Mechanistic insights into single-stranded DNA degradation by lysosomal exonucleases PLD3 and PLD4 from structural snapshots

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: minor_revision

Abstract
Lysosomal exonuclease phospholipase D (PLD) family PLD3 and PLD4 degrade single-stranded RNA or DNA and regulate TLR7 or TLR9 responses. Polymorphisms of these enzymes are associated with human diseases: PLD4 is associated with inflammatory diseases, and PLD3 is associated with neurodegenerative diseases. Here, we determine the structures of substrate-bound PLD3 and PLD4 by cryo-electron microscopy. Our structures reveal that PLD3 rebuilds a substrate-binding pocket, depending on the substrate, mainly via motion of the Phe335-containing loop. Furthermore, we captured the structure in a metastable state that appears during substrate rearrangement following product release. Together, our findings identify the residues that underlie the distinct activities of PLD3 and PLD4. This study provides a mechanistic basis for the exonuclease activity of PLD3 and PLD4 in single-stranded DNA degradation.

Introduction
Nucleic acids (NAs) are fundamental molecules in living organisms that carry genetic information, yet exogenous NAs and their metabolites also play essential roles in host defense1,2. In innate immunity, pathogenic NAs are detected by NA sensors including Toll-like receptor (TLR)3,7-9 and 13 in the endosomal compartment3, as well as retinoic acid-inducible gene-I (RIG-I)-like receptors and cyclic GMP-AMP synthase (cGAS) in the cytoplasm4. These NA sensors trigger the NF-κB and IRF signaling to induce cytokine and type-I interferon production, promoting inflammatory and antiviral responses.

Nucleases restrict NA availability or produce activating ligands, playing indispensableroles in regulating innate immunity. Some sensors recognize double-stranded RNA (dsRNA), double-stranded DNA (dsDNA) or ribosomal RNA, while others recognize DNA/RNA degradation products, complicating the regulation mediated by nucleases.

DNase II, an endonuclease for ssDNA with low sequence specificity, is required for generating TLR9 ligands5. However, DNase II deficiency leads to constitutive activation of cGAS-STING and AIM26, resulting in type I interferonopathy in human7 and embryonic lethality due to type I IFN-dependent anemia in mice8. RNase T2, which digests single-stranded RNA, regulates TLR7 and TLR8 but negatively regulates TLR39–11.

PLD3 and PLD4 are endolysosomal exonucleases that degrade single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA)12,13. Genome-wide association studies have shown that PLD4 polymorphisms are associated with autoimmune diseases, including systemic lupus erythematosus14, systemic sclerosis15 and rheumatoid arthritis16 whereas PLD3 polymorphisms are associated with neurodegenerative diseases such as Alzheimer’s disease17, spinocerebellar ataxia18 and leukoencephalopathy syndrome19. Reflecting these distinct disease associations, PLD4 is expressed in limited tissues, mainly within immune cells, whereas PLD3 is ubiquitously expressed across various tissues. A recent study revealed that PLD3 degrades mitochondrial DNA (mtDNA) as a major substrate within lysosomes of neuronal cells20. In PLD3-deficient cells, mtDNA leakage into the cytosol is elevated, activating cGAS-STING-linked autophagy, suggesting a potential link between PLD3 dysfunction and APP metabolism20.

PLD3 and PLD4 negatively regulate TLR9 responses in immune cells12. They also contribute to the generation of TLR7 ligand in collaboration with RNase T221 or negatively regulate TLR13 responses13. PLD3 and PLD4 are specific for 5’-OH nucleotides, but not for 5’-phosphorylated nucleotides. Since RNase T2 and DNase II generate 3’-phosphorylated nucleotide and 5’-OH nucleotides, PLD3 and PLD4 can degrade the products generated by these nucleases. PLD3 and PLD4 belong to the phospholipase D (PLD) family, which contains the conserved HxK(x) D/E (HKD/E) motif (Fig. 1a, Supplementary Fig. 1). Two HKD/E motifs, brought together either through homodimerization or internal duplication, form a catalytic cavity, in which His and Lys are involved in catalysis, while the Asp/Glu is located away from the catalytic center and contributes to stabilizing protein folding22,23. Despite the functional diversity within the PLD family, including lipid phospholipase, endonuclease, and exonuclease activities, members share a common catalytic mechanism. This mechanism proceeds via a ping-pong type reaction involving a covalent phosphohistidine intermediate24–26. One of the two conserved His in the HKD/E motif functions as a nucleophile to attack the substrate, resulting in the formation of a phosphohistidine intermediate. In the subsequent step, the other His serves as a general acid to activate a water molecule that attacks the phosphohistidine to cleave the phosphodiester bond and regenerate the free catalytic His residue.

Recently, the crystal structures of PLD3 and PLD4 have been reported27,28, revealing the overall architecture of its luminal domain and dimeric organization mediated by inter-helical interactions. The structure of PLD3 co-crystallized with oligonucleotide (ODN) represents the product-bound structure, as the ODN was degraded within the crystal27. PLD3 and PLD4 can function as a 5’-phosphatase for 5’-phosphorylated ssDNA (5’-Pi ssDNA), although their catalytic rate is more than 10,000-fold slower than that of exonucleases acting on 5’-OH ssDNA. The crystal structures of PLD3 and PLD4 co-crystallized with 5’-Pi ssDNA have also been reported28. In PLD3 structures, due to differences in crystallization times, distinct structures were obtained where the catalytic residue His was either phosphorylated via phosphate transfer from 5’-Pi ssDNA or dephosphorylated. In PLD4 structure, the catalytic residue His was phosphorylated, and the dephosphorylated ssDNA was trapped within the catalytic cavity. However, our understanding of substrate recognition, specificity, and exonuclease mechanism remains limited due to the insufficient electron density for the substrate or ssDNA product-bound form containing phosphohistidine intermediate in the previous works.

Here, we present the structures of PLD3 bound to different substrate ODNs by single particle cryo-EM analysis. The substrate adopts an L-shaped backbone structure at the 5’ end, accommodated within the narrow and deep catalytic cavity. Our structures reveal that PLD3 rebuilds its substrate-binding pocket depending on the binding nucleotides mainly by the motion of the Phe335-containing loop. Structural snapshots in the apo form, substrate-binding, product-binding and metastable states provide the structural basis for the conformational change in the process of degrading ssDNA by PLD3 and PLD4. We also identified the residues responsible the difference in exonuclease activity between PLD3 and PLD4, offering potential targets for drug design targeting PLD3 and PLD4.

