Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4

Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflam- matory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phospho- diester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5 0 -to -3 0 manner and are blocked by 5 0 -phosphorylation. We captured structures in apo, intermediate, and product states and revealed a "link- and-release"two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3-phosphohistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease -associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.