• Biodesign
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• v.5 no.4
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• pp.136-140
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• 2017

Lam6 is a member of sterol-specific ${\underline{l}ipid$ transfer proteins ${\underline{a}}nchored$ at ${\underline{m}ebrane$ contact sites (LAMs). Lam6 localizes to the ER-mitochondria contact sites by its PH-like domain and the C-terminal transmembrane helix. Here, we purified and crystallized the Lam6 PH-like domain from Saccharomyces cerevisiae. To aid crystallization of the Lam6 PH-like domain, T4 lysozyme was fused to the N-terminus of the Lam6 PH-like domain with a short dipeptide linker, GlySer. The fusion protein was crystallized under the condition of 0.1 M HEPES-HCl pH 7.0, 10% (w/v) PEG 8000, and 0.1 M $Na_3$ Citrate at 293K. X-ray diffraction data of the crystals were collected to $2.4{\AA}$ resolution using synchrotron radiation. The crystals belong to the orthorhombic space group $P2_12_12_1$ with unit cell parameters $a=59.5{\AA}$, $b=60.1{\AA}$, and $c=105.6{\AA}$. The asymmetric unit contains one T4L-Lam6 molecule with a solvent content of 58.7%. The initial attempt to solve the structure by molecular replacement using the T4 lysozyme structure was successful.

• Molecules and Cells
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• v.40 no.5
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• pp.355-362
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• 2017

The ${\beta}2$ integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of ${\beta}2$ integrin, ${\alpha}M{\beta}2$ and ${\alpha}X{\beta}2$, share the leukocyte distribution profile and integrin ${\alpha}X{\beta}2$ is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. ${\underline{R}}eceptor$ for ${\underline{a}}dvanced$ ${\underline{g}}lycation$ ${\underline{e}}nd$ ${\underline{p}}roducts$ (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and ${\alpha}X{\beta}2$ play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of ${\alpha}X{\beta}2$, we characterize the binding nature and the interacting moieties of ${\alpha}X$ I-domain and RAGE. Their binding requires divalent cations ($Mg^{2+}$ and $Mn^{2+}$) and shows an affinity on the sub-micro molar level: the dissociation constant of ${\alpha}X$ I-domains binding to RAGE being $0.49{\mu}M$. Furthermore, the ${\alpha}X$ I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of ${\alpha}X$ I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to ${\alpha}X$ I-domain. In conclusion, the main mechanism of ${\alpha}X$ I-domain binding to RAGE is a charge interaction, in which the acidic moieties of ${\alpha}X$ I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.25-25
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• 1997

Cyclic nucleotide-gated channels (CNGC's) contain a putative N-glycosylation site (Asn-X-Ser/Thr) in the linker regions connecting the fourth transmembrane domain (S4) and the ion conduction pore (P-region). This putative N-glycosylation site is highly conserved and thus found in many different CNGC in various organisms, from fruit to human.(omitted)

• Molecules and Cells
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• v.21 no.1
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• pp.1-6
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• 2006

Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a mitochondrial pro-apoptotic protein that has a single Bcl-2 homology 3 (BH3) domain and a COOH-terminal transmembrane (TM) domain. Although it belongs to the Bcl-2 family and can heterodimerize with Bcl-2, its pro-apoptotic activity is distinct from those of other members of the Bcl-2 family. For example, cell death mediated by BNIP3 is independent of caspases and shows several characteristics of necrosis. Furthermore, the TM domain, but not the BH3 domain, is required for dimerization, mitochondrial targeting and pro-apoptotic activity. BNIP3 plays an important role in hypoxia-induced death of normal and malignant cells. Its expression is markedly increased in the hypoxic regions of some solid tumors and appears to be regulated by hypoxia-inducible factor (HIF), which binds to a site on the BNIP3 promoter. Silencing, followed by methylation, of the BNIP3 gene occurs in a significant proportion of cancer cases, especially in pancreatic cancers. BNIP3 also has a role in the death of cardiac myocytes in ischemia. Further studies of BNIP3 should provide insight into hypoxic cell death and may contribute to improved treatment of cancers and cardiovascular diseases.

• Applied Biological Chemistry
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• v.49 no.4
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• pp.293-297
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• 2006

Angiotensin converting enzyme(ACE) is a zinc-containing dipeptidase widely distributed in mammalian tissues and is thought to play a significant role in blood pressure regulation by hydrolyzing angiotensin I to the potent vasoconstrictor, angiotensin II. Recently, the presence of ACE in pig ovary was reported and the ACE from pig kidney was isolated and characterized. However no nucleotide sequence of the ACE gene from pig is yet known. We report here the cloning of the ACE cDNA from pig kidney by using the reverse transcriptase-polymerase chain reaction. The complete amino acid sequence deduced from the cDNA contains 1309 residues with a molecular mass of 150 kDa, beginning with a signal peptide of 33 amino acids. Amino acid sequence analysis showed that pig kidney ACE is also probably anchored by a short transmembrane domain located near the C-terminus. This protein contains a tandem duplication of the two homologous amino acid peptidase domain. Each of these two domains bears a putative metal-binding site (His-Glu-Met-Gly-His) identified in mammalian somatic ACE. The alignment of pig ACE amino acid sequence with human, rabbit, and mouse reveals that both two domains have been highly conserved during evolution.

