• Journal of the Korean Magnetic Resonance Society
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• v.21 no.2
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• pp.72-77
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• 2017

${\beta}-catenin$ is a key signaling protein which regulates cell signaling and gene transcription. Abnormal activation of ${\beta}-catenin$ is linked to many cancers, particularly with colorectal cancers. Although many genetic and biological studies on $Wnt/{\beta}-catenin$ have been reported and structures of the complex between ${\beta}-catenin$ and its diverse binding partners have been published, many of them have focused on armadillo repeat domain of ${\beta}-catenin$. Both N- and C-terminal domains have been suggested to regulate interactions of ${\beta}-catenin$ with other molecules, but still little is known about the C-terminal unstructured domain. To investigate the structure of this domain, construct of C-terminus was designed and structural studies were performed using size exclusion chromatography (SEC), circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy. We observed that not only the purified full-length construct but the purified C-terminal construct also dimerizes in solution by SEC, suggesting that this domain involves in dimerization of ${\beta}-catenin$. CD and fluorescence data indicate its flexibility and structural formation in the presence of membrane environments.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.234-238
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• 2012

Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.

• BMB Reports
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• v.34 no.4
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• pp.305-309
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• 2001

MatR in Rhizobium trifolii is a malonate-responsive transcription factor that regulates the expression of genes, matABC, enabling decarboxylation of malonyl-CoA into acetyl-CoA, synthesis of malonyl-CoA from malonate and CoA, and malonate transport. According to an analysis of the amino acid sequence homology, MatR belongs to the GntR family The proteins of this family have two-domain folds, the N-terminal helix-turn-helix DNA-binding domain and the C-terminal ligand-binding domain. In order to End the malonate binding site and amino acid residues that interact with RNA polymerase, a site-directed mutagenesis was performed. Analysis of the mutant MatR suggests that Arg-160 might be involved in malonate binding, whereas Arg-102 and Arg-174 are critical for the repression activity by interacting with RNA polymerase.

• Bulletin of the Korean Chemical Society
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• v.34 no.4
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• pp.1089-1095
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• 2013

The tumor necrosis factor-receptor 1 (TNFR1)-associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain. TRADD is known to interact directly with TNF receptor 2 (TNFR2) and the Fas-associated death domain protein (FADD), which are signal transducers that activate NF-${\kappa}B$ and induce apoptosis, respectively. To date, there has been no structural information on the TRADD and FADD death domain (DDs) complex. In this study, the death domains of TRADD and FADD were co-expressed and purified from Escherichia coli for structural characterization. We found that human TRADD (hTRADD) interacted strongly with mouse FADD (mFADD) via their DDs and interacted weakly with human FADD (hFADD)-DD. Moreover, the structures of the TRADD-DD:FADD-DD complexes were separately modeled from predicted structures in the protein data bank (PDB). The results of this study will have important applications in human diseases such as cancer, AIDS, degenerative and autoimmune diseases, and infectious diseases.

• BMB Reports
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• v.50 no.7
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• pp.367-372
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• 2017

Monoacylglycerol acyltransferase 1 (MGAT) is a microsomal enzyme that catalyzes the synthesis of diacylglycerol (DAG) and triacylglycerol (TAG). However, the subcellular localization and catalytic function domain of this enzyme is poorly understood. In this report, we identified that murine MGAT1 localizes to the endoplasmic reticulum (ER) under normal conditions, whereas MGAT1 co-localize to the lipid droplets (LD) under conditions of enriching fatty acids, contributing to TAG synthesis and LD expansion. For the enzyme activity, both the N-terminal transmembrane domain and catalytic HPHG motif are required. We also show that the transmembrane domain of MGAT1 consists of two hydrophobic regions in the N-terminus, and the consensus sequence FLXLXXXn, a putative neutral lipid-binding domain, exists in the first transmembrane domain. Finally, MGAT1 interacts with DGAT2, which serves to synergistically increase the TAG biosynthesis and LD expansion, leading to enhancement of lipid accumulation in the liver and fat.

• BMB Reports
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• v.40 no.4
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• pp.532-538
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• 2007

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.407-409
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• 2000

Outer envelope membrane proteins of chloroplasts encoded by the nuclear genome are transported without the N-terminal transit peptide. Here, we investigated the targeting mechanism of AtOEP7, an Arabidopsis homolog of small outer envelope membrane proteins in vivo. AtOEP7 was expressed transiently in protoplasts or stably in transgenic plants as fusion proteins with GFP. In both cases AtOEP7:GFP was targeted to the outer envelope membrane when assayed under a fluorescent microscope or by Western blot analysis. Except the transmembrane domain, deletions of the N- or C-terminal regions of AtOEP7 did not affect targeting although a region closed to the C-terminal side of the transmembrane domain affected the targeting efficiency. Targeting experiments with various hybrid transmembrane mutants revealed that the amino acid sequence of the transmembrane domain determines the targeting specificity The targeting mechanism was further studied using a fusion protein, AtOEP7：NLS：GFP, that had a nuclear localization signal. AtOEP7：NLS：GFP was efficiently targeted to the chloroplast envelope despite the presence of the nuclear localization signal. Taken together, these results suggest that the transmembrane domain of AtOEP7 functions as the sole determinant of targeting specificity and that AtOEP7 may be associated with a cytosolic component during translocation to the chloroplast envelope membrane.