Results
ssDNA binding to PLD3 is enhanced under acidic conditions
Human PLD3 (hPLD3) has been reported to undergo proteolytic cleavage after expression as a type-II transmembrane protein (Fig. 1a), yielding a luminal soluble protein in the lysosome29 that retains exonuclease activity comparable to that of the full-length protein30. The luminal domain of hPLD3 was purified from mammalian cells. We employed electrophoretic mobility shift assay (EMSA) to screen the conditions that form the stable complex of hPLD3 and its substrate prior to cryo-EM analysis. Unexpectedly, during this screening, we found that PLD3 H201A/H416A mutant (hPLD3_HA), in which the catalytic residues were mutated, nearly abolished the exonuclease activity, but a small residual activity remained (Supplementary Fig. 2a). This residual exonuclease activity was suppressed by the addition of sodium vanadate which serves as an inhibitor of phosphoryl transfer enzymes. We conducted EMSA assay with varying concentrations of hPLD3 (0, 2.5, 5.0, 10, and 20 μM) incubated with ssDNA. A band corresponding to ssDNA bound to hPLD3 was detected only at 20 μM at pH 6.0; however, at pH 5.5 or below, the band was observed at 10 μM (Supplementary Fig. 2b), indicating that acidic conditions strengthen the interaction between hPLD3 and its substrate. This observation is consistent with previous reports that hPLD3 and hPLD4 require acidic environments for their exonuclease activity12.

Structure determination of PLD3 and PLD4 bound to ssDNA
We successfully determined the structures of hPLD3_HA bound to several different substrates, poly(T) ODN (hPLD3_poly(T)) and poly(A) ODN (hPLD3_poly(A)) at 2.9 Å resolution in addition to hPLD3 WT apo-form (hPLD3_apo) structure at 2.9 Å resolution (Supplementary Fig. 3-5, Supplementary Table 1). We also determined the cryo-EM structures of hPLD4 WT apo-form (hPLD4_apo) and in the presence of poly(T) substrate at 3.3 Å and 3.1 Å resolution, respectively (Supplementary Table 1, Supplementary Figs. 6, 7). Sodium vanadate was added at a low concentration (0.1 mM sodium vanadate) to the sample of hPLD3 HA bound to substrate. However, vanadate was not observed in the cryo-EM map, suggesting that it is unlikely to bind to hPLD3 under the cryo-EM conditions.

PLD3 forms a homodimer through two α-helices, and each protomer consists of HKD1 and HKE2, which adopt the α/β hydrolase fold. The conserved His and Lys residues of each HKD/E motif (His201 and Lys203 in HKD1 and His416 and Lys418 in HKE2) are positioned facing each other to form the catalytic site. In contrast, the Asp/Glu (Asp208 in HKD1 and Glu423 in HKE2) stabilizes hPLD3 structure by forming hydrogen bonds with the main chain amides (Supplementary Fig. 8a). The overall structure of hPLD3_apo is similar to the recently reported crystal structure of hPLD327 (Supplementary Fig. 8b).

The protomer structure of hPLD4 is also similar to that of hPLD3, and both proteins possess essentially the same dimer interface (Supplementary Fig. 9a). In sharp contrast to PLD3, PLD4 forms a tetramer in our structure, adopting a face-to-face configuration with a twist, mediated by the additional interactions between two dimers (A-B dimer and C-D dimer) (Supplementary Fig. 9). The tetramer formation of PLD4 is also supported by size exclusion chromatography, in which PLD4 eluted faster than PLD3 (Supplementary Fig. 10), consistent with a larger molecular assembly. The cryo-EM samples were adjusted to approximately 1 mg/mL (~18 μM), at which concentration hPLD3 was predominantly observed as a dimer, whereas hPLD4 was mainly observed as a tetramer in the cryo-EM analysis. The two PLD4 dimers contact each other through two interfaces with a total area of ~900 Å2. One interface involves the hydrogen bonds between Gln290 and the main chain of His293, and π-π stacking interactions between Phe289 and His285 or His285 and His293. The other interface is essentially the same, but the interacting pairs in each protomer are inverted (Supplementary Fig. 9b). Either the A–B dimer or the C–D dimer is flipped across the two-fold axis. This leads to a misalignment with the opposite dimer (C–D or A–B, respectively), indicating that strict two-fold symmetry is absent between the two dimers (Supplementary Fig. 9c).

It has been reported that hPLD3 can be N-glycosylated at five sites based on mass spectrometry analysis27. In previously determined crystal structures, only two N-linked glycans (linked to Asn236 and Asn284 in hPLD3) were modeled for both human and mouse PLD327,28. In contrast, four N-linked glycans, corresponding to all sites except for Asn97, were observed in our cryo-EM structure (Fig. 1a). For PLD4, no experimental data are available to identify specific glycosylation sites. In the crystal structure of human PLD4, no glycans were modeled28, whereas in our cryo-EM structure, six N-linked glycans were observed (Fig. 1a).

L-shaped substrate captured in the catalytic cavity of PLD3
We determined the structures of hPLD3_poly(T) and hPLD3_poly(A). In both structures, the substrates are captured within a narrow and deep catalytic cavity surrounded by a negatively charged surface (Supplementary Fig. 11). The substrates enter the cavity almost perpendicularly, rather than extending along the protein surface (Fig. 1b). A prominent feature of the binding mode is a sharp bend in the backbone between the 5′-end and the second nucleotide, producing an L-shaped conformation in which the sugar-phosphate backbone is directed inward and the base moiety of the 5′-end nucleotide is oriented outward (Fig. 1c). This L-shaped configuration positions the 5′-end nucleotide near the cavity exit, facilitating the release of the 3′-phosphorylated mononucleotide, the product of PLD3 cleavage. It is widely accepted that ssDNA exhibits considerable flexibility. Therefore, the observed L-shaped conformation of the ssDNA substrate is a consequence of structural constraints imposed by the binding pocket. Interestingly, our structures also revealed a substrate-dependent conformational change in both PLD3 and the bound substrate, which will be discussed in a later section.