• Genomics & Informatics
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• v.7 no.2
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• pp.131-135
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• 2009

Domains are the building blocks of proteins. Exon shuffling is an important mechanism accounting for combination of a limited repertoire of protein domains in the evolution of multicellular species. A relative excess of domains encoded by symmetric exons in metazoan phyla has been presented as evidence of exon shuffling, and symmetric domains can be divided into old and new domains by determining the ages of the domains. In this report, we compare the spread, versatility, and subcellular localization of old and new domains by analyzing eight metazoan genomes and their respective annotated proteomes. We found that new domains have been expanding as multicellular organisms evolved, and this expansion was principally because of increases in class 1-1 domains amongst several classes of domain families. We also found that younger domains have been expanding in membranes and secreted proteins along with multi-cellular organism evolution. In contrast, old domains are located mainly in nuclear and cytoplasmic proteins. We conclude that the increasing mobility and versatility of new domains, in contrast to old domains, plays a significant role in metazoan evolution, facilitating the creation of secreted and transmembrane multidomain proteins unique to metazoa.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.33-33
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• 1996

An ectodomain of gp41, the transmembrane fusion protein of HIV, without the fusion peptide region was expressed using pET system in E. coli. The expressed protein gp41core, was isolated as inclusion body and was purified by ion-exchange chromatography after solubilized in 6M urea. The purified denatured protein was renaturated and the folded domain of gp41core was identified by the presence of the proteolysis resistence domain and a high content of ${\alpha}$-helical secondary structure. (omitted)

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.181-181
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• 1996

The (Na, K) ATPase is a membrane ion transporting ATPase composed of an ${\alpha}$ catalytic subunit and a ${\beta}$ glycoprotein subunit. The topology of the rat ${\alpha}$1 and ${\beta}$1 subunits has been studied by insertion of epitope(s) : at the NH2-terminus and COOH-terminus and between Glu117 and Glul18, Lys828 and Arg829, Gln900 and Trp901, and Va1939 and Phe940 of the ${\alpha}$ subunit; and at the NH2-terminus and COOH-terminus and between Glu228 and Tyr229 of the ${\beta}$ subunit. The epitope-tagged ${\alpha}$l, constructs were expressed in HeLa cells to select for stable cell lines expressing a functional (Na, K)ATPase. All constructs, except for the one tagged between Gln900 and Trp901, resulted in ouabain-resistant colonies indicating that modified proteins retained functional integrity. The epitope-tagged ${\beta}$ constructs were transiently expressed in Cos-7 cells. The orientation of the epitopes with respect to the cell membrane was revealed by indirect immunofluorescence performed on permeabilized and non-permeabilized cells expressing the (Na, K)ATPase chains. The results indicate that the ${\alpha}$ subunit has 4 transmembrane segments in the COOH terminal membrane bound domain between residues 760 and 938, and that both the NH2-terminus and the COOH-terminus are in the cytosol; it was not determined whether there are more transmembrane segments between residue 938 and the COOH-terminus. The ${\beta}$ subunit has only one transmembrane spanning region with the NH2-terminus in the cytosol and the COOH-terminus on the extracytoplasmic surface of the plasma membrane.

• Molecules and Cells
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• v.27 no.6
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• pp.673-680
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• 2009

Conversion of the normal soluble form of prion protein, PrP ($PrP^C$), to proteinase K-resistant form ($PrP^{Sc}$) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from ${\alpha}$-helix to ${\beta}$-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.153-159
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• 2001

In order to clone the peptide synthetase gene form Lysobacter lactamgenus IFO 14,288, the gene fragments were amplified using primers for the adenylation domain and the thionylation domain of the peptide synthetase genes in other organisms by polymerase chain reaction (PCR). The resulting 0.5-kb fragment was cloned in a pGEM-T vector, and the nucleotide sequences were determined. Six different PCR products were obtained; three were identified to be a part of L-$\alpha$-aminoadipyl-L-cysteinyl-D-valine (ACV) synthetase and three to be other peptide synthetases. Using each of the two different classes of PCR products as mixed probes, a cosmid library of L. lactamgenus chromosomal DNA constructed in a pHC79 vector was screened by an in situ hybridization procedure, and one positive clone was selected which was bound by peptide synthetase gene fragments as well as ACV synthetase gene fragments. The partial sequence analysis formt he obtained pPTS-5 cosmid showed th presence of more than two open reading frames. These were for two putative membrane transporters, which were homologous with several integral membrane proteins including the ABC transporter ATP-binding protein of E. coli (YbjZ) and the metal ion uptake protein of Bacillus subtilis (YvrN). A 45% homology was also found between the two transporter proteins at the carboxy terminus. Through a hydropathy analysis and transmembrane analysis. 4-5 transmembrane domains were found in these two proteins. When the genes were expressed in Escherichia coli, the gene products inhibited the hose cell growth, probably due to the disturbance of the membrane transport system.