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A Novel Function of Karyopherin Beta3 Associated with Apolipoprotein A-I Secretion
정경민,차선신,장승기 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.3
Human karyopherin beta3, highly homologous to a yeast protein secretion enhancer (PSE1), has often been reported to be associated with a mediator of a nucleocytoplasmic transport pathway. Previously, we showed that karyopherin beta3 complemented the PSE1 and KAP123 double mutant. Our research suggested that karyopherin beta has an evolutionary function similar to that of yeast PSE1 and/or KAP 123. In this study, we performed yeast two-hybrid screening to find a protein which would interact with karyopherin beta3 and identified apolipoprotein A-I (apo A-I), a secretion protein with a primary function in cholesterol transport. By using in vitro binding assay, co-immunoprecipitation, and colocalization studies, we defined an interaction between karyopherin 3 and apo A-I. In addition, overexpression of karyopherin 3 significantly increased apo A-I secretion. These results suggest that karyopherin beta3 plays a crucial role in apo A-I secretion. These findings may be relevant to the study of a novel function of karyopherin beta3 and coronary artery diseases associated with apo A-I.
In-House Zinc SAD Phasing at Cu K alpha Edge
김민규,차선신,이상민,안영준,정창숙,지창준,이진원 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1
De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu K X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of syn-chrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indi-cate that in-house Zn-SAD phasing can be widely applicable to structure determination.
Experimental Phasing Using Zinc and Sulfur Anomalous Signals Measured at the Zinc Absorption Peak
이상민,김민규,지창준,이진원,차선신 한국미생물학회 2013 The journal of microbiology Vol.51 No.5
Iron is an essential transition metal required for bacterial growth and survival. Excess free iron can lead to the generation of reactive oxygen species that can cause severe damage to cellular functions. Cells have developed iron-sensing regulators to maintain iron homeostasis at the transcription level. The ferric uptake regulator (Fur) is an iron-responsive regulator that controls the expression of genes involved in iron homeostasis, bacterial virulence, stress resistance, and redox metabolism. Here, we report the expression, purification, crystallization,and phasing of the apo-form of Bacillus subtilis Fur (BsFur) in the absence of regulatory metal ions. Crystals were obtained by microbatch crystallization method at 295 K and diffraction data at a resolution of 2.6 Å was collected at the zinc peak wavelength (λ=1.2823 Å). Experimental phasing identified the positions of one zinc atom and four sulfur atoms of cysteine residues coordinating the zinc atom, indicating that the data contained a meaningful anomalous scattering originating from the ordered zinc-coordinating sulfur atoms, in spite of the small anomalous signals of sulfur atoms at the examined wavelength.
Dissection of the Dimerization Modes in the DJ-1 Superfamily
Hoi Jong Jung,차선신,Sangok Kim,Yun Jae Kim,김민규,Sung Gyun Kang,Jung-Hyun Lee,김완규 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.2
The DJ-1 superfamily (DJ-1/ThiJ/PfpI superfamily) is distributed across all three kingdoms of life. These pro-teins are involved in a highly diverse range of cellular functions, including chaperone and protease activity. DJ-1 proteins usually form dimers or hexamers in vivo and show at least four different binding orientations via distinct interface patches. Abnormal oligomerization of human DJ-1 is related to neurodegenerative disorders including Parkinson’s disease, suggesting important functional roles of quaternary structures. However, the quaternary structures of the DJ-1 superfamily have not been extensively studied. Here, we focus on the diverse oligomerization modes among the DJ-1 superfamily proteins and investigate the functional roles of quaternary structures both computationally and experimentally. The oligomerization modes are classified into 4 types (DJ-1, YhbO, Hsp, and YDR types) depending on the distinct interface patches (I-IV) upon dimerization. A unique, rotated interface via patch I is reported, which may potentially be related to higher order oligomerization. In general, the groups based on sequence similarity are consistent with the quaternary structural classes, but their biochemical functions cannot be directly inferred using sequence information alone. The observed phyletic pattern suggests the dynamic nature of quaternary structures in the course of evolution. The amino acid residues at the interfaces tend to show lower mutation rates than those of non-interfacial surfaces.
안영준,김민규,송정민,강미혜,이윤호,차선신 한국구조생물학회 2015 Biodesign Vol.3 No.3
The CaCel gene product from Antarctic springtail Cryptopygus antarcticus (CaCel) belongs to the glycoside hydrolasefamily 45 (GH45) type endo-β-1,4-glucanase. Since the production of soluble recombinant CaCel was not successful at thetemperature range of 15-37oC, we further lowered the expression temperature. The Escherichia coli Rosetta-gami2 (DE3)strain harbouring an expression vector including the CaCel gene was cultured at 10oC. Due to the extremely low growthrate, the induction time was expanded to 9 days and the 18-liter culture volume was necessary to get enough solubleprotein for crystallization. Crystals of CaCel were grown in droplets under Al’s Oil that allows vapor diffusion. In spite ofsmall size, the crystal of CaCel, which belonged to the space group P3121, with unit-cell parameters a = 73.57, b = 83.93,c = 163.77 Å, diffracted to 2.6 Å resolution.
Kim-Hung Huynh,홍명기,Clarice Lee,Huyen-Thi Tran,이상희,안예진,차선신,강린우 한국미생물학회 2015 The journal of microbiology Vol.53 No.11
Acinetobacter baumannii, which is emerging as a multidrugresistant nosocomial pathogen, causes a number of diseases, including pneumonia, bacteremia, meningitis, and skin infections. With ATP hydrolysis, the D-alanine-D-alanine ligase (DDL) catalyzes the synthesis of D-alanyl-D-alanine, which is an essential component of bacterial peptidoglycan. In this study, we determined the crystal structure of DDL from A. baumannii (AbDDL) at a resolution of 2.2 Å. The asymmetric unit contained six protomers of AbDDL. Five protomers had a closed conformation in the central domain, while one protomer had an open conformation in the central domain. The central domain with an open conformation did not interact with crystallographic symmetry-related protomers and the conformational change of the central domain was not due to crystal packing. The central domain of AbDDL can have an ensemble of the open and closed conformations before the binding of substrate ATP. The conformational change of the central domain is important for the catalytic activity and the detail information will be useful for the development of inhibitors against AbDDL and putative antibacterial agents against A. baumannii. The AbDDL structure was compared with that of other DDLs that were in complex with potent inhibitors and the catalytic activity of AbDDL was confirmed using enzyme kinetics assays.