PLD3 adjusts the catalytic cavity size to recognize substrates with distinct conformations
In the cryo-EM map of hPLD3_poly(T), four nucleotides at the 5’ end (dT1-dT4) are clearly resolved and modeled (Supplementary Fig. 12a). The first three nucleotides are accommodated in the catalytic cavity of PLD3, while the fourth nucleotide lies on the cavity (Fig. 1c). The subsequent nucleotides are not well resolved due to the dynamic nature in the PLD3-bound state. The 5’-end dT1 is captured at the innermost region of the cavity, and its 3’-phosphate group faces inward, forming interactions with Lys203, Asn218, Lys418 and Asn432, while the base group faces outward, engaging in π-π stacking with Tyr411 (Fig. 1d). The H201A and H416A mutants also orient their side chains toward the phosphate group of dT1, which is suitable for hydrolyzing 5’ nucleotide of the substrate. Additionally, the 5’-hydroxyl group interacts with the phenolic group of Tyr126 and the main chain carbonyl group of Tyr411. This moiety is confined within a narrow internal space, indicating that 5’-phosphorylation of substrates would cause the steric clash with PLD3 (Fig. 1c, d), thereby preventing accommodation. This structural observation aligns with previous study13 and current findings showing that PLD3 lacks exonuclease activity against 5’-phosphorylated ODN (Supplementary Fig. 13a). The 3’-phosphate group of dT2 interacts with the side chains of Asn328, Trp367 and Tyr437, while the 3’-phosphate group of the third nucleotide interacts with the side chains of His369 and Arg488 (Fig. 1d). The base moiety of dT2, that of dT3 and Phe335 are stacked up in this order through a π-π stacking interaction. dT4 is largely exposed from the catalytic cavity of PLD3 and forms an off-plane interaction with the aromatic ring of Phe335.

In the cryo-EM map of hPLD3_poly(A), five nucleotides at the 5’ end (dA1-dA5) are observed (Supplementary Fig. 12b). The 5’-end dA1 interacts with PLD3 in essentially the same way as dT1 in the poly(T) substrate, and dA2 also adopts the same position as that in the poly(T) substrate (Fig. 1c, e). Strikingly, the base moiety of dA3 is oriented in the opposite direction relative to dA2. As a result, the base moiety of dA2 directly forms an aromatic stacking interaction with Phe335, along with additional polar interactions with Thr441 (Fig. 1c, e). The flipped dA3 forms an aromatic stacking interaction with His369. The following nucleotides, dA4 and dA5, do not interact directly with PLD3. In addition to the distinct conformations of the substrates, exonuclease assay revealed that PLD3 exhibits higher exonuclease activity toward poly(T) than poly(A), suggesting that PLD3 has a sequence preference in its exonuclease activity (Fig. 1f).

The PLD3 structures of the apo form and the substrate-bound forms are well superimposed, suggesting that no large conformational changes occur upon substrate binding, except for the loop containing Phe335. This loop forms a π-π stacking interaction with dT3 in the poly(T) substrate or dA2 in poly(A) substrate. When the poly(A) binds to PLD3, Phe335 retains the same orientation as in the apo-form, allowing dA2 to be snugly accommodated within a confined space (Fig. 1e), formed by Tyr437 and Thr441 at the base, and flanked by Phe125 and Val199 on one side and Phe335 on the other (Supplementary Fig. 14). In contrast, when poly(T) binds PLD3, the benzene ring of Phe335 flips outward to create space for dT3, enabling the formation of a π-π stacking interaction network involving dT2, dT3 and Phe335 (Supplementary Fig. 14). If poly(A) were to adopt the same arrangement as poly(T), steric clashes would occur between dA2 and dA3 due to the larger purine base, indicating that PLD3 can recognize purine and pyrimidine base in a different manner by the motion of Phe335.

Recognition of the 5’ three to four nucleotides by PLD3 is important for the regulation of TLR9 signaling
To validate the functional importance of residues that interact with the substrate, we conducted an end-labeled fluorescence-quenched oligonucleotide (EFQO) assay to monitor the exonuclease activity, using purified PLD3 and an ODN labeled with 5’-FAM and 3’-quencher. ODN1668, which is one of type B CpG ODN serving as a TLR9 ligand, was used in the EFQO assay. We mutated the residues involved in the catalytic reaction (His201/His416, and Glu229 which is predicted to accept proton from His416 to initiate nucleophile attack by His416), in phosphate group interactions (Lys203, Asn328, Trp367, His369, Lys418 and Tyr437) and in π-π stacking interactions (Phe335 and Tyr411). All of the above mutants reduced activity, supporting the importance of phosphate-mediated and π-π stacking interactions in substrate recognition (Fig. 2a, b). The EFQO assay revealed that the HA mutant retained only 0.2% of the specific activity of the wild type, indicating that only a minimal level of activity remains.

To further examine the functional importance in cellulo, Ba/F3 cells were stimulated with CpG-B and NFκB activation was evaluated. Overexpression of wild type PLD3 (PLD3 WT) attenuated TLR9-dependent NFκB activation. In contrast, the response was restored to near-control level when PLD3 mutants K203A, E229A, H369A or K418A were overexpressed. PLD3 H201A/H416A, F335A or W367A mutants showed weaker attenuation of the response than PLD3 WT, whereas N328A, Y411A or Y437F mutants attenuated NFκB activation to a similar extent as PLD3 WT (Fig. 2c, Supplementary Fig. 15). These results further support the importance of PLD3 exonuclease activity in the regulation of TLR9 signaling.

Structural comparison identified residues that contribute to the difference in exonuclease activity between PLD3 and PLD4
Our detailed analysis confirmed that PLD3 exhibits higher exonuclease activity than PLD4 (Supplementary Fig. 13b), consistent with the previous report28. The structures of PLD3 bound substrates allow us to re-examine the difference in activity between PLD3 and PLD4. Consistent with the sequence similarity, the PLD3_apo and PLD4_apo structures are superimposed well, with a root-mean-square deviation (r.m.s.d.) of 0.76 Å, although several loop regions exhibit structural differences (Fig. 3a). Similarly, PLD3_poly(T) is well aligned with PLD3_apo (r.m.s.d. of 0.74 Å), and each protomer of PLD4, whether in the apo form or in the presence of poly(T), also aligns well, with an r.m.s.d. ranging from 0.3 to 0.5 Å, suggesting that PLD3 and PLD4 show no significant conformational change upon substrate binding. Poly(T) substrate fits into PLD4 with only a little steric clash, strongly suggesting that PLD4 can recognize the substrate in a manner similar to PLD3 (Fig. 3b). Structure-based sequence alignment revealed that several substrate-interacting residues in PLD3 are not conserved in PLD4. Asn328, His369 and Arg488 in hPLD3 (Supplementary Fig. 1), which interact with the 3’-phosphates of the substrate, correspond to Glu341, Asn382 and Val503 in hPLD4, respectively. To test the functional consequences of these differences, these residues are converted or vice versa (Fig. 3b). E341N mutation in PLD4 may eliminate the electrostatic repulsion with 3’-phosphate of the second nucleotide. Similarly, V503R mutation in hPLD4 may enhance interaction with 3’-phosphate of the third nucleotide. In our enzymatic assay, E341N mutant of hPLD4 exhibited higher activity than WT and E341N/V503R exhibited markedly increased activity (Fig. 3c). These mutants retained activity even at neutral pH (pH 7.0). Conversely, hPLD3 N328E mutant, which is supposed to cause the electrostatic repulsion with 3’-phosphate of the second nucleotide, exhibited reduced activity compared to WT (Fig. 3c). These results suggest that these sequence discrepancies contribute to the difference in exonuclease activity between PLD3 and PLD4.

