Publications by authors named "Hyun Ho Park"

167 Publications

Molecular basis of IRGB10 oligomerization and membrane association for pathogen membrane disruption.

Commun Biol 2021 Jan 19;4(1):92. Epub 2021 Jan 19.

College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea.

Immunity-related GTPase B10 (IRGB10) belongs to the interferon (IFN)-inducible GTPases, a family of proteins critical to host defense. It is induced by IFNs after pathogen infection, and plays a role in liberating pathogenic ligands for the activation of the inflammasome by directly disrupting the pathogen membrane. Although IRGB10 has been intensively studied owing to its functional importance in the cell-autonomous immune response, the molecular mechanism of IRGB10-mediated microbial membrane disruption is still unclear. In this study, we report the structure of mouse IRGB10. Our structural study showed that IRGB10 bound to GDP forms an inactive head-to-head dimer. Further structural analysis and comparisons indicated that IRGB10 might change its conformation to activate its membrane-binding and disruptive functions. Based on this observation, we propose a model of the working mechanism of IRGB10 during pathogen membrane disruption.
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http://dx.doi.org/10.1038/s42003-020-01640-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815755PMC
January 2021

Phytochemicals as Anti-Inflammatory Agents in Animal Models of Prevalent Inflammatory Diseases.

Molecules 2020 Dec 15;25(24). Epub 2020 Dec 15.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 52828, Korea.

Phytochemicals are known to have anti-inflammatory effects in vitro and in vivo, such as in inflammatory disease model systems. Inflammation is an essential immune response to exogenous stimuli such as infection and injury. Although inflammation is a necessary host-defense mechanism, chronic inflammation is associated with the continuous local or systemic release of inflammatory mediators, non-cytokine mediators, such as ROS and NO, and inflammatory cytokines are strongly implicated in the pathogenesis of various inflammatory disorders. Phytochemicals that exhibit anti-inflammatory mechanisms that reduce sustained inflammation could be therapeutic candidates for various inflammatory diseases. These phytochemicals act by modulating several main inflammatory signaling pathways, including NF-κB, MAPKs, STAT, and Nrf-2 signaling. Here, we discuss the characteristics of phytochemicals that possess anti-inflammatory activities in various chronic inflammatory diseases and review the molecular signaling pathways altered by these anti-inflammatory phytochemicals, with a focus on transcription factor pathways. Furthermore, to evaluate the phytochemicals as drug candidates, we translate the effective doses of phytochemicals in mice or rat disease models into the human-relevant equivalent and compare the human-relevant equivalent doses of several phytochemicals with current anti-inflammatory drugs doses used in different types of chronic inflammatory diseases.
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http://dx.doi.org/10.3390/molecules25245932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765227PMC
December 2020

Structural and biochemical analyses of an aminoglycoside 2'-N-acetyltransferase from Mycolicibacterium smegmatis.

Sci Rep 2020 12 9;10(1):21503. Epub 2020 Dec 9.

Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon, 21990, Republic of Korea.

The expression of aminoglycoside-modifying enzymes represents a survival strategy of antibiotic-resistant bacteria. Aminoglycoside 2'-N-acetyltransferase [AAC(2')] neutralizes aminoglycoside drugs by acetylation of their 2' amino groups in an acetyl coenzyme A (CoA)-dependent manner. To understand the structural features and molecular mechanism underlying AAC(2') activity, we overexpressed, purified, and crystallized AAC(2') from Mycolicibacterium smegmatis [AAC(2')-Id] and determined the crystal structures of its apo-form and ternary complexes with CoA and four different aminoglycosides (gentamicin, sisomicin, neomycin, and paromomycin). These AAC(2')-Id structures unraveled the binding modes of different aminoglycosides, explaining the broad substrate specificity of the enzyme. Comparative structural analysis showed that the α4-helix and β8-β9 loop region undergo major conformational changes upon CoA and substrate binding. Additionally, structural comparison between the present paromomycin-bound AAC(2')-Id structure and the previously reported paromomycin-bound AAC(6')-Ib and 30S ribosome structures revealed the structural features of paromomycin that are responsible for its antibiotic activity and AAC binding. Taken together, these results provide useful information for designing AAC(2') inhibitors and for the chemical modification of aminoglycosides.
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http://dx.doi.org/10.1038/s41598-020-78699-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725843PMC
December 2020

The crystal structure of mouse IRG1 suggests that cis-aconitate decarboxylase has an open and closed conformation.

PLoS One 2020 1;15(12):e0242383. Epub 2020 Dec 1.

College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.

