ABSTRACT
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.
Subject(s)
Superoxide Dismutase , Superoxides , Superoxide Dismutase/metabolism , Superoxides/metabolism , Oxidation-Reduction , Muramidase/metabolism , Copper/chemistryABSTRACT
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.
Subject(s)
Bile Pigments , Oxidoreductases , Bile Pigments/biosynthesis , Bile Pigments/chemistry , Biliverdine/chemistry , Catalysis , Crystallography , Oxidoreductases/genetics , Oxidoreductases/chemistry , MutationABSTRACT
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 (â¼260 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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Drug Design , Enzyme Inhibitors/pharmacology , Protein-Arginine Deiminase Type 3/chemistry , Protein-Arginine Deiminase Type 3/metabolism , Crystallography, X-Ray , Filaggrin Proteins , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein-Arginine Deiminase Type 3/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Interferon-alpha/chemistry , Interferon-alpha/metabolism , Adaptor Proteins, Vesicular Transport/chemistry , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Chromatography, Gel/methods , Crystallography, X-Ray/methods , Humans , Interferon-alpha/genetics , Mice , Molecular Docking Simulation/methods , Mutation , Protein Binding , Protein TransportABSTRACT
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 â¼1.5-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.
ABSTRACT
TRPV1, a member of the transient receptor potential (TRP) channels family, has been found to be involved in redox sensing. The crystal structure of the human TRPV1 ankyrin-repeat domain (TRPV1-ARD) was determined at 4.5â Å resolution under nonreducing conditions. This is the first report of the crystal structure of a ligand-free form of TRPV1-ARD and in particular of the human homologue. The structure showed a unique conformation in finger loop 3 near Cys258, which is most likely to be involved in inter-subunit disulfide-bond formation. Also, in human TRPV1-ARD it was possible for solvent to access Cys258. This structural feature might be related to the high sensitivity of human TRPV1 to oxidants. ESI-MS revealed that Cys258 did not form an S-OH functionality even under nonreducing conditions.
Subject(s)
Ankyrin Repeat/physiology , TRPV Cation Channels/chemistry , TRPV Cation Channels/genetics , Ankyrins/chemistry , Ankyrins/genetics , Ankyrins/metabolism , Crystallization/methods , Humans , Protein Structure, Secondary , TRPV Cation Channels/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Hemerythrin/chemistry , Phosphoric Diester Hydrolases/chemistry , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Betaproteobacteria/enzymology , Catalysis , Cyclic GMP/analogs & derivatives , Cyclic GMP/chemistry , Enzyme Assays , Hemerythrin/isolation & purification , Hydrolysis , Iron/chemistry , Oxidation-Reduction , Phosphoric Diester Hydrolases/isolation & purification , Protein Domains , Sequence AlignmentABSTRACT
Hair is one of the defining characteristics of mammals. The hair shaft has a two-layer structure comprising the cortex, which is the inner layer and is composed of cortical cells, and the cuticle, which is the outermost layer. S100 calcium-binding protein A3 (S100A3) is expressed at high levels in the human hair cuticle. Arginine 51 of S100A3 protein is citrullinated specifically by peptidylarginine deiminase 3 (PAD3), and this citrullination is related to maturation of the cuticle. However, the detailed evolutionary processes of S100A3 and PAD3 during mammalian evolution are unknown. Here, we show that nonsynonymous changes in S100A3 accelerated in the common ancestral branch of mammals, probably as a result of positive selection that returned after the acquisition of hair cuticle-specific function in mammals. Later, pseudogenisation or nonfunctionalisation of S100A3 and PAD3 occurred in some species, such as the cetaceans. Our results show that positive selection and relaxation of the functional constraints of genes played important roles in the evolution of mammalian hair.
Subject(s)
Evolution, Molecular , Hair/chemistry , Mammals/genetics , Protein-Arginine Deiminases/genetics , S100 Proteins/genetics , Selection, Genetic , Amino Acid Sequence , Animals , Phylogeny , Sequence HomologyABSTRACT
In mammals, the green heme metabolite biliverdin is converted to a yellow anti-oxidant by NAD(P)H-dependent biliverdin reductase (BVR), whereas in O2-dependent photosynthetic organisms it is converted to photosynthetic or light-sensing pigments by ferredoxin-dependent bilin reductases (FDBRs). In NADP+-bound and biliverdin-bound BVR-A, two biliverdins are stacked at the binding cleft; one is positioned to accept hydride from NADPH, and the other appears to donate a proton to the first biliverdin through a neighboring arginine residue. During the FDBR-catalyzed reaction, electrons and protons are supplied to bilins from ferredoxin and from FDBRs and waters bound within FDBRs, respectively. Thus, the protonation sites of bilin and catalytic residues are important for the analysis of site-specific reduction. The neutron structure of FDBR sheds light on this issue.
