Structural and functional elucidations of functionally diverse macrodomain family proteins

• Main Authors: Chao-Cheng Cho, 卓昭成
• Other Authors: 徐駿森
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/3gwhbt

博士 === 國立臺灣大學 === 基因體與系統生物學學位學程 === 106 === Macrodomain is a ubiquitous protein module found in organisms of eukaryotes, bacteria, and archaea and well-known for its interaction with adenosine diphosphate ribose (ADP-ribose), a metabolite of protein ADP-ribosylation. Macrodomain-containing proteins exhibit diverse enzymatic activities to process ADP-ribose derivatives such as poly ADP-ribose (PAR), ADP-ribose 1’’-phosphate (Appr1p), and protein-linked mono ADP-ribose, and often participate in regulating various cellular functions including DNA repair, gene expression, and cellular signaling. In this study, structural and biochemical approaches are taken to study the structure and function of two representatives of macrodomain family, viral macrodomain and bacterial poly ADP-ribose glycohydrolase (PARG). The genomes of coronaviruses and a few other RNA viruses encode macrodomains. The non-structural protein of the newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) harbors the conserved macrodomain with unknown function. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macrodomain as a more efficient ADP-ribose binding module than macrodomains from other CoVs. Furthermore, the crystal structure of MERS-CoV macrodomain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macrodomains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved aspartic acid at position 20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macrodomain to ADP-ribose. In eukaryotes, PARG is the major enzyme catalyzing the breakdown of PAR chains and responsible for the turnover of poly ADP-ribosylation (PARylation). However, PARylation of prokaryotic organisms remains poorly understood. We performed immunoblotting using the specific antibody against PAR and detected endogenous PAR signals in the radioresistant bacterium Deinococcus radiodurans. The result of immunoblotting showed that PAR level is upregulated after UV irradiation and disruption of the PARG homologue of D. radiodurans (DrPARG) causes accumulation of PAR in cell. Furthermore, we determined the crystal structures of apo and ADP-ribose bound DrPARG belonging to various space groups. Comparison of apo and bound DrPARG structures revealed conformational changes of the protein during ADP-ribose binding, which may be resulted from lacking structural supports of the flexible N-terminus. The bacterial PARG, unlike its eukaryotic counterpart, was reported to be obligate exo-glycohydrolase. Surprisingly, a solvent accessible 2’-hydroxy group of ADP-ribose was found in the bound form structure, suggesting that DrPARG may possess endo-glycohydrolase activity. In consistent with this finding, in vitro cleavage assay detected PAR in DrPARG processed products. Comparison of DrPARG-PAR model and the obligate exo-PARG structure suggested that the structural flexibility along with the threonine at position 267 (T267) contribute to the endo-glycohydrolase activity. Structural and enzymatic activity analyses were performed and showed that T267 substitution in DrPARG affects endo-glycohydrolase activity. Finally, we showed that DrPARG is capable of processing endogenous PAR of D. radiodurans. Our study of MERS-CoV macrodomain and DrPARG will provide new insights into the structure and function of diverse members of macrodomain family and help to understand their roles in individual organism.