Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges

S100A3, a member of the EF-hand-type Ca²⁺-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn²⁺ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca²⁺/Zn²⁺ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca²⁺ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca²⁺ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca²⁺-binding loop flexible, which are essential for its Ca²⁺ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn²⁺ by (Cys)₃His residues that is compatible with SS2 formation in S100A3.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

Methylation of arginine residues interferes with citrullination by peptidylarginine deiminases in vitro

Peptidylarginine deiminase (PAD) enzymes catalyze the conversion of arginine residues in proteins to citrulline residues. Citrulline is a non-standard amino acid that is not incorporated in proteins during translation, but can be generated post-translationally by the PAD enzymes. Although the existence of citrulline residues in proteins has been known for a long time, only a few proteins have been reported to contain this amino acid under normal conditions. These include the nuclear histones, which also contain a wide variety of other post-translational modifications, as for instance methylation of arginine residues. It has been suggested that citrullination and methylation of arginine residues are competing processes and that PAD enzymes might "reverse" the methylation of arginine residues by converting monomethylated arginine into citrulline. However, conflicting data have been reported on the capacity of PADs to citrullinate monomethylated peptidylarginine. Using synthetic peptides that contain either arginine or methylated arginine residues, we show that the human PAD2, PAD3 and PAD4 enzymes and PAD enzyme present in several mouse tissues in vitro can only convert non-methylated peptidylarginine into peptidylcitrulline and that hPAD6 does not show any deiminating activity at all. A comparison of bovine histones either treated or untreated with PAD by amino acid analysis also supported the interference of deimination by arginine methylation. Taken together, these data indicate that it is unlikely that methyl groups at the guanidino position of peptidylarginine can be removed by peptidylarginine deiminases, which has important implications for the recently reported role of these enzymes in gene regulation.     

Citrullination: a small change for a protein with great consequences for rheumatoid arthritis

A new autoantibody activity, which is almost 100% specific for rheumatoid arthritis (RA), has been found. The essential part of the B-cell epitope is a modified form of arginine (ie citrulline). The conversion of protein-contained arginine to citrulline is an enzymatic process that is carried out by peptidylarginine deiminase (PAD), an enzyme that appears to be hormonally controlled. Because of its remarkable specificity, citrullination and related processes might open new possibilities for studying the aetiology of RA.     

Kinetics of human peptidylarginine deiminase 2 (hPAD2)--reduction of Ca2+ dependence by phospholipids and assessment of proposed inhibition by paclitaxel side chains

Multiple sclerosis is a complex human neurodegenerative disease, characterized by the active destruction of the insulating myelin sheath around the axons in the central nervous system. The physical deterioration of myelin is mediated by hyperdeimination of myelin basic and other proteins, catalysed by the Ca2+ -dependent enzyme peptidylarginine deiminase 2 (PAD2). Thus, inhibition of PAD2 may be of value in treatment of this disease. Here, we have first characterized the in vitro kinetic properties of the human peptidylarginine deiminase isoform 2 (hPAD2). Phosphatidylserine and phosphatidylcholine reduced its Ca2+ dependence by almost twofold. Second, we have explored the putative inhibitory action of the methyl ester side chain of paclitaxel (TSME), which shares structural features with a synthetic PAD substrate, viz., the benzoyl-L-arginine ethyl ester (BAEE). Using the known crystallographic structure of the homologous enzyme hPAD4 and in silico molecular docking, we have shown that TSME interacted strongly with the catalytic site, albeit with a 100-fold lower affinity than BAEE. Despite paclitaxel having previously been shown to inhibit hPAD2 in vitro, the side chain of paclitaxel alone did not inhibit this enzyme's activity.     

The origin of citrulline-containing proteins in the hair follicle and the chemical nature of trichohyalin, an intracellular precursor.

The present studies have demonstrated that the medulla and inner root sheath cells develop within their cytoplasm a protein that is unique in composition and is present in the trichohyalin granules. The protein is rich in arginine residues, some of which undergo a side-chain conversion in situ into citrulline residues. An unusual Ca2+-dependent enzyme activity distinguishable from cross-linking transamidase has been detected in the hair follicle and will act in vitro on trichohyalin protein as the natural substrate. The conversion in vivo must occur during the time that the medullary and inner root sheath cells move up the follicle and their cytoplasm fills with cross-linked protein containing citrulline. The function of citrulline in these proteins is not understood but its formation is a major process during hair growth.     

