0428同步年報-2021-全

056 ACTIVITY REPORT 2021 The interaction network of the ADPR binding groove consists of four binding surfaces (S1- S4) and thus provides effective hydrophobic interaction and hydrogen-bond networks with ADPR. Of note, the steric position of water molecule W7 appears to be the corresponding occupant of a conceivable nucleophilic water site by the acetyl group of 2”- O AADPR. Establishment of the steric hindrance (Gly87, Gly88, and Phe152) in the vicinity of the ribose 3”-hydroxyl group seems to retain the proper orientation of 3”- O AADPR for catalysis. The side chain of Tyr50 from the α2-helix neighboring Asn26 evidently points toward the 3”-hydroxyl groups of the distal ribose moiety, which seems reasonable to concede a nearby nucleophilic water for catalysis in stereochemistry. Since the discovery of the non- canonical substrate specificity of Poa1p, crucial residues involved in the intriguing activity remain obscure. To gain insight into the relevance of the substrate specificity and activity of Poa1p, potentially critical residues for substrate binding and catalysis were designated for mutational study. Kinetic analysis of the designed Poa1p mutants showed a decreased turnover rate ( K cat ) to certain degrees. In addition, the Poa1pF152A- ADPR structure revealed that the phenyl group of Phe152 established a steric hindrance to stabilize the orientation of the distal ribose of O AADPR for catalysis. Of note, the RP-HPLC results of these mutants confirmed their catalytic roles in distinct substrate specificities of O AADPR isomers. His23 was engaged in catalyzing 1”- O AADPR hydrolysis, which indicates its potential role in protein de-mono- ADP-ribosylation, whereas Asn26 and Tyr50 were mainly contributed to 3”- O AADPR hydrolysis, in line with the authors' hypothesis from the structural information. To elaborate the catalytic specificity of 3”- O AADPR hydrolysis from RP-HPLC and kinetic assays, we present a structural comparison side by side of the active site of the Poa1p- ADPR complex ( Fig. 2 ). The α2 helix in Poa1p adopts a core structure more compact than other macro domains, thus providing the best chance for the non-conserved tyrosine residue (Tyr50 in Poa1p) to engage in 3”- O AADPR catalysis. As compared with the isostructural residues of other macro domains, all residues are non-catalytic and far from the distal ribose of the ADPR moiety. The biological consequence of the O AADPR hydrolysis via Poa1p was further explored. A disruption of Poa1p expression in yeast showed a striking sensitivity to transcriptional stress, which implies a physiological role in response to nucleotide depletion. In summary, five conclusions were drawn from the structural and biochemical data. (1) Poa1p is the only catalytically active macro domain in yeast toward deacetylation of O AADPR. (2) The first complex structure of Poa1p with ADPR was determined. (3) Poa1p exhibits the non-canonical 3”-and 1”- O AADPR deacetylase activities that are distinct from canonical 2”- O AADPR deacetylase. (4) The specialized residue Tyr50 and compact folding of the α2 helix in Poa1p contribute to the distinct substrate specificity toward 3”- O AADPR. (5) Phenotypic consequences of the transcriptional effect in yeast might serve as a model organism to discover further physiological functions of O AADPR deacetylases. Altogether, our studies provide a new picture for the diverse catalytic properties of POA1-like macro domains but decipher its biological roles Fig. 1 : (a) Overall structure and topology of Poa1p in a complex with ADP-ribose (ADPR). The disordered region is shown as dashed lines. (b) ADPR binding surfaces (S1-S4) and interaction network of ADPR with protein residues of Poa1p. Residues within S1-S4 are shown as sticks with carbon in blue, yellow, purple and salmon, respectively. [Reproduced from Ref. 5]