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Structural determination of toxin-antitoxin system from Shigella flexneri
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 서민덕 | - |
| dc.contributor.author | 윤원수 | - |
| dc.date.accessioned | 2022-11-29T02:32:43Z | - |
| dc.date.available | 2022-11-29T02:32:43Z | - |
| dc.date.issued | 2021-02 | - |
| dc.identifier.other | 30811 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/20297 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2021. 2 | - |
| dc.description.tableofcontents | General Introduction 1 Shigella flexneri 1 Persistent cells and toxin-antitoxin system 2 Multidrug resistance and multidrug tolerance 5 Six types of toxin-antitoxin system 7 Conditional cooperativity 10 Chapter 1. Structural characterization of antitoxin apo-SfHigA 12 1.1 Introduction 12 1.2 Experimental Procedures 14 1.2.1. Cloning, overexpression, and purification 14 1.2.2. Structure determination and refinement of apo-SfHigA 16 1.2.3. Multi Angle Light Scattering (MALS) 16 1.2.4. Crystallization and data collection 19 1.2.5. Structure determination of V-shape apo-SfHigA 20 1.3 Results and discussion 23 1.3.1. Expression, purification and crystallization of apo-SfHigA 23 1.3.2. Structure determination of apo-SfHigA 27 1.3.3. Validation of apo-SfHigA dimer structure 30 1.3.4. Overall structure of apo-SfHigA 33 1.3.5. DNA binding mode of apo-SfHigA 36 1.4 Summary 40 Chapter 2. Structural change of antitoxin SfHigA by binding of the cognate toxin SfHigB 41 2.1 Introduction 41 2.2 Experimental Procedures 43 2.2.1. Cloning, overexpression, and purification 43 2.2.2. Multi Angle Light Scattering (MALS) 45 2.2.3. Crystallization and data collection 45 2.2.4. Structure determination and refinement 46 2.2.5. Electrophoretic mobility shift assay (EMSA) 46 2.2.6. Surface plasmon resonance (SPR) 47 2.3 Results and discussion 48 2.3.1. Expression and purification of SfHigBA 48 2.3.2. Structure determination of SfHigBA 52 2.3.3. Overall structure of the SfHigBA 57 2.3.4. Ribosome dependent toxin SfHigB 60 2.3.5. Structural comparison with ribosome dependent TA complex 63 2.3.6. Conformational change of SfHigA upon SfHigB binding 66 2.3.7. Different DNA binding Modes between apo-SfHigA and the SfHigBA complex 70 2.4 Summary 74 Discussion 76 Reference 78 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Structural determination of toxin-antitoxin system from Shigella flexneri | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2021. 2 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 1218633 | - |
| dc.identifier.uci | I804:41038-000000030811 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000030811 | - |
| dc.subject.keyword | Shigella flexneri | - |
| dc.subject.keyword | Toxin-antitoxin | - |
| dc.description.alternativeAbstract | Bacterial toxin-antitoxin systems are known to play an essential role in maintaining cells when they are in a difficult environment such as malnutrition, antibiotic treatment, and oxidative stress. There are six kinds of toxin-antitoxin systems according to the toxin-neutralizing mechanism of antitoxin. The type II toxin-antitoxin system which has been the most studied consists of a relatively unstable antitoxin protein and a stable toxin protein. In a normal cellular environment, toxin and antitoxin form a stable complex, which results in neutralization of toxin by antitoxin. However, when cells are placed in barren environments relatively unstable antitoxins are degraded by specific proteolytic enzymes, and free toxins show toxic activities causing cell death or growth inhibition. In the type II toxin-antitoxin system, antitoxin contains a DNA binding domain and it binds to a specific sequence in the promoter region to regulate the transcription of toxins and antitoxins. In this study, we have determined the crystal structures of antitoxin HigA from Shigella flexneri (SfHigA) and toxin-antitoxin complex HigBA from Shigella flexneri (SfHigBA). Apo-SfHigA adopts a V-shaped homodimer formed by N-terminal dimerization. Through structural comparison with known HigA structures, it could be predicted that DNA would bind through the HTH (helix-turn-helix) motif in the C-terminal region of the antitoxin SfHigA. SfHigBA appears as a heterotetramer formed by N-terminal dimerization of SfHigB-bound SfHigA molecules. When SfHigB binds, the structural change of SfHigA is mediated by rigid-body movements of the C-domain accompanied by a conformational change from a wide V-shaped to a narrow V-shaped dimer. As a result, two putative DNA binding helices (α7, α7’) are rearranged in a form that is more compatible with the standard homodimeric DNA binding protein containing the HTH motif. In addition, when SfHigB was bound, the structural changes of the C-domain of SfHigA results in a different DNA binding ability. The DNA binding affinity of SfHigBA, a toxin-antitoxin complex, was higher than that of apo- SfHigA. The structures of apo-SfHigA and SfHigBA covered in this study are expected to contribute to elucidating the biological function of TA system and also developing the antibiotics to overcome the drug tolerance. | - |
| dc.title.subtitle | conformational changes of antitoxin HigA by binding of toxin HigB | - |
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