MacroD1 is an enzyme that hydrolyzes protein mono-ADP-ribosylation. However, the key catalytic residues of MacroD1 in these biochemical reactions remain elusive. Here, we present the crystal structure of MacroD1 in a complex with ADP-ribose (ADPR). The β5-α10-loop functions as a switch loop to mediate substrate recognition and right orientation. The conserved Phe²⁷² in the β5-α10-loop plays a crucial role in the orientation of ADPR distal ribose, and a conserved hydrogen-bond network contributes significantly to hold and orient the catalytic water12, which mediates ADPR hydrolysis. Moreover, we found that MacroD1 was recruited to the sites of DNA damage via recognition of ADP-ribosylation at DNA lesions. The MacroD1-mediated ADPR hydrolysis is essential for DNA damage repair. Taken together, our study provides structural and functional insights into the molecular mechanism of MacroD1-mediated ADPR hydrolysis and its role in DNA damage repair.

MacroD1是一种水解蛋白质单ADP核糖基化的酶。但是，MacroD1在这些生化反应中的关键催化残基仍然难以捉摸。在这里，我们介绍MacroD1与ADP-核糖（ADPR）形成复合物的晶体结构。β5-α10环用作转换环，以介导底物识别和正确定向。保守的Phe ²⁷²β5-α10环中的α在ADPR远端核糖的定向中起关键作用，并且保守的氢键网络对保持和定向催化水12起到了重要作用，水介导了ADPR水解。此外，我们发现MacroD1通过识别DNA损伤处的ADP-核糖基化而被募集到DNA损伤的位点。MacroD1介导的ADPR水解对于DNA损伤修复至关重要。综上所述，我们的研究为MacroD1介导的ADPR水解的分子机制及其在DNA损伤修复中的作用提供了结构和功能方面的见解。