Structure of PLD4 in the presence of poly(T) substrate revealed dynamic nature of substrate
In the C1 symmetry cryo-EM map of the hPLD4 tetramer in the presence of poly(T) substrate, poly(T) molecules are observed in both catalytic sites of one dimer, while densities corresponding to poly(T) are ambiguous in the other dimer. Interestingly, the poly(T) densities differ between the two PLD4 protomers. We were able to model two unconnected 3’-phospho dT nucleotides in one protomer and connected three dT nucleotides in the other protomer (Supplementary Fig. 16). Since PLD4 digests ssDNA to generate deoxynucleotide 3’-phosphate, unconnected two 3’-phospho dT nucleotides are assigned as digestion products, including the newly formed 5’ end of the poly(T) substrate, suggesting that this structure represents the product bound form (referred to as “product-bound hPLD4”). The three connected dT nucleotides are assigned as the 5’ portion of poly(T) substrate, indicating that the other protomer represents the substrate-bound form (referred to as “substrate-bound hPLD4”).

In product-bound hPLD4, one 3’-phospho dT forms salt bridges with His214, Lys216, His428 and Lys430 in the conserved HKD/E motif through its 3’ phosphate group and its base moiety is sandwiched between Phe423 and Leu183 via a π-π stacking interaction (Fig. 4a). These interactions are similar to those observed for the 5’-end dT1 of the poly(T) substrate bound to PLD3, suggesting that this nucleotide corresponds to the product dT3-phosphate. The newly formed 5’ end of the substrate, generated by PLD4 digestion, is located adjacent to the product and its 5’ hydroxyl group faces the 5’ hydroxyl group of the product dT 3-phosphate. The base moiety is sandwiched between Val212 and Phe348, while the 3’-phosphate group does not participate in interactions with PLD4 (Fig. 4a). The nucleotide trailing the newly formed 5’ end dT1 were not modeled due to ambiguous density, which may reflect a conformational flexibility (Supplementary Fig. 16a).

In substrate-bound hPLD4, dT1 is sandwiched between Phe423 and Leu183 in a manner similar to product-bound hPLD4. Importantly, in sharp contrast to product-bound hPLD4 and hPLD3_poly(T)/hPLD3_poly(A), the dT1 3’ phosphate group is positioned away from the catalytic residues. His214 forms a salt bridge with the dT1 3’-phosphate group; however, the side chains of Lys216, His428 or Lys430 are more than 5.5 Å away from the 3’-phosphate group of dT1. Instead, phenolic group of Tyr449 forms bifurcated hydrogen bonds with the 3’ phosphate of dT1, contributing to substrate binding. dT2 is sandwiched between Val212 and Phe348 and dT3 stacks on Phe348 (Fig. 4b). These observations strongly suggest that the observed substrate-bound hPLD4 represents a metastable state rather than a substrate-cleaving state, in which the substrate is properly positioned for catalysis. We speculate that this state represents a transient intermediate during substrate rearrangement following product release, reflecting the repositioning of the substrate after initial digestion by PLD4.

Reconstructing catalytic dynamics from structural snapshots
Structural comparisons were performed among hPLD3_apo, ribose-ring intermediate (PDB ID; 8V06) form (mPLD3_intermediate), mouse PLD3 phosphate-bound form (PDB ID; 8V07) (phosphate-bound mPLD3), and human PLD3 product-bound form (PDB ID; 8Q1X) (product-bound hPLD3) to gain further insights into the catalytic mechanism. The conformations of the catalytic residues, as well as those interacting with the substrate, are well conserved between human and mouse, and across various catalytic states. The catalytic residue His416 in human PLD3 (corresponding to His414 in mouse) exhibits a rotamer shift only in the mPLD3_intermediate and the product-bound hPLD3, moving toward His201 in human (His199 in mouse) (Fig. 5a). Tyr411 in human (Tyr409 in mouse) which stacks with the base of the first nucleotide, exhibits structural variability. Phe335 in human (Phe333 in mouse), which stacks with the base of the second or third nucleotide, undergoes conformational changes. The other residues involved in the substrate binding show no significant changes. The first nucleotide of the bound substrate (poly(T) or poly(A)) in the PLD3_poly(T)/poly(A) occupies the same site as that in the product-bound hPLD3, and their phosphate groups are well aligned with that of the mPLD3_intermediate. Taken together, the structural changes during the enzymatic cycle appear to be minor except for the Phe335-containing loop as described above.

We also examined structural differences among substrates bound to PLD3 and PLD4. The structural comparison between hPLD3_poly(T) and product-bound hPLD4 can reflect conformational changes associated with substrate cleavage. The 3’-phosphate group of the product remains in approximately the same position as in hPLD3_poly(T), whereas the 5’-OH group of the product is shifted by 8.1 Å due to ribose-ring flipping after cleavage (Fig. 5b left). Accompanying this conformational change, the phosphate group of the newly formed 5’-end nucleotide is displaced outward (~6.9 Å) to avoid steric clashes with the 5’-OH group of the product (Fig. 5b left). In addition, the 5’-OH of the newly formed nucleotide is displaced outward (~4.9 Å) and forms a hydrogen bond with the 5’-OH group of the product. In contrast, the structural comparison between the substrate-bound hPLD4 and hPLD3_poly(T) reveals conformational changes associated with the transition toward a substrate-cleaving state. The 3’-phosphate of dT1 or dT2 in the PLD4 complex is displaced by 4.8 or 7.9 Å, respectively, relative to the complex with PLD3, whereas the base moiety of dT1 or dT2 remains almost properly aligned due to π-π stacking interaction with Phe423 or Phe348 (Fig. 5b right). These observations suggest that the base moiety may serve as a structural scaffold to guide the phosphate into its proper position. In the case of dT3, the base moiety and the phosphate group exhibit substantial displacement (4.9 and 10.6 Å, respectively). In summary, unlike PLD3/PLD4, which remains largely unchanged in overall structure, the substrate undergoes significant conformational changes across different states of the enzymatic reaction.

