Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.

PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis.

Structures of human peptidylarginine deiminase type III provide insights into substrate recognition and inhibitor design.

Peptidylarginine deiminase type III (PAD3) is an isozyme belonging to the PAD enzyme family that converts arginine to citrulline residue(s) within proteins. PAD3 is expressed in most differentiated keratinocytes of the epidermis and hair follicles, while S100A3, trichohyalin, and filaggrin are its principal substrates. In this study, the X-ray crystal structures of PAD3 in six states, including its complex with the PAD inhibitor Cl-amidine, were determined. This structural analysis identified a large space around Gly374 in the PAD3-Ca2+-Cl-amidine complex, which may be used to develop novel PAD3-selective inhibitors. In addition, similarities between PAD3 and PAD4 were found based on the investigation of PAD4 reactivity with S100A3 in vitro. A comparison of the structures of PAD1, PAD2, PAD3, and PAD4 implied that the flexibility of the structures around the active site may lead to different substrate selectivity among these PAD isozymes.

Identification and Characterization of a Redox Sensor Phosphodiesterase from Ferrovum sp. PN-J185 Containing Bacterial Hemerythrin and HD-GYP Domains.

The second messenger bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates numerous important physiological functions in bacteria. In this study, we identified and characterized the first dimeric, full-length, non-heme iron-bound phosphodiesterase (PDE) containing bacterial hemerythrin and HD-GYP domains (Bhr-HD-GYP). We found that the amino acid sequence encoded by the FV185_09380 gene from Ferrovum sp. PN-J185 contains an N-terminal bacterial hemerythrin domain and a C-terminal HD-GYP domain, which is characteristic of proteins with PDE activity toward c-di-GMP. Inductively coupled plasma optical emission spectroscopy analyses showed that Bhr-HD-GYP contains 4 equiv of iron atoms per subunit, suggesting both hemerythrin and HD-GYP domains have non-heme di-iron sites. A redox-dependent spectral change expected for oxo-bridged non-heme iron with carboxylate ligands was observed, and this redox interconversion was reversible. However, unlike marine invertebrate hemerythrin, which functions as an oxygen-binding protein, Bhr-HD-GYP did not form an oxygen adduct because of rapid autoxidation. The reduced ferrous iron complex of the protein catalyzed the hydrolysis of c-di-GMP to its linearized product, 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), whereas the oxidized ferric iron complex had no significant activity. These results suggest that Bhr-HD-GYP is a redox and oxygen sensor enzyme that regulates c-di-GMP levels in response to changes in cellular redox status or oxygen concentration. Our study may lead to an improved understanding of the physiology of iron-oxidizing bacterium Ferrovum sp. PN-J185.

QM/MM Investigation for Protonation States in a Bilin Reductase PcyA-Biliverdin IXα Complex.

Herein we report quantum mechanical/molecular mechanical (QM/MM) studies to investigate the most probable protonation states of active site amino acids and bound substrate based on a recently reported neutron diffraction structure of phycocyanobilin:ferredoxin oxidoreductase (PcyA) by Unno et al. This structure was considered to be bound in its initial state of biliverdin IXα (BV), which has the C-pyrrole ring in the deprotonated state. The protonation state of BV suggested by neutron and spectroscopic studies is a stable, two-electron reduced complex with a bound hydronium ion. Several ambiguities in the neutron structure were observed which prompted a further theoretical analysis of the structure. This structural investigation provides new understanding of the PcyA and BV protonation states not previously reported in the literature. Our calculations suggest that the hydronium ion (H3 O+ ) is energetically unfavorable, preferentially protonating the neighboring His88 residue and that the C-ring of BV is not protonated.

Insights into the Proton Transfer Mechanism of a Bilin Reductase PcyA Following Neutron Crystallography.

Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH(+), a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial" water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 Nδ was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via H3O(+). These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis.

Structures of the substrate-free and product-bound forms of HmuO, a heme oxygenase from corynebacterium diphtheriae: x-ray crystallography and molecular dynamics investigation.

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe(3+)-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe(3+)-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe(3+)-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe(3+)-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.

Human S100A3 tetramerization propagates Ca(2+)/Zn(2+) binding states.

