핵자기공명 분광학을 이용한 S100-RAGE 복합체와 anti-CRISPR AcrIIA4의 구조 및 동력학 연구

학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2018. 2. 서정용.Distinct protein-protein interactions were investigated using NMR spectroscopy as well biophysical characterization tools such as calorimetry and size exclusion chromatography. In Chapter I, calcium binding of S100A5 and the interaction between S100A5 and receptor for advanced glycation end product (RAGE) were investigated using calorimetry and NMR spectroscopy. S100A5 exhibits a sequential manner to bind calcium ions using the C-terminal and N-terminal calcium binding EF-hands with the equilibrium dissociation constants (KD) of 1.3 µM and 3.5 µM, respectively. S100A5 interacts with the V domain of RAGE (RAGEv) to form a heterotrimeric complex with KD of 5.9 µM in a calcium-dependent manner, which is a distinct stoichiometry from the S100 family proteins. Chemical shift perturbation data from NMR titration experiments indicates that S100A5 employs the periphery of the dimer interface to interact with RAGEv. Distinct binding mode and stoichiometry of against different S100 family proteins could be important to modulate diverse RAGE signaling. In Chapter II, solution structure and dynamics of AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9) widely used for genome editing were investigated. The structure of AcrIIA4 is a compact monomeric αβββαα fold consisting of three α helices flanked against three antiparallel β strands and short 310 helix. The backbone dynamics of AcrIIA4 reveals that distinct backbone dynamics in fast and slow timescales at loop regions is related to interaction surface for SpyCas9. Furthermore, AcrIIA4 binds to apo-SpyCas9 with KD ~4.8 µM and ~0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9, which infers that a binary complex between AcrIIA4 and SpyCas9 can associate with sgRNA to form a tight ternary complex, avoiding competition with the DNA substrate for SpyCas9 binding. This studies provide insights into anti-CRISPR-mediated inhibition mechanisms for disabling the SpyCas9, thereby broadening CRISPR-Cas regulatory tools for genome editing.CHAPTER I. BIOPHYSICAL CHARACTERIZATION OF CALCIUM-BINDING OF S100A5 AND CALCIUM INDUCED S100A5‒RAGE COMPLEX 1 1. Abstract 2 2. Introduction 3 2. 1. Receptor for advanced glycation end product (RAGE) 3 2. 2. S100A5 3 3. Materials and Methods 9 3. 1. Cloning 9 3. 2. Protein expression 9 3. 2. 1. Luria bertani medium 9 3. 2. 2. Isotope labeling medium 9 3. 3. Purification 12 3. 3. 1. S100A5 and S100A5m 12 3. 3. 2. RAGE 12 3. 4. Size exclusion chromatography and MALS analysis 18 3. 5. NMR experiment 18 3. 6. Isothermal titration calorimetry (ITC) 19 4. Results 20 4. 1. Thermodynamics of calcium binding events in S100A5 20 4. 2. Resonance assignment of S100A5m and RAGE 28 4. 3. Calcium-dependent binding and the stoichiometry between S100A5 and RAGEv 43 4. 4. Characterization of the complex of S100A5 and RAGEv 54 5. Discussion 62 6. Conclusion 65 CHAPTER II. SOLUTION STRUCTURE AND DYNAMICS OF ANTI-CRISPR ACRIIA4, CAS9 INHIBITOR 66 1. Abstract 67 2. Introduction 68 2. 1. CRISPR-Cas9 68 2. 2. Anti-CRISPR AcrIIA4 68 3. Materials and Methods 74 3. 1. Cloning 74 3. 2. Protein expression 74 3. 3. Sample preparation 77 3. 3. 1. AcrIIA4 77 3. 3. 2. SpyCas9 77 3. 3. 3. sgRNA 78 3. 4. NMR spectroscopy 84 3. 5. Structure calculations 85 3. 6. Isothermal titration calorimetry (ITC) 85 4. Results 87 4. 1. Resonance assignment of AcrIIA4 87 4. 2. Determination of solution structure of AcrIIA4 95 4. 3. Backbone dynamics of AcrIIA4 100 4. 4. Interaction of AcrIIA4 to SpyCas9 107 5. Discussion 117 6. Conclusion 122 III. References 123 IV. Summary in Korean 127Docto