Increased protein citrullination is linked to various diseases including rheumatoid arthritis (RA), lupus, and cancer. Citrullinated autoantigens, a hallmark of RA, are recognized by anti-citrullinated protein antibodies (ACPAs) which are used to diagnose RA. ACPA-recognizing citrullinated enolase, vimentin, keratin, and filaggrin are also pathogenic. Here, we used a chemoproteomic approach to define the RA-associated citrullinome. The identified proteins include numerous serine protease inhibitors (Serpins), proteases and metabolic enzymes. We demonstrate that citrullination of antiplasmin, antithrombin, t-PAI, and C1 inhibitor (P1-Arg-containing Serpins) abolishes their ability to inhibit their cognate proteases. Citrullination of nicotinamide N-methyl transferase (NNMT) also abolished its methyltransferase activity. Overall, these data advance our understanding of the roles of citrullination in RA and suggest that extracellular protein arginine deiminase (PAD) activity can modulate protease activity with consequent effects on Serpin-regulated pathways. Moreover, our data suggest that inhibition of extracellular PAD activity will be therapeutically relevant.

Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy 30a. Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated.

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we solved the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and cupferron, to 2.2-Å resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state/intermediate since its binding affinity to 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, cupferron, and the ground state analogue (S)-atrolacatate reveal that the para-carbon of the substrate phenyl ring moves by 0.8–1.2 Å between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to MR with bound (S)-atrolactate, the intermediate-Mg2+ distance shortens, suggesting a tighter complex with the catalytic Mg2+. In addition, Tyr 54 moves nearer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the enantiomers of mandelic acid and a variety of aryl- and heteroaryl-substituted mandelate derivatives, suggesting that β,γ-unsaturation is a requisite feature of substrates for the enzyme. We show that β,γ-unsaturation is not an absolute requirement for catalysis and that mandelate racemase can bind and catalyze the racemization of (S)-trifluorolactate (k(cat) = 2.5 ± 0.3 s(-1), K(m) = 1.74 ± 0.08 mM) and (R)-trifluorolactate (k(cat) = 2.0 ± 0.2 s(-1), K(m) = 1.2 ± 0.2 mM). The enzyme was shown to catalyze hydrogen-deuterium exchange at the α-postion of trifluorolactate using (1)H NMR spectrocsopy. β-Elimination of fluoride was not detected using (19)F NMR spectroscopy. Although mandelate racemase bound trifluorolactate with an affinity similar to that exhibited for mandelate, the turnover numbers (k(cat)) were markedly reduced by ∼318-fold, resulting in catalytic efficiencies (k(cat)/K(m)) that were ~400-fold lower than those observed for mandelate. These observations suggested that chemical steps on the enzyme were likely rate-determining, which was confirmed by demonstrating that the rates of mandelate racemase-catalyzed racemization of (S)-trifluorolactate were not dependent upon the solvent microviscosity. Circular dichroism spectroscopy was used to measure the rates of nonenzymatic racemization of (S)-trifluorolactate at elevated temperatures. The values of ΔH(‡) and ΔS(‡) for the nonenzymatic racemization reaction were determined to be 28.0 (±0.7) kcal/mol and -15.7 (±1.7) cal K(-1) mol(-1), respectively, corresponding to a free energy of activation equal to 33 (±4) kcal/mol at 25 °C. Hence, mandelate racemase stabilizes the altered trifluorolactate in the transition state (ΔG(tx)) by at least 20 kcal/mol.

Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the posttranslational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor (ER) target genes. However, PAD2 lacks a canonical Nuclear Localization Sequence (NLS), and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Calcium binding to PAD2 weakens this cytoplasmic interaction, which generating a pool of calcium bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus.