Methods
Protein expression and purification
The cDNAs of human PLD3 (hPLD3) and human PLD4 (hPLD4) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. The DNA sequence corresponding to the luminal domain (residues 61-490 for hPLD3 or residues 91-506 for hPLD4) were amplified by the polymerase chain reaction (PCR) and cloned into the pEZT-BM vector. These proteins were fused with an N-terminal secretion signal sequence derived from rabbit IgG heavy chain and a C-terminal HRV3C protease cleavage site and protein A tag. Site-directed mutations were introduced by PCR-based mutagenesis. All plasmids were verified by DNA sequencing. Baculoviruses were generated in Spodoptera frugiperda Sf9 cells using the Bac-to-Bac system (Invitrogen). For protein expression, Expi293F cells (Invitrogen) were cultured in Expi293 expression medium at 37°C under 8% CO2 in a CO2 incubator (PHC). When the cell density reached 3.0−6.0 × 106 cells per mL, cells were diluted to 3.0 × 106 cells per mL and P4 virus was added at a final concentration of 7% (v/v). Cells were cultured at 30°C for 5 days in the presence of 10 mM sodium butyrate to enhance protein expression.

The cell culture supernatant was obtained by centrifugation at 7,000g for 10 min. The collected supernatant was applied to IgG Sepharose 6 Fast Flow (Cytiva), which specifically binds to protein A fusion conjugates. The mixture was gently stirred at 4°C for an hour. The resin was washed with buffer A (20 mM Tris-HCl pH 7.0 containing 150 mM NaCl) and subsequently, proteins were eluted using buffer B (100 mM glycine pH 3.5, 150 mM NaCl). The eluted proteins were instantly neutralized using a pH adjustment buffer (400 mM Tris-HCl pH 7.0, 150 mM NaCl). After cleavage of protein A tag by HRV3C protease, the eluted proteins were further purified by gel filtration chromatography (Superdex 200 increase, Cytiva) in buffer A. The peak fractions were concentrated using Amicon Ultra centrifugal filter (Merk Millipore), frozen in liquid nitrogen and stored at −80°C until use.

Cryo-EM sample preparation and data acquisition
The hPLD3 samples (WT or HA+ssDNA) were adjusted to a concentration of 1.0 mg ml–1, For sample preparation in the presence of ssDNA, 40 μM of 49-mer ssDNA (poly(T) or poly(A)) was mixed with protein samples in buffer C (20 mM MES-NaOH buffer (pH 5.5), 150 mM NaCl) containing 0.1 mM sodium vanadate. Three-microliter aliquots of samples were placed onto a freshly glow-discharged Quantifoil holey carbon grids (R1.2/1.3, Cu, 300 mesh). After 3s of blotting in 100% humidity at 6°C, the grid was plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). For hPLD4, hPLD4 (WT) was adjusted to concentrations of 4 mg ml–1. For sample preparation of hPLD4 in the presence of ssDNA, 40 μM of 49-mer ssDNA was mixed with 1 mg ml-1 protein samples in buffer C (20 mM MES-NaOH buffer (pH 5.5) containing 150 mM NaCl) for 3 min on ice and immediately placed onto a freshly glow-discharged Quantifoil holey carbon grids (R1.2/1.3, Cu, 300 mesh). After 2.5s of blotting in 100% humidity at 6°C, the grid was plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM data collection was performed using a Titan Krios G4 microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy filter (GIF) and a Gatan K3 camera in electron-counting mode, at the Cryo-EM facility of the University of Tokyo, Japan. Imaging was performed at a nominal magnification of ×105,000, which corresponded to a calibrated pixel size of 0.83 Å px–1. Each movie was recorded for 5.0s and subdivided into 64 frames at the specimen. Cryo-EM data were analyzed using CryoSPARC v434. Raw movie stacks were motion-corrected using patch motion correction. The CTF parameters were determined using patch CTF estimation. The data processing workflow is summarized in Supplementary Fig. 3-7. Particles were picked using the blob picker or template picker. Multiple rounds of 2D classification, ab initio reconstruction, and heterogeneous refinement were performed to select particle stacks for final 3D map reconstruction using non-uniform refinement35. The map was binned to a pixel size of 1.09 Å for hPLD4_apo while the other maps were not binned. The final resolution was estimated by gold-standard Fourier shell correlation (FSC) between the two independently refined half maps (FSC=0.143)36. The atomic model was manually accomplished using the Coot program37. The built model was refined through alternating cycles using the Coot and PHENIX programs38. The refinement statistics are summarized in Supplementary Table 1.

Structure and sequence comparison
Multiple sequence alignments of the PLD family proteins were performed using CLUSTAL Omega. Structure figures were prepared using the PyMOL Molecular Graphics System, Schrödinger, L., & DeLano, W or UCSF Chimera39.

Electrophoretic mobility shift assay
A ssDNA (5’-ACGTTCCTGATGCTAAGTATGCACTTCATCGTCAAGCAATGCTATGCAT, Substrate-I) was used. Varying concentrations of hPLD3 (0, 2.5, 5.0, 10, 20 μM) were incubated with 5 μM of Substrate-I at 4°C for 1 h. Sodium vanadate was added two equimolar amounts of hPLD3 when indicated. After incubation for 45-60 minutes, the mixtures were subjected to electrophoresis in Tris-Acetate buffer prepared at the indicated pH.

End-labeled fluorescence-quenched oligonucleotide (EFQO) assays
A reaction mixture was prepared by combining a purified protein sample at a concentration of 5 ng μl-1 with 0.5 μM of 5’ FAM-ssDNA-BHQ1 (FASMAC) with the sequence TCCATGACGTTCCTGAtgct (ODN1668) in which lowercase indicates a phosphorothioate linkage in a volume of 100 μl using a buffer solution containing 20 mM MES-NaOH at pH 5.5 and 150 mM NaCl. The fluorescence emission, detected at wavelengths between 500-550 nm following excitation at 485 nm, was continuously measured using Glomax Discover microplate reader (Promega). The measurements were taken at room temperature over a duration of 100 minutes at 1 minute intervals. Activity was calculated based on the initial rate, which was estimated by fitting a linear regression to the data from 1 to 11 minutes.

