Human Cytosolic Aspartyl-tRNA Synthetase에 대한 구조 연구

학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2014. 2. 한병우.Human cytosolic aspartyl-tRNA synthetase (DRS)는 단백질의 translation 과정에 관여하는 aminoacyl-tRNA synthetase (aaRS)의 한 요소로써, aspartic acid와 그것을 인식하는 특정 tRNA를 연결하는 효소이다. 또한 DRS는 체내에서 multi-tRNA synthetase complex (MSC)를 이루는 단백질로 알려져 있으며 이들 MSC는 체내에서 aaRS들의 저장소의 역할을 하면서 translation과 non-canonical function을 조절한다고 알려져 있으나 DRS의 non-canonical function은 아직 밝혀지지 않은 실정이다. 본 연구에서는 X선을 이용하여 DRS의 결정으로부터 2.25 Å resolution의 회절자료를 얻어 DRS의 구조를 규명했다. DRS의 결정구조로부터 DRS는 homodimer이며 mitochondrial aspartyl-tRNA synthetase와 비교해 보았을 때, sequence상에서는 22.9%의 낮은 유사성을 띠나 구조적으로는 r.m.s.d. distance가 1.7 Å으로 유사한 것을 밝혔다. 그리고 DRS의 N-terminus 부분에서 N-helix를 유추할 수 있었고 이 구조가 DRS와 tRNAAsp간의 상호작용을 조절할 것으로 예상했다. 또한 post-translational modification (PTM) sites를 분석하여 새로운 PTM sites를 발견했으며 이 부분들이 DRS 와 tRNAAsp사이에 위치할 것으로 보아 이들간의 결합에 영향을 미칠 것으로 예상했다. 146번 serine은 dimeric interface이면서 인산화하는 부분으로 146번 serine의 인산화가 DRS의 구조 변화를 유도하여 DRS의 기능을 조절할 것으로 본다. DRS의 결정구조는 MSC상에서의 DRS의 결합상태를 확인하고 DRS의 새로운 기능을 밝히는데 도움이 될 것으로 기대한다.Human cytosolic aspartyl-tRNA synthetase (DRS), a component of aminoacyl-tRNA synthetase (aaRS), is an enzyme that attaches the aspartic acid to its cognate tRNA in RNA translation process. Also it composes multi-tRNA synthetase complex (MSC) in combination with 8 cytosolic aaRSs and 3 aminoacyl tRNA synthetase complex-interacting multifunctional proteins, which is known as a reservoir for aaRSs and regulates aaRSs between their translational functions and non-canonical functions. Non-canonical function of DRS has not been revealed yet. We solved the crystal structure of DRS at 2.25 Å resolution. From the crystal structure, we revealed that DRS is a homodimer with a dimer interface 3,750.5 Å2 which is 16.6% of the monomeric surface area. When compared with mitochondrial DRS, they share only 22.9% sequence identity but are structurally similar to each other with r.m.s.d. distance of 1.7 Å. Our structure supports the switching model of the N-helix in DRS which was proposed to control the binding affinity between DRS and tRNAAsp, by showing the C-terminal end of the N-helix. And post-translational modification (PTM) of DRS analyses found new PTM sites that seem to affect the interaction of DRS and tRNAAsp. Ser146 residue located in dimeric interface of DRS is one of phosphorylation sites, and we imply that the phosphorylation of Ser146 triggers the conformational change of DRS which could be related with unforseen function. Our structural study might help to elucidate its interactions in MSC and shed light on its non-canonical functions.Contents Abstract i Contents ii List of figures iii List of table iii I. Introduction II. Materials and Methods 1. Materials 2. Methods 2.1. Cloning 2.2. Overexpression 2.3. Purification 2.4. Crystallization 2.5. X-ray data collection and structure determination 2.6. Post-translational modification analysis 2.7. Mutagenesis 2.8. Structure determination of DRS S146D mutant III. Results and Discussion 1. Cloning 2. Overexpression and Purification 3. Crystallization 4. X-ray data collection and structure determination 5. Structural comparison between DRS and DRS2 6. N-terminal extension of DRS 7. PTM studies of DRS 8. Key intermolecular interaction of DRS dimer. 9. Site-directed mutations in DRS 10. Structure determination of DRS S146D mutant IV. References V. Acknowledgment Abstract in KoreanMaste