Protein Data Bank Accession Codes: Coordinate and structural factors were deposited with the Protein Data Bank under PDB ID: 7BR9.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242383PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707506PMC
January 2021

Wide-open conformation of UDP-MurNc-tripeptide ligase revealed by the substrate-free structure of MurE from Acinetobacter baumannii.

FEBS Lett 2021 Jan 3;595(2):275-283. Epub 2020 Dec 3.

Department of Global Innovative Drugs, Graduate School of Chung-Ang University, Seoul, Korea.

MurE ligase catalyzes the attachment of meso-diaminopimelic acid to the UDP-MurNAc- -Ala- -Glu using ATP and producing UDP-MurNAc- -Ala- -Glu-meso-A pm during bacterial cell wall biosynthesis. Owing to the critical role of this enzyme, MurE is considered an attractive target for antibacterial drugs. Despite extensive studies on MurE ligase, the structural dynamics of its conformational changes are still elusive. In this study, we present the substrate-free structure of MurE from Acinetobacter baumannii, which is an antibiotic-resistant superbacterium that has threatened global public health. The structure revealed that MurE has a wide-open conformation and undergoes wide-open, intermediately closed, and fully closed dynamic conformational transition. Unveiling structural dynamics of MurE will help to understand the working mechanism of this ligase and to design next-generation antibiotics targeting MurE.
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http://dx.doi.org/10.1002/1873-3468.14007DOI Listing
January 2021

A high-resolution (1.2 Å) crystal structure of the anti-CRISPR protein AcrIF9.

FEBS Open Bio 2020 Dec 5;10(12):2532-2540. Epub 2020 Nov 5.

Department of Global Innovative Drugs, Graduate School of Chung-Ang University, Seoul, Korea.

Prokaryotic adaptive immunity by CRISPR-Cas systems, which confer resistance to foreign genetic elements, has been used by bacteria to combat viruses. To cope, viruses evolved multiple anti-CRISPR proteins, which can inhibit system function through various mechanisms. Although the structures and mechanisms of several anti-CRISPR proteins have been elucidated, those of the AcrIF9 family have not yet been identified. To understand the molecular basis underlying AcrIF9 anti-CRISPR function, we determined the 1.2 Å crystal structure of AcrIF9. Structural and biochemical studies showed that AcrIF9 exists in monomeric form in solution and can directly interact with DNA using a positively charged cleft. Based on analysis of the structure, we suggest part of the anti-CRISPR molecular mechanism by AcrIF9.
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http://dx.doi.org/10.1002/2211-5463.12986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714069PMC
December 2020

A 1.3 Å high-resolution crystal structure of an anti-CRISPR protein, AcrI E2.

Biochem Biophys Res Commun 2020 Dec 25;533(4):751-757. Epub 2020 Sep 25.

Department of Global Innovative Drugs, Graduate School of Chung-Ang University, Seoul, 06974, Republic of Korea; College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea. Electronic address:

As a result of bacterial infection with viruses, bacteria have developed CRISPR-Cas as an adaptive immune system, which allows them to destroy the viral genetic material introduced via infection. However, viruses have also evolved to develop multiple anti-CRISPR proteins, which are capable of inactivating the CRISPR-Cas adaptive immune system to combat bacteria. In this study, we aimed to elucidate the molecular mechanisms associated with anti-CRISPR proteins by determining a high-resolution crystal structure (1.3 Å) of Type I-E anti-CRISPR protein called AcrIE2. Our structural analysis revealed that AcrIE2 was composed of unique folds comprising five antiparallel β-sheets (β1∼β5) surrounding one α-helix (α1) in the order, ββαβββ. Structural comparison of AcrIE2 with a structural homolog called AcrIF9 showed that AcrIE2 contained a long and flexible β4-β5 connecting loop and a distinct surface feature. These results indicated that the inhibitory mechanism of AcrIE2 might be different from that of AcrIF9. This unique structure of AcrIE2 indicates its special mode of CRISPR-Cas inhibitory activity. Therefore, this study helps us understand the diversity in the inhibitory mechanisms of Acr family.
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http://dx.doi.org/10.1016/j.bbrc.2020.09.067DOI Listing
December 2020

Structural analysis of a novel substrate-free form of the aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.

Acta Crystallogr F Struct Biol Commun 2020 Aug 28;76(Pt 8):364-371. Epub 2020 Jul 28.

College of Pharmacy, Chung-Ang University, Dongjak-gu, Seoul 06974, Republic of Korea.

Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl-CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6')-Ii enzymes target the amino group linked to the 6' C atom in an aminoglycoside. Several structures of the AAC(6')-Ii from Enterococcus faecium [Ef-AAC(6')-Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef-AAC(6')-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6')-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6')-Ii.
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http://dx.doi.org/10.1107/S2053230X20009735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397467PMC
August 2020

Enzymatic reaction mechanism of cis-aconitate decarboxylase based on the crystal structure of IRG1 from Bacillus subtilis.

Sci Rep 2020 07 9;10(1):11305. Epub 2020 Jul 9.

College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea.

Itaconate, which is formed by decarboxylation of cis-aconitate-an intermediate metabolite in the tricarboxylic acid cycle-has been used as a building block in polymer synthesis and is an important chemical in several biomedical and industrial applications. Itaconate is an immunometabolite with antibacterial, antiviral, immunoregulatory, and tumor-promoting activities. Recent focus has been on the role of itaconate in the field of immunology, with immune-responsive gene 1 (IRG1) being identified as the cis-aconitate decarboxylase responsible for itaconate production. We solved the structure of IRG1 from Bacillus subtilis (bsIRG1) and showed that IRG1 adopts either a closed or an open conformation; bsIRG1 was in the open form. A1 and A2 loops around the active site are flexible and can control the formation of the open and closed forms of IRG1. An in silico docking simulation showed that only the open form of IRG1 can accommodate the substrate. The most energetically favorable position of cis-aconitate in the active site of bsIRG1 involved the localization of C2 and C5 of cis-aconitate into the H102 region and H151 region of bsIRG1, respectively. Based on the structural study of bsIRG1, compared with IDS epimerase, and in silico docking simulation, we proposed two tentative enzymatic reaction mechanisms of IRG1, a two-base model and a one-base model.
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http://dx.doi.org/10.1038/s41598-020-68419-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347537PMC
July 2020

Heat dissipative mechanical damping properties of EPDM rubber composites including hybrid fillers of aluminium nitride and boron nitride.

Soft Matter 2020 Aug 7;16(29):6812-6818. Epub 2020 Jul 7.

School of Chemical Engineering, Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea.

As highly integrated electronic devices and automotive parts are becoming used in high-power and load-bearing systems, thermal conductivity and mechanical damping properties have become critical factors. In this study, we applied two different fillers of aluminium nitride (AlN) and boron nitride (BN), having polygonal and platelet shapes, respectively, into ethylene-propylene-diene monomer (EPDM) rubber to ensure improved thermo-mechanical properties of EPDM composites. These two different shapes are considered advantageous in providing effective pathways of phonon transfer as well as facilitating sliding movement of packed particles. When the volume ratio of AlN : BN was 1 : 1, the thermal conductivity of the hybrid-filler system (EPDM/AlN/BN) increased in comparison to that of the single-filler system (EPDM/AlN) of 3.03 to 4.76 W m K. The coefficient of thermal expansion (CTE) and thermal distortion parameter (TDP) substantially decreased from 59.3 ppm °C and 17.5 m K of EPDM/AlN, to 39.7 ppm °C and 8.4 m K of EPDM/AlN/BN, representing reductions of 33 and 52%, respectively. Moreover, the damping coefficient of EPDM/AlN/BN was greatly increased to 0.5 of at 50 °C, compared to 0.03 of neat EPDM. These excellent performances likely stem from the effective packing of AlN/BN hybrid fillers, which could induce facile energy transfer and effective energy dissipation by the sliding movement of the adjacent hybrid fillers in the EPDM matrix.
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http://dx.doi.org/10.1039/c9sm02123jDOI Listing
August 2020

Crystal structure of the human NLRP9 pyrin domain reveals a bent N-terminal loop that may regulate inflammasome assembly.

FEBS Lett 2020 Aug 28;594(15):2396-2405. Epub 2020 Jun 28.

College of Pharmacy, Chung-Ang University, Seoul, Korea.

Members of the NLR family pyrin domain containing (NLRPs) are pattern recognition receptors that participate in innate immunity. They form inflammasomes, which are platforms for caspase-1 recruitment and activation. The NLRP pyrin domain (PYD) is critical for the assembly of inflammasomes due to its ability to mediate protein interactions. Despite intensive structural studies on inflammasomes with PYDs, the structure of the PYD of NLRP9-the least studied member of the family-remains unknown. Herein, we report the crystal structure of the human NLRP9 PYD at 2.1 Å resolution, which reveals a kinked N-terminal loop oriented toward the interior of the helical bundle. Based on our findings, we propose a regulatory role for the kinked N-terminal loop of NLRP9 PYD in inflammasome assembly.
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http://dx.doi.org/10.1002/1873-3468.13866DOI Listing
August 2020

Comparison of Target Recognition by TRAF1 and TRAF2.