The S100A3 homotetramer assembles upon citrullination of a specific symmetric Arg51 pair on its homodimer interface in human hair cuticular cells. Each S100A3 subunit contains two EF-hand-type Ca(2+)-binding motifs and one (Cys)3His-type Zn(2+)-binding site in the C-terminus. The C-terminal coiled domain is cross-linked to the presumed docking surface of the dimeric S100A3 via a disulfide bridge. The aim of this study was to determine the structural and functional role of the C-terminal Zn(2+)-binding domain, which is unique to S100A3, in homotetramer assembly. The binding of either Ca(2+) or Zn(2+) reduced the α-helix content of S100A3 and modulated its affinity for the other cation. The binding of a single Zn(2+) accelerated the Ca(2+)-dependent tetramerization of S100A3 while inducing an extensive unfolding of helix IV. The Ca(2+) and Zn(2+) binding affinities of S100A3 were enhanced when the other cation bound in concert with the tetramerization of S100A3. Small angle scattering analyses revealed that the overall structure of the S100A3 tetramer bound both Ca(2+) and Zn(2+) had a similar molecular shape to the Ca(2+)-bound form in solution. The binding states of the Ca(2+) or Zn(2+) to each S100A3 subunit within a homotetramer appear to be propagated by sensing the repositioning of helix III and the rearrangement of the C-terminal tail domain. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Structure analysis and characterization of the cytochrome c-554 from thermophilic green sulfur photosynthetic bacterium Chlorobaculum tepidum.

The cytochrome (Cyt) c-554 in thermophilic green photosynthetic bacterium Chlorobaculum tepidum serves as an intermediate electron carrier, transferring electrons to the membrane-bound Cyt c z from various enzymes involved in the oxidations of sulfide, thiosulfate, and sulfite compounds. Spectroscopically, this protein exhibits an asymmetric α-absorption band for the reduced form and particularly large paramagnetic (1)H NMR shifts for the heme methyl groups with an unusual shift pattern in the oxidized form. The crystal structure of the Cyt c-554 has been determined at high resolution. The overall fold consists of four α-helices and is characterized by a remarkably long and flexible loop between the α3 and α4 helices. The axial ligand methionine has S-chirality at the sulfur atom with its C(ε)H3 group pointing toward the heme pyrrole ring I. This configuration corresponds to an orientation of the lone-pair orbital of the sulfur atom directed at the pyrrole ring II and explains the lowest-field (1)H NMR shift arising from the 18(1) heme methyl protons. Differing from most other class I Cyts c, no hydrogen bond was formed between the methionine sulfur atom and polypeptide chain. Lack of this hydrogen bond may account for the observed large paramagnetic (1)H NMR shifts of the heme methyl protons. The surface-exposed heme pyrrole ring II edge is in a relatively hydrophobic environment surrounded by several electronically neutral residues. This portion is considered as an electron transfer gateway. The structure of the Cyt c-554 is compared with those of other Cyts c, and possible interactions of this protein with its electron transport partners are discussed.

Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase type I.

Peptidylarginine deiminase (PAD) catalyzes the post-translational conversion of peptidylarginine to peptidylcitrulline in the presence of calcium ions. Among the five known human PAD isozymes (PAD1-4 and PAD6), PAD1 exhibits the broadest substrate specificity. Crystals of PAD1 obtained using polyethylene glycol 3350 as a precipitant diffracted to 3.70 Å resolution using synchrotron radiation. Two PAD1 molecules were contained in the asymmetric unit and the crystals belonged to space group P6(1), with unit-cell parameters a = b = 90.3, c = 372.3 Å. The solvent content was 58.2%.

A novel hybrid protein composed of superoxide-dismutase-active Cu(II) complex and lysozyme.

A novel hybrid protein composed of a superoxide dismutase-active Cu(II) complex (CuST) and lysozyme (CuST@lysozyme) was prepared. The results of the spectroscopic and electrochemical analyses confirmed that CuST binds to lysozyme. We determined the crystal structure of CuST@lysozyme at 0.92 Å resolution, which revealed that the His15 imidazole group of lysozyme binds to the Cu(II) center of CuST in the equatorial position. In addition, CuST was fixed in position by the weak axial coordination of the Thr89 hydroxyl group and the hydrogen bond between the guanidinium group of the Arg14 residue and the hydroxyl group of CuST. Furthermore, the combination of CuST with lysozyme did not decrease the superoxide dismutase activity of CuST. Based on the spectral, electrochemical, structural studies, and quantum chemical calculations, an O2- disproportionation mechanism catalyzed by CuST@lysozyme is proposed.

Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase type III.