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR-TFHTP complex to 1.68 Å resolution. Rather than chelating the active site Mg(2+) with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Brønsted acid-base catalysts Lys 166 and His 297. Recognizing that positioning a carboxylate between the Brønsted acid-base catalysts could yield an inhibitor, we showed that tartronate was a competitive inhibitor of MR (Ki = 1.8 ± 0.1 mM). The X-ray crystal structure of the MR-tartronate complex (1.80 Å resolution) revealed that the glycolate moiety of tartronate chelated the Mg(2+) and that the carboxylate bridged Lys 166 and His 297. Models of tartronate in monomers A and B of the crystal structure mimicked the binding orientations of (S)-mandelate and that anticipated for (R)-mandelate, respectively. For the latter monomer, the 20s loop appeared to be disordered, as it also did in the X-ray structure of the MR triple mutant (C92S/C264S/K166C) complexed with benzilate, which was determined to 1.89 Å resolution. These observations indicate that the 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate. In general, our observations suggest that inhibitors of other enolase superfamily enzymes may be designed to capitalize on the recognition of the active site Brønsted acid-base catalysts as binding determinants.

Protein arginine deiminases (PADs) play an important role in the pathogenesis of various diseases, including rheumatoid arthritis, multiple sclerosis, lupus, ulcerative colitis, and breast cancer. Therefore, the development of PAD inhibitors has drawn significant research interest in recent years. Herein, we describe the development of the first photoswitchable PAD inhibitors. These compounds possess an azobenzene photoswitch to optically control PAD activity. Screening of a series of inhibitors structurally similar to BB-Cl-amidine afforded compounds 1 and 2 as the most promising candidates for the light-controlled inhibition of PAD2; the cis isomer of 1 is 10-fold more potent than its trans isomer, whereas the trans isomer of 2 is 45-fold more potent than the corresponding cis isomer. The altered inhibitory potency upon photoisomerization has been confirmed in a competitive activity-based protein profiling (ABPP) assay. Further investigations indicate that the trans isomer of 2 is an irreversible inhibitor, whereas the cis isomer acts as a competitive inhibitor. In cells, the trans isomer of compound 1 is completely inactive, whereas the cis isomer inhibits histone H3-citrullination in a dose-dependent manner. Taken together, 1 serves as the foundation for developing photopharmaceuticals that can be activated at the desired tissue, using light, to treat diseases where PAD activity is dysregulated.

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate and serves as a paradigm for understanding the enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. The enzyme utilizes a two-base mechanism with Lys 166 and His 297 acting as Brønsted acid and base catalysts, respectively, in the R → S reaction direction. In the S → R reaction direction, their roles are reversed. Using isothermal titration calorimetry (ITC), MR is shown to bind the intermediate/transition state (TS) analogue inhibitor benzohydroxamate (BzH) in an entropy-driven process with a value of ΔCp equal to -358 ± 3 cal mol(-1) K(-1), consistent with an increased number of hydrophobic interactions. However, MR binds BzH with an affinity that is ∼2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions [St. Maurice, M., and Bearne, S. L. (2004) Biochemistry 43, 2524], suggesting that additional specific interactions contribute to binding. To test the hypothesis that cation-π/NH-π interactions between the side chains of Lys 166 and His 297 and the aromatic ring and/or the hydroxamate/hydroximate moiety of BzH contribute to the binding of BzH, site-directed mutagenesis was used to generate the MR variants K166M, K166C, H297N, and K166M/H297N and their binding affinity for various ligands determined using ITC. Comparison of the binding affinities of these MR variants with the intermediate/TS analogues BzH and cyclohexanecarbohydroxamate revealed that cation-π/NH-π interactions between His 297 and the hydroxamate/hydroximate moiety and the phenyl ring of BzH contribute approximately 0.26 and 0.91 kcal/mol to binding, respectively, while interactions with Lys 166 contribute approximately 1.74 and 1.74 kcal/mol, respectively. Similarly, comparison of the binding affinities of these mutants with substrate analogues revealed that Lys 166 contributes >2.93 kcal/mol to the binding of (R)-atrolactate, and His 297 contributes 2.46 kcal/mol to the binding of (S)-atrolactate. These results are consistent with Lys 166 and His 297 playing dual roles in catalysis: they act as Brønsted acid-base catalysts, and they stabilize both the enolate moiety and phenyl ring of the altered substrate in the TS.