Int J Mol Sci 2020 Apr 21;21(8). Epub 2020 Apr 21.

College of Pharmacy, Chung-Ang University, Dongjag-gu, Seoul 06974, Korea.

Although TRAF1 and TRAF2 share common receptors and have extremely conserved amino acid residues, recent studies have shown that key differences in receptor binding preferences with different affinities exist, which might be important for their different functions in TRAF-mediated signal transduction. To better understand TRAF1 and TRAF2 signaling, we analyzed and compared their receptor binding-affinities. Our study revealed that TRADD, TANK, and caspase-2 bind to both TRAF1 and TRAF2 with different affinities in vitro. Sequence and structural analyses revealed that S454 on TRAF2 (corresponding to A369 of TRAF1) is critical for the binding of TRADD, and F347 on TRAF1 (corresponding to L432 of TRAF2) is a critical determinant for high affinity binding of TANK and caspase-2.
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http://dx.doi.org/10.3390/ijms21082895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215387PMC
April 2020

Structural and sequence comparisons of bacterial enoyl-CoA isomerase and enoyl-CoA hydratase.

J Microbiol 2020 Jul 22;58(7):606-613. Epub 2020 Apr 22.

Unit of Research for Practical Application, Korea Polar Research Institute, Incheon, 21990, Republic of Korea.

Crystal structures of enoyl-coenzyme A (CoA) isomerase from Bosea sp. PAMC 26642 (BoECI) and enoyl-CoA hydratase from Hymenobacter sp. PAMC 26628 (HyECH) were determined at 2.35 and 2.70 Å resolution, respectively. BoECI and HyECH are members of the crotonase superfamily and are enzymes known to be involved in fatty acid degradation. Structurally, these enzymes are highly similar except for the orientation of their C-terminal helix domain. Analytical ultracentrifugation was performed to determine the oligomerization states of BoECI and HyECH revealing they exist as trimers in solution. However, their putative ligand-binding sites and active site residue compositions are dissimilar. Comparative sequence and structural analysis revealed that the active site of BoECI had one glutamate residue (Glu135), this site is occupied by an aspartate in some ECIs, and the active sites of HyECH had two highly conserved glutamate residues (Glu118 and Glu138). Moreover, HyECH possesses a salt bridge interaction between Glu98 and Arg152 near the active site. This interaction may allow the catalytic Glu118 residue to have a specific conformation for the ECH enzyme reaction. This salt bridge interaction is highly conserved in known bacterial ECH structures and ECI enzymes do not have this type of interaction. Collectively, our comparative sequential and structural studies have provided useful information to distinguish and classify two similar bacterial crotonase superfamily enzymes.
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http://dx.doi.org/10.1007/s12275-020-0089-1DOI Listing
July 2020

Structural and biochemical characterization of TRAF5 from Notothenia coriiceps and its implications in fish immune cell signaling.

Fish Shellfish Immunol 2020 Jul 10;102:56-63. Epub 2020 Apr 10.

College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea. Electronic address:

Conserved immune cell signaling in fish was recently highlighted by the identification of various immune cell signaling molecules. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are critical adaptor molecules in immune cell signaling and contain E3 ubiquitin ligase activity. Here, we report the first crystal structure of the TRAF5 TRAF domain from the black rockcod (Notothenia coriiceps; ncTRAF5). Our structure revealed both similarities and differences with mammalian TRAF5. Structural and biochemical analyses indicated that ncTRAF5 forms a functional trimer unit in solution, with a structural flexibility that might be critical for imparting resistance to cold temperature-induced stress. We also found conserved surface residues on ncTRAF5 that might be critical binding hot spots for interaction with various receptors.
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http://dx.doi.org/10.1016/j.fsi.2020.04.016DOI Listing
July 2020

Structures of Human Transglutaminase 2: Finding Clues for Interference in Cross-linking Mediated Activity.

Int J Mol Sci 2020 Mar 23;21(6). Epub 2020 Mar 23.

College of Pharmacy, Chung-Ang University, Seoul 06974, Korea.

Human transglutaminase 2 (TGase2) has various functions, including roles in various cellular processes such as apoptosis, development, differentiation, wound healing, and angiogenesis, and is linked to many diseases such as cancer. Although TGase2 has been considered an optimized drug target for the treatment of cancer, fibrosis, and neurodegenerative disorders, it has been difficult to generate TGase2-targeted drugs for clinical use because of the relatively flat and broad active site on TGase2. To design more specific and powerful inhibitors, detailed structural information about TGase2 complexed with various effector and inhibitor molecules is required. In this review, we summarized the current structural studies on TGase2, which will aid in designing drugs that can overcome the aforementioned limitations.
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http://dx.doi.org/10.3390/ijms21062225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139744PMC
March 2020

Assembly of platforms for signal transduction in the new era: dimerization, helical filament assembly, and beyond.