Gel-based nuclease activity assay
A reaction mixture was created by combining a purified protein sample at a concentration of 20 nM for PLD3 or 40 nM for PLD4 with 2.5 μM of substrates. The mixture was then adjusted to the specified pH and incubated at 37°C for a designated period. Following the incubation, the mixtures were heated at 95°C for 2 minutes to halt the nuclease activity, and subsequently analyzed by electrophoresis in Tris-Borate-EDTA buffer.

Analysis of TLR9-dependent NFκB activation
TLR9 and Unc93B1 genes were amplified from mouse genomic DNA via PCR, while wild-type and mutant forms of hPLD3 were amplified from cDNA obtained from RIKEN. The TLR9 and Unc93B1 cDNAs were subsequently cloned into retroviral pMX puro vectors provided by Dr. T. Kitamura. Both wild-type and mutant hPLD3 cDNAs were cloned into pMX-IRES-BFP vectors. Cloning procedures were performed using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) and the Rapid DNA Ligation Kit (Roche Applied Science). For retroviral packaging, the pMX vectors were transfected into Plat-E packaging cells using FuGENE 6 (Roche Applied Science). Following 24 hours of incubation, viral supernatants were collected. Ba/F3 cells harboring an NF-κB GFP reporter plasmid were then infected with these viral suspensions in the presence of DOTAP (Roche Applied Science). To standardize hPLD3 expression levels, BFP-positive cells were isolated using FACS Aria (BD Bioscience). Post-stimulation with CpG B, cell analysis was conducted using an ID7000 spectral flow cytometer (Sony Biotechnology). The method of normalization involved subtracting the MFI of unstimulated samples from the MFI of stimulated samples.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Lysosomal phospholipase 3 and 4 (PLD3/PLD4) degrade single stranded RNA and DNA oligonucleotides. Several groups recently reported crystal and cryo-EM structures of these enzymes in the absence of substrate, in a phospho-intermediate state of the reaction and in the presence of DNA products. In the current manuscript, Hirano and colleagues now report the cryo-EM structure of PLD3 and PLD4 in the presence of an un-cleaved DNA substrate. In this way, they elucidate residues and conformational changes in the active site important for substrate binding and catalysis. Based on their data, they suggest a refined model for PLD3 and PLD4-mediated single strand DNA degradation.\n\nThe structural data described in this manuscript appear overall sound. Indeed, they provide some novel mechanistic insights into the catalytic mechanism of PLD3/PLD4. However, in light of the previously published structural and mechanistic data on PLD3/PLD4, in particular from the Nemazee group (Yuan et al, Structure 2024), the overall scientific progress of this work appears rather limited. In my view, the manuscript is therefore not suited for the general audience of Nat Comm, but rather for a more specialized journal.\n\nSpecific comments:\nSome more work could be done to the figures:\nFig. 1b: Please carefully label the figure (PLD3, poly(T) substrate, two-fold axis, etc).\nFig. 1d (and many others) are too crowded and it is difficult to see what the authors aim to show; reduce complexity, for example, by reducing background, and shadows, etc.\nFig. 2b: Please mention in the legend how the activity has been determined. Ideally, it should be a linear fit to the initial rate.\nFig. 3: Please properly label the figures, e.g. add a color code for PLD3 (polyT), PLD4 (apo). This comparison is not very meaningful since the indicated structural changes could be related to the different PLD isoforms (PLD3 vs PLD4) or the substrate (apo vs. polyT) binding. A meaningful comparison would be PLD3 (apo) vs PLD3 (polyT); PLD4 (apo) vs PLD4 (polyT); PLD3 (polyT) vs PLD3 (polyA); PLD3 (polyT) vs PLD4 (polyT); some of these figures are already included in the manuscript.\nFig. 4a is again rather complex. Again, it may be worth removing the shades in Fig. 4a, b.\nFig. 5, line 274 seq. should be described/included in the Discussion, not in the Results. The model is based on data not only from the authors but also from other groups (like the phospho-intermediate) and this analysis represents an interpretation of the data. A (properly labelled) movie showing the transitions between the states would help to explain the suggested mechanism.\nFig. 5c could be reduced in complexity while Fig. 5b is very neat, but should be better labelled.\nTable S1, glycans:\nThe glycans are only mentioned in the data processing report, although they are of potential interest. It would be neat to report and illustrate which glycans are observed in these structures and how to they compare to the previously published crystal/cryo-EM structures.\nFig. S1.\nSome labels could be included in the alignment (for example, green boxes, pink boxes). Human, mouse and bovine sequences from each isoform are essentially identical and the few differences are not mentioned. Would it make sense to provide sequences from more distantly related species to identify conserved/non-conserved areas?\nFig. S2:\nWhat does the triangle on top of the gels indicate? Increasing protein concentrations or increasing time intervals? What is one supposed to see in the band labelled with ‘bound to hPLD3’? Is the intensity of this band changing in the different conditions? Please add some more information to the legend. In fact, has vanadate been added to the cryo-EM conditions? If yes, was the vanadate binding site determined? This may (or may not) reveal an interesting structural intermediate.\nFig. S8: Please properly label the isoforms in the figure!\nFig. S9a/c: I do not understand from the figure how the tetramer assembles, please simply the presentation! Beyond the dimers, the tetrameric assembles does likely not bear any functional relevance? Could the tetramer form if PLD4 still contained a transmembrane region, is there actually a proper two-fold symmetry between the two dimers? Please add some discussion.\nFig. S10: The difference in running behavior is small and it is unclear whether it may be related to different assemblies or slightly different protein concentrations applied to the column. A parallel RALS/MALS analysis would be helpful to decide between these scenarios.\n\nIntroduction\nThe previously published structural data on PLD3/PLD4 should be shortly summarized that one can do a meaningful comparison to the new structures. For example, shortly describe the dimer interface, the phospho-intermediate, the structure of the product-bound PLD3. In particular, the phospho-intermediate structure of PLD4 bound to an uncleaved substrate (pdb: 8v08) should be described in some more detail.