In the presence of calcium ions, human peptidylarginine deiminase (PAD) converts arginine residues in proteins to citrulline. Of the five known human PAD enzymes, the type III isozyme (PAD3) exhibits the highest specificity for synthetic and natural substrates. This study aimed to determine the structure of PAD3 in order to elucidate its selective citrullination mechanism. Crystals of PAD3 obtained using polyethylene glycol 400 as a precipitant diffracted to 2.95 Å resolution using synchrotron radiation. They belonged to space group R3, with unit-cell parameters a = b = 114.97, c = 332.49 Å (hexagonal axes). Assuming two molecules were contained in an asymmetric unit, the calculated Matthews coefficient was 2.83 Å(3) Da(-1), corresponding to a solvent content of 56.6%. Initial phases were determined using PAD4 as a molecular-replacement model.

Autocitrullination and Changes in the Activity of Peptidylarginine Deiminase 3 Induced by High Ca2+ Concentrations.

Peptidylarginine deiminases (PADs) are enzymes that catalyze the Ca2+-dependent conversion of arginine residues into proteins to citrulline residues. Five PAD isozymes have been identified in mammals. Several studies have shown that the active-site pockets of these isozymes are formed when Ca2+ ions are properly bound. We previously characterized the structures of PAD3 in six states. Among these, we identified a "nonproductive" form of PAD3 in which the active site was disordered even though five Ca2+ ions were bound. This strange structure was probably obtained as a result of either high Ca2+ concentration (∼260 mM)-induced denaturation during the crystallization process or high Ca2+-concentration-induced autocitrullination. While autocitrullination has been reported in PAD2 and PAD4 for some time, only a single report on PAD3 has been published recently. In this study, we investigated whether PAD3 catalyzes the autocitrullination reaction and identified autocitrullination sites. In addition to the capacity of PAD3 for autocitrullination, the autocitrullination sites increased depending on the Ca2+ concentration and reaction time. These findings suggest that some of the arginine residues in the "nonproductive" form of PAD3 would be autocitrullinated. Furthermore, most of the autocitrullinated sites in PAD3 were located near the substrate-binding site. Given the high Ca2+ concentration in the crystallization condition, it is likely that Arg372 was citrullinated in the "nonproductive" PAD3 structure, the structure was slightly altered from the active form by citrulline residues, and probably inhibited Ca2+-ion binding at the proper position. Following Arg372 citrullination, PAD3 enters an inactive form; however, the Arg372-citrullinated PAD3 are considered minor components in autocitrullinated PAD3 (CitPAD3), and CitPAD3 does not significantly decrease the enzyme activity. Autocitrullination of PAD3 could not be confirmed at the low Ca2+ concentrations seen in vivo. Future experiments using cells and animals are needed to verify the effect of Ca2+ on the PAD3 structure and functions in vivo.

Heme oxygenase reveals its strategy for catalyzing three successive oxygenation reactions.

Heme oxygenase (HO) is an enzyme that catalyzes the regiospecific conversion of heme to biliverdin IXalpha, CO, and free iron. In mammals, HO has a variety of physiological functions, including heme catabolism, iron homeostasis, antioxidant defense, cellular signaling, and O(2) sensing. The enzyme is also found in plants (producing light-harvesting pigments) and in some pathogenic bacteria, where it acquires iron from the host heme. The HO-catalyzed heme conversion proceeds through three successive oxygenations, a process that has attracted considerable attention because of its reaction mechanism and physiological importance. The HO reaction is unique in that all three O(2) activations are affected by the substrate itself. The first step is the regiospecific self-hydroxylation of the porphyrin alpha-meso carbon atom. The resulting alpha-meso-hydroxyheme reacts in the second step with another O(2) to yield verdoheme and CO. The third O(2) activation, by verdoheme, cleaves its porphyrin macrocycle to release biliverdin and free ferrous iron. In this Account, we provide an overview of our current understanding of the structural and biochemical properties of the complex self-oxidation reactions in HO catalysis. The first meso-hydroxylation is of particular interest because of its distinct contrast with O(2) activation by cytochrome P450. Although most heme enzymes oxidize exogenous substrates by high-valent oxo intermediates, HO was proposed to utilize the Fe-OOH intermediate for the self-hydroxylation. We have succeeded in preparing and characterizing the Fe-OOH species of HO at low temperature, and an analysis of its reaction, together with mutational and crystallographic studies, reveals that protonation of Fe-OOH by a distal water molecule is critical in promoting the unique self-hydroxylation. The second oxygenation is a rapid, spontaneous auto-oxidation of the reactive alpha-meso-hydroxyheme; its mechanism remains elusive, but the HO enzyme has been shown not to play a critical role in it. Until recently, the means of the third O(2) activation had remained unclear as well, but we have recently untangled its mechanistic outline. Reaction analysis of the verdoheme-HO complex strongly suggests the Fe-OOH species as a key intermediate of the ring-opening reaction. This mechanism is very similar to that of the first meso-hydroxylation, including the critical roles of the distal water molecule. A comprehensive study of the three oxygenations of HO highlights the rational design of the enzyme architecture and its catalytic mechanism. Elucidation of the last oxygenation step has enabled a kinetic analysis of the rate-determining step, making it possible to discuss the HO reaction mechanism in relation to its physiological functions.

