0428同步年報-2021-全

Life Science 055 This report features the work of Yu-Yuan Hsiao and his collaborators published in Nat Commun. 12 , 601 (2021). TLS 13B1 Protein Crystallography TLS 15A1 Biopharmaceuticals Protein Crystallography TPS 05A Protein Microcrystallography • Protein Crystallography • Biological Macromolecules, Protein Structures, Life Science References 1 T.-C. Liu, C.-T.g Lin, K.-C. Chang, K.-W. Guo, S. Wang, J.-W. Chu, Y.-Y. Hsiao, Nat. Commun. 12 , 601 (2021). 2 T. C. Liu, K. W. Guo, J. W. Chu, Y. Y. Hsiao, Comput. Struct. Biotechnol. J. 19 , 3682 (2021). Poa1p Macro Domain Structure: Deciphering a Non-Canonical 3"- O AADPR Deacetylase In this report, the undefined enzymatic activity and substrate specificity of the Poa1p macro domain were revealed on deciphering the crystal structures in a combination of biochemical approaches and guided a study of biological effects of the OAADPR metabolism in epigenetics. O -acetyl-ADP-ribose ( O AADPR) is a fascinating signaling molecule that has been implicated in the regulation of numerous cellular processes, including the formation of silent information regulator complexes and gene silencing. This molecule was first identified from the conserved NAD- dependent histone/protein deacetylase reaction catalyzed by sirtuins. In cells, the ratio of 2”- and 3”- O AADPR exists in equilibrium as 48:52 at slightly alkaline pH through a non- enzymatic intermolecular transesterification. 1 There is accumulating evidence that a group of macro domain proteins efficiently catalyzes the deacetylation of 2”- O AADPR, including MacroD-like proteins (human MacroD1, human MacroD2, E. coli Ymdb, O. iheyensis MacroD), the sirtuin-linked macro domain SAV0325 from S. aureus , and the human TARG1-like macro domain C6orf130. 2 The catalysis ability of macro domains stems from their physical or genetic link with sirtuins, thus revealing the functional connections with sirtuins and a novel aspect of O AADPR metabolism. 3 To date, only 2”- O AADPR deacetylases but no 3”- O AADPR deacetylase was reported. S. cerevisiae contains sirtuins as a major NAD-consuming family but lacks any poly(ADP-ribose) polymerase (PARP) homologues, which indicates that sirtuin reaction product O AADPR might hold the most potential physiological roles in yeast. Qualitative analysis of yeast cell extracts revealed at least three distinct activities contributing to the metabolism of O AADPR in vivo , but only Nudix hydrolase Ysa1 was reported to hydrolyze O AADPR/ADPR to AMP and ribose phosphate or acetyl-ribose phosphate, thereby lowering the cellular O AADPR/ADPR levels. 4 Protein identities of the unknown deacetylase and acetyl transferase remain obscure. 2,4 Poa1p is a unique fungal-type macro domain protein that shares little sequence homology with other macro domains. To explore the function of the uncharacterized macro domain, a research team led by Chun-Hua Hsu (National Taiwan University) determined the structures of apo , ADPR-bound Poa1p, and its mutants. The diffraction data were collected at TPS 05A , TLS 15A1 and TLS 13B1 . 5 Our biochemical data reveals that, within two macro domains from S. cerevisiae , only Poa1p carried a robust catalytic activity toward deacetylation of O AADPR, yielding a significant reaction product, ADPR. To elucidate enzymatic properties of Poa1p at a molecular level, the crystal structure of ADPR-bound Poa1p was determined ( Fig. 1(a) ). The structure of Poa1p-ADPR complex, at resolution 1.78 Å, adopts a three-layered α/β/α sandwich fold with the ADPR molecule accommodated within the central crevice of Poa1p. This condition strongly indicates a typical macro domain fold for ADPR binding. Having structurally characterized the Poa1p macro domain, the authors then investigated the substrate specificity of Poa1p for O AADPR deacetylation. The RP-HPLC and 18 O-labeling results revealed that Poa1p can hydrolyze both 3”- and 1”- O AADPR isomers, and preferentially facilitate 3”- O AADPR cleavage at neutral pH. To decipher the molecular mechanism of this finding, interactions between Poa1p and ADPR were examined ( Fig. 1(b) , see next page).