Subject(s)
Bile Pigments/chemistry , Enzymes/chemistry , Quantitative Structure-Activity Relationship , Animals , Bile Pigments/metabolism , Catalysis , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Protein ConformationABSTRACT
Sortilin is a multifunctional sorting receptor involved in cytokine production in immune cells. To understand the mechanism of Sortilin-mediated cytokine trafficking, we determined the 2.45-Å structure of the dimerized Sortilin ectodomain (sSortilin or the Vps10-domain) crystallized at acidic pH. Substantial conformational changes upon dimerization lead to the intermolecular hydrophobic interaction between the conserved E455 and F137. Analysis of the electrostatic surface and size-exclusion chromatography revealed that sSortilin dimerization occurs due to an increase in hydrophobic interactions at the neutral dimer interface at acidic pH. The N682-attached N-glycan in the vicinity of the dimer interface implies its involvement in the dimerization. The disruption of Sortilin dimerization by mutations impairs efficient interferon-alpha secretion from cells. These results suggest the functional importance of Sortilin dimerization in cytokine trafficking.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Protein Interaction Domains and Motifs , Protein Multimerization , Acids/pharmacology , Adaptor Proteins, Vesicular Transport/genetics , Animals , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Humans , Hydrogen-Ion Concentration , Interferon-alpha/metabolism , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Protein Interaction Domains and Motifs/drug effects , Protein Interaction Domains and Motifs/genetics , Protein Multimerization/drug effects , Protein Multimerization/genetics , Protein Structure, Quaternary/genetics , Protein Transport/geneticsABSTRACT
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 etâ 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.
ABSTRACT
Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the ß5 subunit of the 20S proteasome, and not to the other active subunits, ß1 and ß2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells.
Subject(s)
Anesthetics, Local/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Animals , Cattle , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Leupeptins/pharmacology , Lidocaine/analogs & derivatives , Lidocaine/pharmacology , Molecular Docking Simulation , Protein Stability/drug effectsABSTRACT
Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the reduction of biliverdin (BV) to produce phycocyanobilin, a linear tetrapyrrole pigment used for light harvesting and light sensing. Spectroscopic and HPLC analyses inidicate that BV bound to the I86D mutant of PcyA is fully protonated (BVH+ ) and can accept an electron, but I86D is unable to donate protons for the reduction; therefore, compared to the wild-type PcyA, the I86D mutant stabilizes BVH+ . To elucidate the structural basis of the I86D mutation, we determined the atomic-resolution structure of the I86D-BVH+ complex and the protonation states of the essential residues Asp105 and Glu76 in PcyA. Our study revealed that Asp105 adopted a fixed conformation in the I86D mutant, although it had dual conformations in wild-type PcyA which reflected the protonation states of BV. Taken together with biochemical/spectroscopic results, our analysis of the I86D-BVH+ structure supports the hypothesis that flexibility of Asp105 is essential for the catalytic activity of PcyA.
Subject(s)
Bacterial Proteins/chemistry , Biliverdine/chemistry , Molecular Docking Simulation , Oxidoreductases/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biliverdine/metabolism , Binding Sites , Mutation , Oxidoreductases/genetics , Oxidoreductases/metabolism , Protein Binding , Synechocystis/enzymologyABSTRACT
Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of arginine in protein molecules to a citrulline residue in a Ca(2+)-dependent manner. In this study, we determined the structure of an active form of human PAD1 crystallized in the presence of Ca(2+) at 3.2-Å resolution. Although human PAD2 and PAD4 isozymes were previously reported to form a head-to-tail homodimer, it is still unknown whether this quaternary structure is common to other PAD isozymes. The asymmetric unit of the crystal contained two PAD1 molecules; however, the head-to-tail dimeric form was not found. Small-angle X-ray scattering analyses revealed PAD1 to be a monomer in solution, while PAD3 was dimerized with a structure similar to PAD2 and PAD4. PAD1 was apparently different from the crystal structures of PAD2 and PAD4, with an elongated N-terminal loop that appears to prevent the formation of the homodimer. Of interest, the N-terminal loop occupied the substrate binding site of the adjacent PAD1 molecules in the crystal. Deimination of S100A3 peptides in vitro implied that PAD isozymes recognize the quaternary structure of S100A3. The substrate-accessible monomeric structure brought about by the extension of its N terminus may partly account for the highest tolerant substrate recognition of PAD1. This is the first ever report on the molecular structure of PAD1 demonstrating the unique monomeric form of the PAD isozyme.
Subject(s)
Hydrolases/chemistry , Arginine/chemistry , Binding Sites/physiology , Crystallography, X-Ray/methods , Humans , Isoenzymes/chemistry , Protein-Arginine Deiminase Type 1 , Protein-Arginine Deiminase Type 2 , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , X-RaysABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biliverdine/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Synechocystis/enzymology , Crystallography , Crystallography, X-Ray , Models, Molecular , Neutron Diffraction , Protons , Synechocystis/chemistry , Synechocystis/metabolismABSTRACT
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%.