Characterization of a Cobalt-Substituted Globin-Coupled Oxygen Sensor Histidine Kinase from Anaeromyxobacter sp. Fw109-5: Insights into Catalytic Regulation by Its Heme Coordination Structure.

Heme-based gas sensors are an emerging class of heme proteins. AfGcHK, a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5, is an oxygen sensor enzyme in which oxygen binding to Fe(II) heme in the globin sensor domain substantially enhances its autophosphorylation activity. Here, we reconstituted AfGcHK with cobalt protoporphyrin IX (Co-AfGcHK) in place of heme (Fe-AfGcHK) and characterized the spectral and catalytic properties of the full-length proteins. Spectroscopic analyses indicated that Co(III) and Co(II)-O2 complexes were in a 6-coordinated low-spin state in Co-AfGcHK, like Fe(III) and Fe(II)-O2 complexes of Fe-AfGcHK. Although both Fe(II) and Co(II) complexes were in a 5-coordinated state, Fe(II) and Co(II) complexes were in high-spin and low-spin states, respectively. The autophosphorylation activity of Co(III) and Co(II)-O2 complexes of Co-AfGcHK was fully active, whereas that of the Co(II) complex was moderately active. This contrasts with Fe-AfGcHK, where Fe(III) and Fe(II)-O2 complexes were fully active and the Fe(II) complex was inactive. Collectively, activity data and coordination structures of Fe-AfGcHK and Co-AfGcHK indicate that all fully active forms were in a 6-coordinated low-spin state, whereas the inactive form was in a 5-coordinated high-spin state. The 5-coordinated low-spin complex was moderately active-a novel finding of this study. These results suggest that the catalytic activity of AfGcHK is regulated by its heme coordination structure, especially the spin state of its heme iron. Our study presents the first successful preparation and characterization of a cobalt-substituted globin-coupled oxygen sensor enzyme and may lead to a better understanding of the molecular mechanisms of catalytic regulation in this family.

Detailed structure of mouse interferon α2 and its interaction with Sortilin.

Interferon α (IFNα) is a type I interferon, an essential cytokine employed by the immune system to fight viruses. Although a number of the structures of type I interferons have been reported, most of the known structures of IFNα are in complex with its receptors. There are only two examples of structures of free IFNα: one is a dimeric X-ray structure without side-chain information; and another is an NMR structure of human IFNα. Although we have shown that Sortilin is involved in the secretion of IFNα, the details of the molecular interaction and the secretion mechanism remain unclear. Recently, we solved the X-ray structure of mouse Sortilin, but the structure of mouse IFNα remained unknown. In this study, we determined the crystal structure of mouse IFNα2 at 2.1 Å resolution and investigated its interaction with Sortilin. Docking simulations suggested that Arg22 of mouse IFNα2 is important for the interaction with mouse Sortilin. Mutation of Arg22 to alanine facilitated IFNα2 secretion, as determined by flow cytometry, highlighting the contribution of this residue to the interaction with Sortilin. These results suggest an important role for Arg22 in mouse IFNα for Sortilin-mediated IFNα trafficking.

Enzymatic ring-opening mechanism of verdoheme by the heme oxygenase: a combined X-ray crystallography and QM/MM study.

