase 2: A Study Involving Multiple Microsecond Long Molecular Dynamics Simulations.

Peptidylarginine deiminase 2 (PAD2) is a Ca2+-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. This kind of structural modification in histone molecules may affect gene regulation, leading to effects that may trigger several diseases, including breast cancer, which makes PAD2 an attractive target for anticancer drug development. To design new effective inhibitors to control activation of PAD2, improving our understanding of the molecular mechanisms of PAD2 using up-to-date computational techniques is essential. We have designed five different PAD2-substrate complex systems based on varying protonation states of the active site residues. To search the conformational space broadly, multiple independent molecular dynamics simulations of the complexes have been performed. In total, 50 replica simulations have been performed, each of 1 µs, yielding a total simulation time of 50 µs. Our findings identify that the protonation states of Cys647, Asp473, and His471 are critical for the binding and localization of the N-α-benzoyl-l-arginine ethyl ester substrate within the active site. A novel mechanism for enzyme activation is proposed according to near attack conformers. This represents an important step in understanding the mechanism of citrullination and developing PAD2-inhibiting drugs for the treatment of breast cancer.

Proximity-Dependent Labeling of Cysteines.

Mapping protein-protein interactions is crucial for understanding various signaling pathways in living cells, and developing new techniques for this purpose has attracted significant interest. Classic methods (e.g., the yeast two-hybrid) have been supplanted by more sophisticated chemical approaches that label proximal proteins (e.g., BioID, APEX). Herein we describe a proximity-based approach that uniquely labels cysteines. Our approach exploits the nicotinamide N-methyltransferase (NNMT)-catalyzed methylation of an alkyne-substituted 4-chloropyridine (SS6). Upon methylation of the pyridinium nitrogen, this latent electrophile diffuses out of the active site and labels proximal proteins on short time scales (≤5 min). We validated this approach by identifying known (and novel) interacting partners of protein arginine deiminase 2 (PAD2) and pyruvate dehydrogenase kinase 1 (PDK1). To our knowledge, this technology uniquely exploits a suicide substrate to label proximal cysteines in live cells.

Citrullination of fibrinogen by peptidylarginine deiminase 2 impairs fibrin clot structure.

BACKGROUND: Citrullination is the post-translational conversion of arginine into citrulline in proteins. The reaction is catalyzed by peptidylarginine deiminase (PAD), of which five isoforms exist. Fibrinogen is a substrate for PAD2 and PAD4, and citrullinated fibrinogen (cFBG) has been detected in patients with inflammatory diseases. In purified systems, cFBG is known to inhibit the release of fibrinopeptide A (FPA) and B (FPB) and impairs fibrin polymerization. However, the effect of cFBG on fibrin structure and fibrinolysis in a plasma environment remains unclear. We hypothesized that citrullination of fibrinogen impairs fibrin properties. METHODS: Fibrinogen was citrullinated by recombinant PAD2 and PAD4. The impact of cFBG on fibrin structure was investigated by turbidity measurements in fibrinogen-deficient plasma spiked with cFBG or native fibrinogen. RESULTS: Citrullination of fibrinogen by PAD2 dose-dependently reduced the rate of fibrin polymerization, as well as the overall hemostasis potential of fibrin, the maximum velocity of fibrin formation, the fibrin mass/length ratio, and the lysis of fibrin clots. CONCLUSION: Citrullination of fibrinogen by PAD2 affects not only fibrin polymerization but also fibrin fiber properties, indicating that the fibrin network formed in the presence of cFBG may influence hemostasis. Our results suggest that citrullination of fibrinogen alters the composition of fibrin fibers which may lead to a looser fibrin network that is more susceptible to fibrinolysis and thereby affecting the hemostatic balance.