Exp Mol Med 2020 03 5;52(3):356-366. Epub 2020 Mar 5.

College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea.

Supramolecular organizing center (SMOC)-mediated signal transduction is an emerging concept in the field of signal transduction that is ushering in a new era. The formation of location-specific, higher-order SMOCs is particularly important for cell death and innate immune signaling processes. Several protein interaction domains, including the death domain (DD) superfamily and the CIDE domain, are representative mediators of SMOC assembly in cell death and innate immune signaling pathways. DD superfamily- and CIDE domain-containing proteins form SMOCs that activate various caspases and provide signaling scaffold platforms. These assemblies can lead to signal transduction and amplification during signaling events. In this review, we summarize recent findings on the molecular basis of DD superfamily- and CIDE domain-mediated SMOC formation.
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http://dx.doi.org/10.1038/s12276-020-0391-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156525PMC
March 2020

Loss-of-function of EBP50 is a new cause of hereditary peripheral neuropathy: EBP50 functions in peripheral nerve system.

Glia 2020 Sep 20;68(9):1794-1809. Epub 2020 Feb 20.

Department of Pharmacology, Brain Science and Engineering Institute, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu, Republic of Korea.

Finding causative genetic mutations is important in the diagnosis and treatment of hereditary peripheral neuropathies. This study was conducted to find new genes involved in the pathophysiology of hereditary peripheral neuropathy. We identified a new mutation in the EBP50 gene, which is co-segregated with neuropathic phenotypes, including motor and sensory deficit in a family with Charcot-Marie-Tooth disease. EBP50 is known to be important for the formation of microvilli in epithelial cells, and the discovery of this gene mutation allowed us to study the function of EBP50 in the nervous system. EBP50 was strongly expressed in the nodal and paranodal regions of sciatic nerve fibers, where Schwann cell microvilli contact the axolemma, and at the growth tips of primary Schwann cells. In addition, EBP50 expression was decreased in mouse models of peripheral neuropathy. Knockout mice were used to study EBP50 function in the peripheral nervous system. Interestingly motor function deficit and abnormal histology of nerve fibers were observed in EBP50 heterozygous mice at 12 months of age, but not 3 months. in vitro studies using Schwann cells showed that NRG1-induced AKT activation and migration were significantly reduced in cells overexpressing the I325V mutant of EBP50 or cells with knocked-down EBP50 expression. In conclusion, we show for the first time that loss of function due to EBP50 gene deficiency or mutation can cause peripheral neuropathy.
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http://dx.doi.org/10.1002/glia.23805DOI Listing
September 2020

Competitive Binding of Magnesium to Calcium Binding Sites Reciprocally Regulates Transamidase and GTP Hydrolysis Activity of Transglutaminase 2.

Int J Mol Sci 2020 Jan 25;21(3). Epub 2020 Jan 25.

Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul 03080, Korea.

Transglutaminase 2 (TG2) is a Ca-dependent enzyme, which regulates various cellular processes by catalyzing protein crosslinking or polyamination. Intracellular TG2 is activated and inhibited by Ca and GTP binding, respectively. Although aberrant TG2 activation has been implicated in the pathogenesis of diverse diseases, including cancer and degenerative and fibrotic diseases, the structural basis for the regulation of TG2 by Ca and GTP binding is not fully understood. Here, we produced and analyzed a Ca-containing TG2 crystal, and identified two glutamate residues, E437 and E539, as Ca-binding sites. The enzymatic analysis of the mutants revealed that Ca binding to these sites is required for the transamidase activity of TG2. Interestingly, we found that magnesium (Mg) competitively binds to the E437 and E539 residues. The Mg binding to these allosteric sites enhances the GTP binding/hydrolysis activity but inhibits transamidase activity. Furthermore, HEK293 cells transfected with mutant TG2 exhibited higher transamidase activity than cells with wild-type TG2. Cells with wild-type TG2 showed an increase in transamidase activity under Mg-depleted conditions, whereas cells with mutant TG2 were unaffected. These results indicate that E437 and E539 are Ca-binding sites contributing to the reciprocal regulation of transamidase and GTP binding/hydrolysis activities of TG2 through competitive Mg binding.
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http://dx.doi.org/10.3390/ijms21030791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037829PMC
January 2020

Crystal structure of the reactive intermediate/imine deaminase A homolog from the Antarctic bacterium Psychrobacter sp. PAMC 21119.

