• Interdisciplinary Bio Central
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• 제1권1호
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• pp.3.1-3.6
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• 2009

Introduction: GTPases known as translation factor play a vital role as ribosomal subunit assembly chaperone. The bacterial Obg proteins ($Spo{\underline{0B}}$-associated ${\underline{G}}TP$-binding protein) belong to the subfamily of P-loop GTPase proteins and now it is considered as one of the new target for antibacterial drug. The majority of bacterial Obgs have been commonly found to be associated with ribosome, implying that these proteins may play a fundamental role in ribosome assembly or maturation. In addition, one of the experimental evidences suggested that Bacillus subtilis Obg (BsObg) protein binds to the L13 ribosomal protein (BsL13) which is known to be one of the early assembly proteins of the 50S ribosomal subunit in Escherichia coli. In order to investigate binding mode between the BsObg and the BsL13, protein-protein docking simulation was carried out after generating 3D structure of the BsL13 structure using homology modeling method. Materials and Methods: Homology model structure of BsL13 was generated using the EcL13 crystal structure as a template. Protein-protein docking of BsObg protein with ribosomal protein BsL13 was performed by DOT, a macro-molecular docking software, in order to predict a reasonable binding mode. The solvated energy minimization calculation of the docked conformation was carried out to refine the structure. Results and Discussion: The possible binding conformation of BsL13 along with activated Obg fold in BsObg was predicted by computational docking study. The final structure is obtained from the solvated energy minimization. From the analysis, three important H-bond interactions between the Obg fold and the L13 were detected: Obg:Tyr27-L13:Glu32, Obg:Asn76-L13:Glu139, and Obg:Ala136-L13:Glu142. The interaction between the BsObg and BsL13 structures were also analyzed by electrostatic potential calculations to examine the interface surfaces. From the results, the key residues for hydrogen bonding and hydrophobic interaction between the two proteins were predicted. Conclusion and Prospects: In this study, we have focused on the binding mode of the BsObg protein with the ribosomal BsL13 protein. The interaction between the activated Obg and target protein was investigated with protein-protein docking calculations. The binding pattern can be further used as a base for structure-based drug design to find a novel antibacterial drug.

• BMB Reports
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• 제39권5호
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• pp.560-570
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• 2006

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the A-chain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.

• Molecules and Cells
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• 제46권8호
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• pp.513-525
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• 2023

Orange carotenoid protein (OCP) of photosynthetic cyanobacteria binds to ketocarotenoids noncovalently and absorbs excess light to protect the host organism from light-induced oxidative damage. Herein, we found that mutating valine 40 in the α3 helix of Gloeocapsa sp. PCC 7513 (GlOCP1) resulted in blue- or red-shifts of 6-20 nm in the absorption maxima of the lit forms. We analyzed the origins of absorption maxima shifts by integrating X-ray crystallography, homology modeling, molecular dynamics simulations, and hybrid quantum mechanics/molecular mechanics calculations. Our analysis suggested that the single residue mutations alter the polar environment surrounding the bound canthaxanthin, thereby modulating the degree of charge transfer in the photoexcited state of the chromophore. Our integrated investigations reveal the mechanism of color adaptation specific to OCPs and suggest a design principle for color-specific photoswitches.

• BMB Reports
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• 제50권5호
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• pp.269-274
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• 2017

The biological activities of macrophage migration inhibitory factor (MIF) might be mediated through a classical receptor-mediated or non-classical endocytic pathway. JAB1 (C-Jun activation domain-binding protein-1) promotes the degradation of the tumor suppressor, p53, and the cyclin-dependent kinase inhibitor, p27. When MIF and JAB1 are bound to each other in various intracellular sites, MIF inhibits the positive regulatory effects of JAB1 on the activity of AP-1. The intestinal parasite, Anisakis simplex, has an immunomodulatory effect. The molecular mechanism of action of As-MIF and human JAB1 are poorly understood. In this study, As-MIF and hJAB1 were expressed and purified with high solubility. The structure of As-MIF and hJAB1 interaction was modeled by homology modeling based on the structure of Ace-MIF. This study provides evidence indicating that the MIF domain of As-MIF interacts directly with the MPN domain of hJAB1, and four structure-based mutants of As-MIF and hJAB1 disrupt the As-MIF-hJAB1 interaction.