The least understood mechanism during heme degradation by the enzyme heme oxygenase (HO) is the third step of ring opening of verdoheme to biliverdin, a process which maintains iron homeostasis. In response to this mechanistic uncertainty, we launched a combined study of X-ray crystallography and theoretical QM/MM calculations, designed to elucidate the mechanism. The air-sensitive ferrous verdoheme complex of HmuO, a heme oxygenase from Corynebacterium diphtheriae, was crystallized under anaerobic conditions. Spectral analysis of the azide-bound verdoheme-HmuO complex crystals assures that the verdoheme group remains intact during the crystallization and X-ray diffraction measurement. The structure offers the first solid evidence for the presence of a water cluster in the distal pocket of this catalytically critical intermediate. The subsequent QM/MM calculations based on this crystal structure explore the reaction mechanisms starting from the FeOOH-verdoheme and FeHOOH-verdoheme complexes, which mimic, respectively, the O(2)- and H(2)O(2)-supported degradations. In both mechanisms, the rate-determining step is the initial O-O bond breaking step, which is either homolytic (for FeHOOH-verdoheme) or coupled to electron and proton transfers (in FeOOH-verdoheme). Additionally, the calculations indicate that the FeHOOH-verdoheme complex is more reactive than the FeOOH-verdoheme complex in accord with experimental findings. QM energies with embedded MM charges are close to and yield the same conclusions as full QM/MM energies. Finally, the calculations highlight the dominant influence of the distal water cluster which acts as a biocatalyst for the conversion of verdoheme to biliverdin in the two processes, by fixing the departing OH and directing it to the requisite site of attack, and by acting as a proton shuttle and a haven for the highly reactive OH(-) nucleophile.

Dioxygen activation for the self-degradation of heme: reaction mechanism and regulation of heme oxygenase.

Heme oxygenase (HO) catalyzes the regiospecific conversion of heme to biliverdin, CO, and free iron through three successive oxygenation reactions. HO catalysis is unique in that all three O(2) activations are performed by the substrate itself. This Forum Article overviews our current understanding on the structural and biochemical properties of HO catalysis, especially its first and third oxygenation steps. The HO first step, regiospecific hydroxylation of the porphyrin alpha-meso-carbon atom, is of particular interest because of its sharp contrast to O(2) activation by cytochrome P450. HO was proposed to utilize the FeOOH species but not conventional ferryl hemes as a reactive intermediate for self-hydroxylation. We have succeeded in preparing and characterizing the FeOOH species of HO at low temperature, and our analyses of its reaction, together with mutational and crystallographic studies, reveal that protonation of FeOOH by a distal water molecule is critical in promoting the unique self-hydroxylation. The second oxygenation is a rapid, spontaneous autooxidation of the reactive alpha-meso-hydroxyheme in which the HO enzyme does not play a critical role. Further O(2) activation by verdoheme cleaves its porphyrin macrocycle to form biliverdin and free ferrous iron. This third step has been considered to be a major rate-determining step of HO catalysis to regulate the enzyme activity. Our reaction analysis strongly supports the FeOOH verdoheme as the key intermediate of the ring-opening reaction. This mechanism is very similar to that of the first meso-hydroxylation, and the distal water is suggested to enhance the third step as expected from the similarity. The HO mechanistic studies highlight the catalytic importance of the distal hydrogen-bonding network, and this manuscript also involves our attempts to develop HO inhibitors targeting the unique distal structure.

Optimal Mutant Model of Human S100A3 Protein Citrullinated at Arg51 by Peptidylarginine Deiminase Type III and Its Solution Structural Properties.

S100A3 protein, a member of the EF-hand-type Ca2+-binding S100 protein family, undergoes a Ca2+-/Zn2+-induced structural change to a tetrameric state upon specific citrullination of R51 in human hair cuticular cells. To elucidate the underlying mechanism, we prepared recombinant mutant S100A3 proteins, including R51A, R51C, R51E, R51K, and R51Q, as potential models of post-translationally modified S100A3 and evaluated their biophysical and biochemical properties relative to wild-type (WT) S100A3 and WT citrullinated in vitro. Size exclusion chromatography (SEC) showed that R51Q formed a tetramer in the presence of Ca2+, while Ca2+ titration monitored by Trp fluorescence indicated that R51Q had Ca2+-binding properties similar to those of citrullinated S1003A. We therefore concluded that R51Q is the optimal mutant model of post-translationally modified S100A3. We compared the solution structure of WT S100A3 and the R51Q mutant in the absence and presence of Ca2+ and Zn2+ by SEC-small-angle X-ray scattering. The radius of gyration of R51Q in the metal-free state was almost the same as that of WT; however, it increased by ∼1.5-fold in the presence of Ca2+/Zn2+, indicating a large expansion in molecular size. By contrast, addition of Ca2+/Zn2+ to WT led to nonspecific aggregation in SEC analysis and dynamic light scattering, suggesting that citrullination of S100A3 is essential for stabilization of the Ca2+-/Zn2+-bound state. These findings will lead to the further development of structural analyses for the Ca2+-/Zn2+-bound S100A3.