Biochem Biophys Res Commun 2020 02 27;522(3):585-591. Epub 2019 Nov 27.

College of Pharmacy, Chung-Ang University, Dongjak-gu, Seoul, 06974, Republic of Korea. Electronic address:

The RidA subfamily proteins catalyze the deamination reaction of enamine/imine intermediates, which are metabolites of amino acids such as threonine and serine. Numerous structural and functional studies have been conducted on RidA isolated from mesophiles and thermophiles. However, little is known about the structure of the RidA proteins isolated from psychrophiles. In the present study, we elucidated the crystal structure of RidA from the Antarctic bacterium Psychrobacter sp. PAMC 21119 (Pp-RidA) at 1.6 Å resolution to identify the structural properties contributing to cold-adaptability. Although the overall structure of Pp-RidA is similar to those of its homologues, it exhibits specific structural arrangements of a loop positioned near the active site, which is assumed to play a role in covering the active site of catalysis. In addition, the surface electrostatic potential of Pp-RidA suggested that it exhibits stronger electrostatic distribution relative to its homologues. Our results provide novel insights into the key determinants of cold-adaptability.
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http://dx.doi.org/10.1016/j.bbrc.2019.11.139DOI Listing
February 2020

Structural insights into the enzyme specificity of a novel ω-transaminase from the thermophilic bacterium Sphaerobacter thermophilus.

J Struct Biol 2019 12 24;208(3):107395. Epub 2019 Sep 24.

College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea. Electronic address:

Transaminases are pyridoxal 5'-phosphate-dependent enzymes that reversibly catalyze transamination reactions from an amino group donor substrate to an amino group acceptor substrate. ω-Transaminases (ωTAs) utilize compounds with an amino group not at α-carbon position as their amino group donor substrates. Recently, a novel ωTA with broad substrate specificity and high thermostability from the thermophilic bacterium Sphaerobacter thermophilus (St-ωTA) has been reported. Although St-ωTA has been biochemically characterized, little is known about its determinants of substrate specificity. In the present study, we determined the crystal structure of St-ωTA at 1.9 Å resolution to clarify in detail its mechanism of substrate recognition. The structure of St-ωTA revealed that it has a voluminous active site resulting from the unique spatial arrangement of residues comprising its active site. In addition, our molecular docking simulation results suggest that substrate compounds may bind to active site residues via electrostatic interactions or hydrophobic interactions that can be induced by subtle rearrangements of active site residues. On the basis of these structural analyses, we propose a plausible working model of the enzymatic mechanism of St-ωTA. Our results provide profound structural insights into the substrate specificity of St-ωTA and extend the boundaries of knowledge of TAs.
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http://dx.doi.org/10.1016/j.jsb.2019.09.012DOI Listing
December 2019

Structural Consideration of the Working Mechanism of Fold Type I Transaminases From Eubacteria: Overt and Covert Movement.

Comput Struct Biotechnol J 2019 23;17:1031-1039. Epub 2019 Jul 23.

College of Pharmacy, Chung-Ang University, Dongjak-gu, Seoul 06974, Republic of Korea.

Transaminases (TAs) reversibly catalyze the transfer reaction of an amino group between an amino group donor and an amino group acceptor, using pyridoxal 5'-phosphate (PLP) as a cofactor. TAs are categorized according to the amino group position of the donor substrate and respective TAs recognize their own specific substrates. Over the past decade, a number of TA structures have been determined by X-ray crystallography. On the basis of the structural information, the detailed mechanism of substrate recognition by TAs has also been elucidated. In this review, fold type I TAs are addressed intensively. Comparative studies on structural differences between the apo and holo forms of fold type I TAs have demonstrated that regions containing the active site exhibit structural plasticity in the apo form, facilitating PLP insertion into the active site. In addition, given that TAs recognize two different kinds of substrates, they possess dual substrate specificity. It is known that spatial rearrangements of active site residues occur upon binding of the substrates. Intriguingly, positively charged residues are predominantly distributed at the active site cavity. The electric field generated by such charge distributions may attract negatively charged molecules, such as PLP and amino group acceptors, into the active site. Indeed, TAs show remarkable dynamics in diverse aspects. In this review, we describe the comprehensive working mechanism of fold type I TAs, with a focus on conformational changes.
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http://dx.doi.org/10.1016/j.csbj.2019.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698932PMC
July 2019

Domain swapping of death domain superfamily: Alternative strategy for dimerization.

