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This dissertation, Structural Studies of RIG-I Like Receptors and DAK, the Negative Regulator of MDA-5, a Member of the RLR Family by Hei-ching, Jane, Kwok, 郭希晴, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: i Abstract of thesis entitled Structural studies of RIG-I-like receptors and DAK, the negative regulator of MDA-5 - a member of the RLR family Submitted by Hei Ching Jane KWOK for the degree of Doctor of Philosophy at The University of Hong Kong in August 2014 RIG-I like receptors (RIG-I, MDA-5 and LGP-2) are responsible for the recognition of intracellular viral RNA and initiate innate immune response in human. RIG-I can recognize viral RNA from various viruses including influenza A virus and ebola virus whereas MDA-5 triggers the responses from piconoviruses RNA specifically. Structures were solved with human RIG-I ΔCARD and MDA-5 ΔCARD in the presence of dsRNA and ATP, yet the structure of the apo inactivated RIG-I remained unclear. This thesis presented the X-ray diffraction data of full length RIG-I crystals with a resolution of 2.85A, at spacegroup of F23 and a s unit cell of a= b= c= 216.43A, α= β= γ= 90. Our work indicated a promising solution towards the solving of RIG- I structure using MAD by I3C phasing. Once the structure of apo inactivated RIG-I is solved, together with the structures solved previously, we would be able to derive the full mechanism for RIG-I activation upon viral RNA binding and ATP hydrolysis. This will be beneficial for the future in controlling viral infection towards innate immune system in human. ii DAK, the negative regulator of MDA-5, is initially discovered for its function for Dha phosphorylation: Dha + ATP  DhaP + ADP. It also has a secondary enzymatic function as a FMN cyclase: FAD  AMP + cFMN, which only exists in mammalian DAK. It is the only enzyme known to produce cFMN which the fate is still unknown. The regulation between the dual-function enzyme is uncertain but experimental data indicated that the substrates of one reaction inhibit the activities of the other (Cabezas, Costas, Pinto, Couto, & Cameselle, 2005). We investigated the FMN cyclase properties of hDAK by crystallizing full length human DAK and soaking the crystals with FAD in different conditions. Our structural studies reveal the binding sites for FAD, AMP and Dha. When superposing hDAK structures and DAK structures in C. freundii and E. coli, it became obvious that FAD, AMP, ADP and ATP molecule occupy the same site 2+ 2 within L domain with Mg or Mn coordinating between the conserved Asp396, Asp401, Asp403 and the phosphate(s) group in the molecule. The isoalloxazine ring of FAD protrudes to the adjacent K domain that is in proximity ((Word count: 447) Subjects: Natural immunityRNA viruses

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