• 생명과학회지
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• 제28권6호
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• pp.656-662
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• 2018

향상된 인디고이드 생산능력을 갖는 코리네박테리움 유래의 변이 플라빈-함유 모노옥시게나제(cFMO)를 개발하기 위하여, cFMO 효소의 상동성모델을 이용하여 말토오스-결합단백질(MBP)과 융합된 4가지 변이체(F170Y, A210G, A210S, T326S)를 제작하고 그 생화학적 특징을 밝혔다. 정제된 MBP-T326S는 최적 활성을 위하여 야생형보다 고농도의 FAD ($100{\mu}M$)를 요구하며, $100{\mu}M$의 FAD 첨가조건에서 $k_{cat}/K_m$이 3.8배 증가되었다. 인돌 옥시게나제 활성은 야생형의 63-77%를 나타냈다. MBP-T326S는 기질이 존재하지 않을 경우 쓸모없는 NADPH 산화효소 활성이 매우 낮은 수준을 보여주었다(21-24%). T326S이외의 변이 단백질들은 야생형에 비하여 $K_m$은 비슷하며 $k_{cat}/K_m$은 증가하였다. MBP-F170Y와 -A210S 변이단백질은 인돌 옥시게나제 활성이 각각 3.1배, 2.9배 증가하였다. 2.5 g/l의 트립토판을 함유한 LB배지에서 인디고이드 생산을 시험했을 때, 야생형 cFMO를 함유한 대장균은 684 mg/l의 인디고와 104 mg/l의 인디루빈을 생산한 반면, T326S를 함유한 세포는 1,040 mg/l의 인디고와 112 mg/l의 인디루빈을 생산하였다. 이전의 결과인 Methylophaga 유래의 FMO를 발현하는 대장균에서 가장 높은 수준인 920 mg/l의 인디고를 생산한 것과 비교하면, 본 연구결과는 인디고 생산이 13% 높은 수준이였다. 상동성 모델링에 기반한 cFMO의 단백질공학은 인디고이드 생산균을 개발하는데 보다 더 논리적인 전략을 제시하였다.

• Parasites, Hosts and Diseases
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• 제54권6호
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• pp.759-768
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• 2016

Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus. We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus. Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens $p38{\alpha}$, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for $p38{\alpha}$. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with $TGF-{\beta}1$ effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus, as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human $TGF-{\beta}1$.

• Genomics & Informatics
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• 제3권4호
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• pp.142-148
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• 2005

The immune response-related genes have been suggested to be the most favorable genes for positive selection during evolution. Comparing the entire DNA sequence of chimpanzee chromosome 22 (PTR22) with human chromosome 21 (HSA21), we have identified 15 orthologs having indel in their coding sequences. Among them, interferon-${\alpha}$ receptor-1 gene (IFNAR1), an immuneresponse-related gene, is subjected to comparative genomic analysis. Chimpanzee IFNAR1 showed the same genomic structure as human IFNAR1 (11 exons and 10 introns) except the 3 bp insertion in exon 4. The sequence alignment of IFNAR1 coding sequence indicated that 'ISPP' amino acid sequence motif is highly conserved in chimpanzee and other animals including mouse and chicken. However, the human IFNAR1 shows that one proline residue is missing in the sequence motif. The homology modeling of the IFNAR1 structures suggests that the proline deletion in human IFNAR1 leads to the formation of the following ${\alpha}$-helix, whereas two sequential prolines in chimpanzee IFNAR1 inhibit it. As a result, human IFNAR1 may adopt a characteristic structure distinct from chimpanzee IFNAR1. This human specific trait could contribute to specific immune response in the most optimized manner for humans. Further molecular biological studies on the IFNAR1 will help us to gain insights into the molecular implication of species-specific host-pathogen interaction in primate evolution.