Authors:
Hyun Ho Park

Int J Biol Macromol 2019 Oct 24;138:565-572. Epub 2019 Jul 24.

College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea. Electronic address:

The role of death domain (DD) protein-mediated inter-protein interactions in cell death and immune cell signaling have been extensively investigated as they are tentative targets for therapeutic interventions and are involved in signal transduction. Structural studies, especially those involving the recent advanced cryo-electron microscopy, indicated that the DD superfamily proteins can assemble into different forms of oligomers, including homo and heterodimer, honey comb-like circular oligomer, and helical filament via three types of interactions, namely type I, type II, and type III. Recently, several structural reports indicated that domain swapping-mediated dimerization of the DD superfamily proteins might be an alternative oligomerization strategy in this family of protein interacting domains. In this review, all the binding strategies associated with the DD superfamily are summarized with a special focus on the novel domain swapping mechanism.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.07.139DOI Listing
October 2019

Various approaches for measurement of synaptic vesicle endocytosis at the central nerve terminal.

Arch Pharm Res 2019 Jun 21;42(6):455-465. Epub 2019 May 21.

College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea.

At the presynaptic terminal, neurotransmitters are stored in synaptic vesicles (SVs), which are released and recycled via exo- and endocytosis. SV endocytosis is crucial for sustaining synaptic transmission by maintaining the SV pool. Many studies have shown that presynaptic dysfunction, particularly impairment of SV endocytosis, is related to neurological disorders. Notably, the presynaptic terminal is considered to be a sensitive structure because certain presynaptic dysfunctions, manifested as impaired SV endocytosis or ultrastructural changes in the presynaptic terminal, can be observed before there is a biochemical or pathological evidence of a neurological disorder. Therefore, monitoring and assessing the presynaptic function by SV endocytosis facilitates the development of early markers for neurological disorders. In this study, we reviewed the current methods for assessing and visualizing SV endocytosis at the central nerve terminal.
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http://dx.doi.org/10.1007/s12272-019-01161-wDOI Listing
June 2019

Structural basis of substrate recognition by a novel thermostable (S)-enantioselective ω-transaminase from Thermomicrobium roseum.

Sci Rep 2019 05 6;9(1):6958. Epub 2019 May 6.

College of Pharmacy, Chung-Ang University, Dongjak-gu, Seoul, 06974, Republic of Korea.

Transaminases catalyze the reversible transfer reaction of an amino group between a primary amine and an α-keto acid, utilizing pyridoxal 5'-phosphate as a cofactor. ω-transaminases (ωTAs) recognize an amino group linked to a non-α carbon of amine substrates. Recently, a novel (S)-enantioselective ωTA from Thermomicrobium roseum (Tr-ωTA) was identified and its enzymatic activity reported. However, the detailed mechanism of (S)-enantioselective substrate recognition remained unclear. In this study, we determined the crystal structure of Tr-ωTA at 1.8 Å resolution to elucidate the mechanism underlying Tr-ωTA substrate (S)-enantioselectivity. A structural analysis of Tr-ωTA along with molecular docking simulations revealed that two pockets at the active site tightly restrict the size and orientation of functional groups of substrate candidates. Based on the structural information and docking simulation results, we propose a comprehensive catalytic mechanism of Tr-ωTA. The present study thus provides structural and functional insights into the (S)-enantioselectivity of Tr-ωTA.
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http://dx.doi.org/10.1038/s41598-019-43490-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502798PMC
May 2019

Crystal structure of a transcription factor, GerE (PaGerE), from spore-forming bacterium Paenisporosarcina sp. TG-14.

Biochem Biophys Res Commun 2019 05 6;513(2):374-379. Epub 2019 Apr 6.

Unit of Polar Genomics, Korea Polar Research Institute, Incheon, 21990, Republic of Korea; Department of Polar Sciences, University of Science and Technology, Incheon, 21990, Republic of Korea. Electronic address:

In cold and harsh environments such as glaciers and sediments in ice cores, microbes can survive by forming spores. Spores are composed of a thick coat protein, which protects against external factors such as heat-shock, high salinity, and nutrient deficiency. GerE is a key transcription factor involved in spore coat protein expression in the mother cell during sporulation. GerE regulates transcription during the late sporulation stage by directly binding to the promoter of cotB gene. Here, we report the crystal structure of PaGerE at 2.09 Å resolution from Paenisporosarcina sp. TG-14, which was isolated from the Taylor glacier. The PaGerE structure is composed of four α-helices and adopts a helix-turn-helix architecture with 68 amino acid residues. Based on our DNA binding analysis, the PaGerE binds to the promoter region of CotB to affect protein expression. Additionally, our structural comparison studies suggest that DNA binding by PaGerE causes a conformational change in the α4-helix region, which may strongly induce dimerization of PaGerE.
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http://dx.doi.org/10.1016/j.bbrc.2019.04.019DOI Listing
May 2019

Preparation of stable recombinant Osm1 noncovalently bound with flavin adenosine dinucleotide cofactor for structural study.