\nLine 132: As a result of the EMSA assay, the bound substrate was likely degraded due to the residual exonuclease activity of PLD3 HA, hindering identification of the bound substrate sequence. To unambiguously assign the nucleotide, we determined hPLD3 structure bound to poly(T) or poly(A) substrate.\nI cannot follow the rationale. How was substrate degradation hindered that the authors could determine the substrate-bound structure?\nSome text revisions, including correction of typos, are recommended.", "reviewer_2": "Reviewer Recommendation: Minor Revision\nGeneral Comments\nThank you to the authors for sharing this valuable study. The manuscript is clearly written and presents a compelling investigation into the catalytic mechanisms of PLD3 and PLD4 using a multidisciplinary approach. The cryoEM structures are particularly significant in elucidating the residues involved in catalysis, and the data appear to be of high quality with appropriate validation. The authors have effectively combined cryoEM, biochemical assays, and comparative modeling to reveal structural details relevant to substrate recognition and catalytic mechanism.\nNonetheless, some points should be addressed prior to acceptance to improve accuracy, completeness, and clarity.\nSpecific Comments\n1. Residue Numbering Error\nThe manuscript refers to a phenylalanine residue in different conformations or rearrangements in the loop as Phe355. However, based on the PDB files, the correct residue is Phe335. Please revise this throughout the main text and figures, and ensure that all other residue annotations are consistent with the deposited structures.\n2. CryoEM Model Quality and Validation\nThe cryoEM maps are of good resolution and appear to support the atomic models well. There is no evident bias in particle picking or 2D classification, and the models appropriately reflect the density maps. However, the following methodological details should be clarified:\no For PDB 9VBJ, please specify in the Methods section that the map was binned to a pixel size of 1.09 Å.\no For PDB 9VBK validation report, please clarify the symmetry imposed during reconstruction.\no For PDB 9VBI validation report please clarify the magnification of 150000x\n3. Biochemical Assay Figures\nThe gels shown in Supplementary Figure 2a are not of optimal quality. Nevertheless, they convey the necessary information to support the claims in the main text. If possible, consider improving the image clarity for publication.\n4. Substrate Comparison and Structural Insights\no The authors should expand the discussion on why the HA mutant retains residual enzymatic activity. Given the functional roles of H416 and H201, this is unexpected. Importantly, H416 forms a covalent intermediate with the substrate in mouse PLD3 structures (PDBs: 8V06, 8V07), which are not discussed in the current manuscript. Including these comparisons, along with an expanded discussion of possible rotamers of His201 and His416, would strengthen the mechanistic interpretation, especially considering the similarity between mouse and human PLD3 active sites.\no A structural comparison between human PLD3–poly(T)/poly(A) complexes (this study), the apo structure (this study), and mouse PLD3 (PDBs 8V06 and 8V07) should be incorporated into Figure 5 to provide a more complete overview of the catalytic mechanism. Also, residues in Figure 5 need to be label.\n5. Supplementary Figure 14\nIt would be beneficial to include the poly(A) substrate in Supplementary Figure 14 in the same representation as poly(T), enabling a more informative and balanced comparison between the two.\n6. Biological Relevance of Substrate Conformation\nThe authors should comment on whether the observed L-shaped conformation of the ssDNA substrates (polyT or polyA) is likely to occur under physiological conditions or whether it is influenced by structural constraints imposed by the binding pocket.\nPlease consider these revisions to enhance the clarity, accuracy, and scientific rigor of the manuscript. The work presents important insights into PLD3/PLD4 function, and I look forward to seeing the final version.\nBest regards,\nReviewer"}, "author_response": "Response to reviewers\nThe comments of all reviewers were very useful in helping us to improve the quality of our manuscript. Our responses to the comments of each reviewer can be found below.\nThe Figures have been substantially reorganized as follows:\nOriginal Revised\nFig. 1b Fig. 1b (labels are added)\nFig. 1d,e Fig. 1d,e (recreated for better viewing)\nFig. 2b Fib. 2b (replace with reanalyzed data)\nFig. 3a,b Fig. 3a,b (replace with a new figure)\nFig. 4 Fig. 4 (recreated for better viewing)\nFig. 5a (newly created)\nFig. 5c Fig. 5b (recreated for better viewing)\nFig. 5b Fig. 5c (labels are added)\nFig. 5a Fig. 5d\nSupplementary Fig. 1 Supplementary Fig. 1 (Two species are added)\nSupplementary Fig. 2 Supplementary Fig. 2 (modified)\nSupplementary Fig. 9 Supplementary Fig. 9 (recreated for better viewing)\nSupplementary Fig. 14b (newly created)\nSupplementary movie (newly created)\nValidation report (9VBI) Validation report (9VBI) (updated)\nValidation report (9VBK) Validation report (9VBK) (updated)\nThe figure legends have been modified accordingly in the revised manuscript.\nThroughout the paper, the notation for protein structures was standardized as follows:\nhPLD3_apo (human PLD3 apo form)\nhPLD3_poly(T) (human PLD3 bound to poly(T) substrate)\nhPLD3_poly(A) (human PLD3 bound to poly(A) substrate)\nhPLD4_apo (human PLD4 apo form)\nproduct-bound hPLD4 (product-bound form of human PLD4)\nsubstrate-bound hPLD4 (substrate-bound form of human PLD4)\nmPLD3_intermediate (mouse PLD3 phospho-histidine intermediate (PDB ID: 8V06))\nphosphate-bound mPLD3 (mouse PLD3 phosphate-bound form (PDB ID: 8V07))\nproduct-bound hPLD3 (human PLD3 product-bound form (PDB ID: 8Q1X))\nReviewer #1 (Remarks to the Author):\nLysosomal phospholipase 3 and 4 (PLD3/PLD4) degrade single stranded RNA and DNA oligonucleotides. Several\ngroups recently reported crystal and cryo-EM structures of these enzymes in the absence of substrate, in a phospho-\nintermediate state of the reaction and in the presence of DNA products. In the current manuscript, Hirano and\ncolleagues now report the cryo-EM structure of PLD3 and PLD4 in the presence of an un-cleaved DNA substrate. In\nthis way, they elucidate residues and conformational changes in the active site important for substrate binding and\ncatalysis. Based on their data, they suggest a refined model for PLD3 and PLD4-mediated single strand DNA\ndegradation.