• Journal of Ginseng Research
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• 제39권4호
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• pp.406-413
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• 2015

Background: Pathogenesis-related 10 (PR-10) proteins are small, cytosolic proteins with a similar three-dimensional structure. Crystal structures for several PR-10 homologs have similar overall folding patterns, with an unusually large internal cavity that is a binding site for biologically important molecules. Although structural information on PR-10 proteins is substantial, understanding of their biological function remains limited. Here, we showed that one of the PgPR-10 homologs, PgPR-10.3, shares binding properties with flavonoids, kinetin, emodin, deoxycholic acid, and ginsenoside Re (1 of the steroid glycosides). Methods: Gene expression patterns of PgPR-10.3 were analyzed by quantitative real-time PCR. The three-dimensional structure of PgPR-10 proteins was visualized by homology modeling, and docking to retrieve biologically active molecules was performed using AutoDock4 program. Results: Transcript levels of PgPR-10.3 expressed in leaves, stems, and roots of 3-wk-old ginseng plantlets were on average 86-fold lower than those of PgPR-10.2. In mature 2-yr-old ginseng plants, the mRNA of PgPR-10.3 is restricted to leaves. Ginsenoside Re production is especially prominent in leaves of Panax ginseng Meyer, and the binding property of PgPR-10.3 with ginsenoside Re suggests that this protein has an important role in the control of secondary metabolism. Conclusion: Although ginseng PR-10.3 gene is expressed in all organs of 3-wk-old plantlets, its expression is restricted to leaves in mature 2-yr-old ginseng plants. The putative binding property of PgPR-10.3 with Re is intriguing. Further verification of binding affinity with other biologically important molecules in the large hydrophobic cavity of PgPR-10.3 may provide an insight into the biological features of PR-10 proteins.

• Molecules and Cells
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• 제39권8호
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• pp.594-602
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• 2016

Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.

• KSBB Journal
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• 제23권1호
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• pp.44-47
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• 2008

Streptomyces peucetius가 생산하는 anthracycline 계열의 doxorubicin은 치료목적으로 사용되는 중요한 항암제 중 하나이다. Doxorubicin은 rhodomycin D에서부터 몇 단계의 생합성 과정을 더 거쳐 생산되는데, 생물학적 활성을 갖기 위해서는 deoxy-sugar의 전이가 반드시 일어나야 한다. 본 논문에서는 이종균주인 Streptomyces venezuelae에 11개의 유전자를 형질 전환하여 TDP-L-daunosamine를 생산하고 이것을 ${\varepsilon}$-rhodomycinone에 전이하여 rhodomycin D를 생산하는 연구를 수행하였다. S. peucetius 유래의 7개 유전자 dnmU, T, J, V, Z, Q, S.를 당 합성 및 전이를 위해 plasmid 형태로 전이하였으며, S. venezuelae의 desIII, IV와 doxorubicin 내성 유전자인 drrA, B는 chromosomal DNA에 삽입하였다. Aglycone 기질인 ${\varepsilon}$-rhodomycinone을 확보하기 위하여 6L의 고체 배지에 S. peucetius를 배양하여 유기용매로 추출하고 preparative HPLC로 분리 정제하였다. 결과적으로 이종균주인 S. venezuelae에서 ${\varepsilon}$-rhodomycinone에 당 전이가 일어난 생산물을 확인함으로써 deoxy-sugar의 생합성 및 전이에 필요한 최소한의 유전적 정보를 확인할 수 있었다. 또한, 유사서열 단백질 모델링을 통하여, 최초로 당 전이 반응에 필수적인 도움효소 DnrQ의 구조를 예측하였다.