Acta Crystallogr F Struct Biol Commun 2019 Mar 20;75(Pt 3):159-165. Epub 2019 Feb 20.

College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea.

Osm1, a soluble fumarate reductase from Saccharomyces cerevisiae, is localized in both the mitochondria and the endoplasmic reticulum (ER). OSM1 genetically interacts with ERO1, which encodes an essential ER oxidoreductase for disulfide-bond formation under anaerobic conditions. However, the detailed enzymatic mechanisms involved in this interaction and the cellular roles of Osm1 are not fully understood. In this study, monomeric and stable recombinant Osm1 was successfully prepared for structural study. During purification, it was realized that the majority of recombinant Osm1 expressed in Escherichia coli lacked the flavin adenosine dinucleotide (FAD) cofactor. However, exogenously introduced FAD could be incorporated into recombinant Osm1, generating stable and homogenous holo Osm1. Moreover, after removing a flexible fragment by limited proteolysis, holo Osm1 formed isotropic crystals that retained catalytic activity. X-ray diffraction data were successfully collected from the Osm1 crystals to a resolution of 1.75 Å.
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http://dx.doi.org/10.1107/S2053230X19000190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404855PMC
March 2019

Caspase recruitment domains for protein interactions in cellular signaling (Review).

Authors:
Hyun Ho Park

Int J Mol Med 2019 Mar 11;43(3):1119-1127. Epub 2019 Jan 11.

Department of Pharmacy, College of Pharmacy, Chung‑Ang University, Seoul 06974, Republic of Korea.

The caspase recruitment domain (CARD), a well‑known protein interaction module, belongs to the death domain (DD) superfamily, which includes DDs, death effector domains, and pyrin domains. The DD superfamily mediates the protein interactions necessary for apoptosis and immune cell signaling pathways. Among these domains, the CARD has been studied extensively as it mediates important cellular signaling events that are associated with various human diseases including cancer, neuro‑degenerative diseases and immune disorders. Homo‑type and hetero‑type CARD‑CARD interactions mediate the formation of large signaling complexes, including caspase‑activating complexes and downstream signaling complexes. The present review summarizes and discusses the results of structural studies of various CARDs and their complexes. These studies shed light on the mechanisms that control the assembly and disassembly of signaling complexes and provide an improved understanding of cellular signaling processes.
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http://dx.doi.org/10.3892/ijmm.2019.4060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365033PMC
March 2019

Corrigendum to: Dual mechanisms for the regulation of brain-derived neurotrophic factor by valproic acid in neural progenitor cells.

Korean J Physiol Pharmacol 2019 Jan 26;23(1):91. Epub 2018 Dec 26.

Departments of Pharmacology and Advanced Translational Medicine, School of Medicine, Konkuk University, Seoul 05029, Korea.

[This corrects the article on p. 679 in vol. 22, PMID: 30402028.].
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http://dx.doi.org/10.4196/kjpp.2019.23.1.91DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315093PMC
January 2019

Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3.

J Struct Biol 2019 02 6;205(2):189-195. Epub 2019 Jan 6.

College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea. Electronic address:

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.
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http://dx.doi.org/10.1016/j.jsb.2018.12.006DOI Listing
February 2019

Crystal Structure and Functional Characterization of a Xylose Isomerase (PbXI) from the Psychrophilic Soil Microorganism, Paenibacillus sp.

J Microbiol Biotechnol 2019 Feb;29(2):244-255

Unit of Polar Genomics, Korea Polar Research Institute, Incheon 21990, Republic of Korea.

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (XI) from the psychrophilic soil microorganism, sp. R4. Surprisingly, activity assay results showed that XI is not a cold-active enzyme, but displays optimal activity at 60°C. We solved the crystal structure of XI at 1.94-Å resolution to investigate the origin of its thermostability. The XI structure shows a (β/α)-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the β4-α6 loop region, was newly identified and is thought to be important for the stability of XI. Compared with previously determined thermostable and mesophilic XI structures, the β1-α2 loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible β1-α2 loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate lowtemperature purpose-specific XI enzymes.
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http://dx.doi.org/10.4014/jmb.1810.10057DOI Listing
February 2019