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine residues in substrate proteins to the neutral, non-standard residue citrulline. PAD family members 1, 2, 3, and 6 have previously been localized to the cell cytoplasm and, thus, their potential to regulate gene activity has not been described. We recently demonstrated that PAD2 is expressed in the canine mammary gland epithelium and that levels of histone citrullination in this tissue correlate with PAD2 expression. Given these observations, we decided to test whether PAD2 might localize to the nuclear compartment of the human mammary epithelium and regulate gene activity in these cells. Here we show, for the first time, that PAD2 is specifically expressed in human mammary gland epithelial cells and that a portion of PAD2 associates with chromatin in MCF-7 breast cancer cells. We investigated a potential nuclear function for PAD2 by microarray, qPCR, and chromatin immunoprecipitation analysis. Results show that the expression of a unique subset of genes is disregulated following depletion of PAD2 from MCF-7 cells. Further, ChIP analysis of two of the most highly up- and down-regulated genes (PTN and MAGEA12, respectively) found that PAD2 binds directly to these gene promoters and that the likely mechanism by which PAD2 regulates expression of these genes is via citrullination of arginine residues 2-8-17 on histone H3 tails. Thus, our findings define a novel role for PAD2 in gene expression in human mammary epithelial cells.     

Proteins Deiminated by Peptidylarginine Deiminase in Mouse Uterus and Their Changes during the Estrous Cycle

Peptidylarginine deiminase (PAD) catalyzes the formation of citrullyl residues by deimination of arginyl residues of proteins in the presence of Ca²⁺. We found several deiminated proteins in mouse uterus using antibodies recognizing the citrulline residue. The levels of these proteins changed during the estrous cycle in parallel with PAD activity.     

Structure of Ca2+-bound S100A4 and its interaction with peptides derived from nonmuscle myosin-IIA

S100A4, also known as mts1, is a member of the S100 family of Ca2+-binding proteins that is directly involved in tumor invasion and metastasis via interactions with specific protein targets, including nonmuscle myosin-IIA (MIIA). Human S100A4 binds two Ca2+ ions with the typical EF-hand exhibiting an affinity that is nearly 1 order of magnitude tighter than that of the pseudo-EF-hand. To examine how Ca2+ modifies the overall organization and structure of the protein, we determined the 1.7 A crystal structure of the human Ca2+-S100A4. Ca2+ binding induces a large reorientation of helix 3 in the typical EF-hand. This reorganization exposes a hydrophobic cleft that is comprised of residues from the hinge region,helix 3, and helix 4, which afford specific target recognition and binding. The Ca2+-dependent conformational change is required for S100A4 to bind peptide sequences derived from the C-terminal portion of the MIIA rod with submicromolar affinity. In addition, the level of binding of Ca2+ to both EF-hands increases by 1 order of magnitude in the presence of MIIA. NMR spectroscopy studies demonstrate that following titration with a MIIA peptide, the largest chemical shift perturbations and exchange broadening effects occur for residues in the hydrophobic pocket of Ca2+-S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca2+ dependence of formation of theS100A4-MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function.     

Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

PADs (peptidylarginine deiminases) are calcium-dependent enzymes that change protein-bound arginine to citrulline (citrullination/deimination) affecting protein conformation and function. PAD up-regulation following chick spinal cord injury has been linked to extensive tissue damage and loss of regenerative capability. Having found that human neural stem cells (hNSCs) expressed PAD2 and PAD3, we studied PAD function in these cells and investigated PAD3 as a potential target for neuroprotection by mimicking calcium-induced secondary injury responses. We show that PAD3, rather than PAD2 is a modulator of cell growth/death and that PAD activity is not associated with caspase-3-dependent cell death, but is required for AIF (apoptosis inducing factor)-mediated apoptosis. PAD inhibition prevents association of PAD3 with AIF and AIF cleavage required for its translocation to the nucleus. Finally, PAD inhibition also hinders calcium-induced cytoskeleton disassembly and association of PAD3 with vimentin, that we show to be associated also with AIF; together this suggests that PAD-dependent cytoskeleton disassembly may play a role in AIF translocation to the nucleus. This is the first study highlighting a role of PAD activity in balancing hNSC survival/death, identifying PAD3 as an important upstream regulator of calcium-induced apoptosis, which could be targeted to reduce neural loss, and shedding light on the mechanisms involved.     

Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes.

Peptidylarginine deiminases (PADs) convert arginine residues in proteins into citrulline residues Ca(2+)-dependently. PAD V was recently found in granulocyte-differentiated HL-60 cells. To find a target of PAD V, we incubated HL-60 granulocytes with the calcium ionophore A23187 and studied deiminated proteins by immunocytochemistry and immunoblotting using a monospecific antibody to modified citrulline residues. Immunocytochemical signals were found in the nucleus upon incubation with A23187. Immunoblotting indicated that 40-, 18-, 17-, and 14-kDa proteins were preferentially deiminated. The 40-kDa protein, which was focused to pI 5.0 on two-dimensional gel electrophoresis, was identified as nucleophosmin/B23 by mass spectrometry. The 18-, 17-, and 14-kDa proteins extracted with 0.4 N H(2)SO(4) comigrated with histones H3, H2A, and H4, respectively, on two-dimensional gel electrophoresis specialized for histones. The citrulline content of histones amounted to about 10% of the histone molecules. We discuss the implications of deimination of these proteins for their nuclear functions.     

Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D

The effect of deimination of arginyl residues in bovine myelin basic protein (MBP) on its susceptibility to digestion by cathepsin D has been studied. Using bovine component 1 (C-1) of MBP, the most unmodified of the components with all 18 arginyl residues intact, we have generated a number of citrullinated forms by treatment of the protein with purified peptidylarginine deiminase (PAD) in vitro. We obtained species containing 0-9.9 mol of citrulline/mol of MBP. These various species were digested with cathepsin D, a metalloproteinase which cleaves proteins at Phe-Phe linkages. The rate of digestion compared to component 1 was only slightly affected when 2.7 or 3.8 mol of citrulline/mol of MBP was present. With 7.0 mol of citrulline/mol of MBP, a large increase in the rate of digestion occurred. No further increase was observed with 9.9 mol of citrulline/mol of MBP. The immunodominant peptide 43-88 (bovine sequence) was released slowly when 2.7 and 3.8 mol of citrulline/mol of MBP was present, but it was released rapidly when 7.0 mol of citrulline/mol of MBP was present. The dramatic change in digestion with 7.0 mol of citrulline/mol of MBP or more could be explained by a change in three-dimensional structure. Molecular dynamics simulation showed that increasing the number of citrullinyl residues above 7 mol/mol of MBP generated a more open structure, consistent with experimental observations in the literature. We conclude that PAD, which deiminates arginyl residues in proteins, decreases both the charge and compact structure of MBP. This structural change allows better access of the Phe-Phe linkages to cathepsin D. This scheme represents an effective way of generating the immunodominant peptide which sensitizes T-cells for the autoimmune response in demyelinating disease.     

A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.     

Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

Conversion of peanut trypsin-chymotrypsin inhibitor B-III to a chymotrypsin inhibitor by deimination of the P1 arginine residues in two reactive sites

The deimination of the arginine residues in peanut trypsin-chymotrypsin inhibitor B-III caused the disappearance of its trypsin-inhibitory activity. Peanut protease inhibitor B-III was incubated with peptidylarginine deiminase, resulting in the conversion of 2.5 mol of arginine to citrulline and in the loss of its trypsin-inhibitory activity. However, the ability of the deiminated inhibitor to inhibit chymotrypsin was as strong as before. Structural analysis of the deiminated B-III indicated that the P1 arginine residues at both reactive sites, Arg(10) and Arg(38), were completely modified to citrulline by the action of peptidylarginine deiminase, and that the Arg(60) in the C-terminal region of B-III was partially deiminated. These residues seem to be exposed on the surface of the molecule. The P1' arginine residue at the first reactive site, Arg(11), was not deiminated at all.     

Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

This study investigated the functional roles of the N-terminal Ca²⁺ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca²⁺-binding site of PAD4 were mutated to disrupt the binding of Ca²⁺ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k _cat/K _m,BAEE values were 0.02, 0.63 and 0.01 s⁻¹mM⁻¹ (20.8 s⁻¹mM⁻¹ for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k _cat value of 0.3 s⁻¹ (13.3 s⁻¹ for wild-type), whereas D176A retained some catalytic power with a k _cat of 9.7 s⁻¹. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k _cat/K _m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca²⁺ indicated that the conformational stability of the enzyme is highly dependent on Ca²⁺ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca²⁺ ions in the N-terminal Ca²⁺-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca²⁺ ions play critical roles in the full activation of the PAD4 enzyme.     

Development of a fluorescent probe for detection of citrulline based on photo-induced electron transfer

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine residues to form citrulline residues. Aberrant citrullination of histones by one of the PAD isozymes, PAD4, is associated with various diseases, including rheumatoid arthritis, so high-throughput screening systems are needed to identify PAD4 inhibitors as chemical tools to investigate the role of PAD4, and as candidate therapeutic agents. Here, we utilized the addition-cyclization reaction between phenylglyoxal and citrulline under acidic conditions to design turn-on fluorescent probes for citrulline based on the donor-excited photoinduced electron transfer (d-PeT) mechanism. Among several derivatives of phenylglyoxal bearing a fluorescent moiety, we found that FGME enabled detection of citrulline without a neutralization process, and we used it to establish a simple methodology for turn-on fluorescence detection of citrulline.