Structural and Mechanistic Insights into Disease-associated Endolysosomal Exonucleases PLD3 and PLD4
Year: 2025
Type: conference-abstract
Abstract: Phospholipases D3 and D4 (PLD3 and PLD4) are exonucleases located in endolysosomal compartments. PLD3 and PLD4 digeste single-stranded nucleic acids in a 5′-to-3′ manner, and are associated with autoinflammatory and autoimmune diseases. Here we demonstrate the crystal structures of PLD3 and PLD4 in apo, intermediate, and product states. Both enzymes form a dimer of pseudo-dimers, where the active site locates at the interface of the intra-chain dimer. The structures demonstrate a 'link- and-release' two-step catalytic mechanism, where a covalent histidine-linked intermediate is involved in this process. Unexpectedly, we captured a covalent phosphohistidine in the active site when co-crystallizing the enzyme with a 5′- phosphorylated oligonucleotide, revealing an extremely slow phosphatase activity of PLD3 and PLD4 against 5′-phosphorylated oligonucleotides. Therefore, these oligonucleotides act as strong inhibitors of the exonucleases. Compared to PLD3, PLD4 contains an extra hydrophobic clamp that stabilizes the substrate and could affect oligonucleotide substrate preference and product release. Additionally, the disease-associated mutants reduce enzyme activity and thermostability, while the structures provide insights into disease association. Overall, our biochemical and structural analyses deepen our understanding of PLD catalytic mechanism and provide insights into therapeutic design.
Source: Structural Dynamics
DOI: 10.1063/4.0000621
Institution: Scripps Research Institute
Language: English
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $2,200
OpenAlex ID: W4409324533