• Archives of Pharmacal Research
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• v.26 no.10
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• pp.846-854
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• 2003

The oligomerization of G-proteincoupled receptors (GPCRs) has been shown to occur by various mechanisms, such as via disulfide covalent linkages, non covalent (ionic, hydrophobic) interactions of the N-terminal, and/or transmembrane and/or intracellular domains. Interactions between GPCRs could involve an association between identical proteins (homomers) or non-identical proteins (heteromers), or between two monomers (to form dimers) or multiple monomers (to form oligomers). It is believed that muscarinic receptors may also be arranged into dimeric or oigomeric complexes, but no systematic experimental evidence exists concerning the direct physical interaction between receptor proteins as its mechanism. We undertook this study to determine whether muscarinic receptors form homomers or a heteromers by direct protein-protein interaction within the same or within different subtypes using a yeast two-hybrid system. Intracellular loops (i1, i2 and i3) and the C-terminal cytoplasmic tails (C) of human muscarinic (Hm) receptor subtypes, Hm1, Hm2 and Hm3, were cloned into the vectors (pB42AD and pLexA) of a two-hybrid system and examined for heteromeric or homodimeric interactions between the cytoplasmic domains. No physical interaction was observed between the intracellular domains of any of the Hm/Hm receptor sets tested. The results of our study suggest that the Hm1, Hm2 and Hm3 receptors do not form dimers or oligomers by interacting directly through either the hydrophilic intracellular domains or the C-terminal tail domains. To further investigate extracellular domain interactions, the N-terminus (N) and extracellular loops (o1 and o2) were also cloned into the two-hybrid vectors. Interactions of Hm2N with Hm2N, Hm2o1, Hm2o2, Hm3N, Hm3o1 or Hm3o2 were examined. The N-terminal domain of Hm2 was found to have no direct interaction with any extracellular domain. From our results, we excluded the possibility of a direct interaction between the muscarinic receptor subtypes (Hm1, Hm2 and Hm3) as a mechanism for homo- or hetero-meric dimerization/oligomerization. On the other hand, it remains a possibility that interaction may occur indirectly or require proper conformation or subunit formation or hydrophobic region involvement.

• Molecules and Cells
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• v.38 no.9
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• pp.781-788
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• 2015

Mutations of MYO15A are generally known to cause severe to profound hearing loss throughout all frequencies. Here, we found two novel MYO15A mutations, c.3871C>T (p.L1291F) and c.5835T>G (p.Y1945X) in an affected individual carrying congenital profound sensorineural hearing loss (SNHL) through targeted resequencing of 134 known deafness genes. The variant, p.L1291F and p.Y1945X, resided in the myosin motor and IQ2 domains, respectively. The p.L1291F variant was predicted to affect the structure of the actin-binding site from three-dimensional protein modeling, thereby interfering with the correct interaction between actin and myosin. From the literature analysis, mutations in the N-terminal domain were more frequently associated with residual hearing at low frequencies than mutations in the other regions of this gene. Therefore we suggest a hypothetical genotype-phenotype correlation whereby MYO15A mutations that affect domains other than the N-terminal domain, lead to profound SNHL throughout all frequencies and mutations that affect the N-terminal domain, result in residual hearing at low frequencies. This genotype-phenotype correlation suggests that preservation of residual hearing during auditory rehabilitation like cochlear implantation should be intended for those who carry mutations in the N-terminal domain and that individuals with mutations elsewhere in MYO15A require early cochlear implantation to timely initiate speech development.

• BMB Reports
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• v.46 no.1
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• pp.37-40
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• 2013

CY-007 and CY-049 pteridine glycosyltransferases (PGTs) that differ in sugar donor specificity to catalyze either glucose or xylose transfer to tetrahydrobiopterin were studied here to uncover the structural determinants necessary for the specificity. The importance of the C-terminal domain and its residues 218 and 258 that are different between the two PGTs was assessed via structure-guided domain swapping or single and dual amino acid substitutions. Catalytic activity and selectivity were altered in all the mutants (2 chimeric and 6 substitution) to accept both UDP-glucose and UDP-xylose. In addition, the wild type activities were improved 1.6-4.2 fold in 4 substitution mutants and activity was observed towards another substrate UDP-N-acetylglucosamine in all the substitution mutants from CY-007 PGT. The results strongly support essential role of the C-terminal domain and the two residues for catalysis as well as sugar donor specificity, bringing insight into the structural features of the PGTs.