\nThe structural data described in this manuscript appear overall sound. Indeed, they provide some novel mechanistic\ninsights into the catalytic mechanism of PLD3/PLD4. However, in light of the previously published structural and\nmechanistic data on PLD3/PLD4, in particular from the Nemazee group (Yuan et al, Structure 2024), the overall\nscientific progress of this work appears rather limited. In my view, the manuscript is therefore not suited for the\ngeneral audience of Nat Comm, but rather for a more specialized journal.\nSeveral structural studies of PLD3 and PLD4, with or without ssDNA oligonucleotide or ssRNA substrates, have been\nreported (NAR 2024; Structure 2024; Immunity 2024). These studies provided the overall structural architecture;\nhowever, our understanding of substrate recognition, specificity, and the exonuclease mechanism remains limited due\nto insufficient information on substrate binding. Our cryo-EM structures of PLD3 and PLD4 successfully capture the\nsubstrate-bound, product-bound, and metastable states. In particular, we determined for the first time the complex\nstructure with the substrate prior to cleavage and revealed that the binding pocket changes depending on the substrate.\nBy successfully capturing other states as well, we were able to propose an integrative model of the enzymatic cycle in\nwhich PLD3 and PLD4 actively function. During the cycle, the protein undergoes minimal conformational changes\nexcept in the Phe335-containing loop, and after cleavage, the bound substrate is repositioned into a conformation\nsuitable for further cleavage through coordinated movements of the product and the remaining substrate. We do believe\nthat we can provide new insights into the structural details relevant to substrate recognition and catalysis by our findings.\nSpecific comments:\nSome more work could be done to the figures:\nFig. 1b: Please carefully label the figure (PLD3, poly(T) substrate, two-fold axis, etc).\nWe have added labels (PLD3, poly(T) and two-fold axis) to the figure.\nFig. 1d (and many others) are too crowded and it is difficult to see what the authors aim to show; reduce complexity,\nfor example, by reducing background, and shadows, etc.\nbackground and shadows).\nFig. 2b: Please mention in the legend how the activity has been determined. Ideally, it should be a linear fit to the\ninitial rate.\nWe appreciate the reviewer’s suggestions. We have re-analyzed the data based on the initial rate as suggested (Originally,\nactivity was defined based on the 30-minute data). As a result, while there are slight changes in the values of activity,\nthe overall findings are consistent with the previous analysis (Please see the figures below). We have replaced the figure\n(Fig. 2b) and the method for calculating activity has been added to the method as follows (page 40, line 565~):\n“Activity was calculated based on the initial rate, which was estimated by fitting a linear regression to the data from 1\nto 11 minutes.”\nFig. 3: Please properly label the figures, e.g. add a color code for PLD3 (polyT), PLD4 (apo). This comparison is not\nvery meaningful since the indicated structural changes could be related to the different PLD isoforms (PLD3 vs PLD4)\nor the substrate (apo vs. polyT) binding. A meaningful comparison would be PLD3 (apo) vs PLD3 (polyT); PLD4 (apo)\nvs PLD4 (polyT); PLD3 (polyT) vs PLD3 (polyA); PLD3 (polyT) vs PLD4 (polyT); some of these figures are already\nincluded in the manuscript.\nWe appreciate the reviewer’s suggestions. To clarify the structural changes, we have replaced Fig. 3 with a superposition\nof the three structures: hPLD3_apo, hPLD4_apo and hPLD3_poly(T). hPLD3_apo and hPLD4_apo are superimposed\nwell, with a root-mean-square deviation (r.m.s.d.) of 0.76 Å, although several loop regions exhibit structural differences.\nSimilarly, hPLD3_ poly(T) is well aligned with hPLD3_apo (r.m.s.d. of 0.74 Å), suggesting that PLD3 undergoes\nminimal conformational change upon substrate binding. We also conducted the structural comparison between\nhPLD4_apo and hPLD4_poly(T). Each protomer of PLD4, whether in the apo form or in the presence of poly(T), aligns\nwell, with an r.m.s.d. ranging from 0.3 to 0.5 Å, showing no significant conformational change upon substrate binding.\n“Consistent with the sequence similarity, the PLD3_apo and PLD4_apo structures are superimposed well, with a root-\nmean-square deviation (r.m.s.d.) of 0.76 Å, although several loop regions exhibit structural differences (Fig. 3a).\nSimilarly, PLD3_poly(T) is well aligned with PLD3_apo (r.m.s.d. of 0.74 Å), and each protomer of PLD4, whether in\nthe apo form or in the presence of poly(T), also aligns well, with an r.m.s.d. ranging from 0.3 to 0.5 Å, suggesting that\nPLD3 and PLD4 show no significant conformational change upon substrate binding.”\nFig. 4a is again rather complex. Again, it may be worth removing the shades in Fig. 4a, b.\nWe appreciate the reviewer’s suggestions. We have revised the figures (Fig. 4a, b) as suggested (reducing background\nand shadows).\nFig. 5, line 274 seq. should be described/included in the Discussion, not in the Results. The model is based on data not\nonly from the authors but also from other groups (like the phospho-intermediate) and this analysis represents an\ninterpretation of the data. A (properly labelled) movie showing the transitions between the states would help to explain\nthe suggested mechanism.\nReviewer2 raised a similar concern regarding the catalytic mechanism. In response to Reviewers’ comments, we have\nrevised the structure of this paragraph. Specifically, to provide a more comprehensive structural comparison, we added\na structural comparison among human PLD3_apo, PLD3_poly(T), PLD3_poly(A), mouse PLD3 phospho-histidine\nintermediate (PDB ID; 8V06) (mPLD3_intermediate), mouse PLD3 phosphate-bound form (PDB ID; 8V07)\n(phosphate-bound mPLD3) and human PLD3 product-bound form (PDB ID; 8Q1X) (product-bound hPLD3) in the\nResults section (page 21, line 307~). According to the reviewer’s suggestion, the discussion of the enzymatic cycle was\nmoved to the Discussion section.\n“Structural comparisons were performed among hPLD3 apo, hPLD3_poly(T), hPLD3_poly(A), mouse PLD3 phospho-\nhistidine intermediate (PDB ID; 8V06) form (mPLD3_intermediate), mouse PLD3 phosphate-bound form (PDB ID;\n8V07) (phosphate-bound mPLD3), and human PLD3 product-bound form (PDB ID; 8Q1X) (product-bound hPLD3) to\ngain further insights into the catalytic mechanism. The conformations of the catalytic residues, as well as those\ninteracting with the substrate are well conserved between human and mouse, and across various catalytic states. The\ncatalytic residue His416 in human PLD3 (corresponding to His414 in mouse) exhibits a rotamer shift only in the\nmPLD3_intermediate and the product-bound hPLD3, moving toward His201 in human (His199 in mouse) (Fig. 5a).\nTyr411 in human (Tyr409 in mouse) which stacks with the base of the first nucleotide, exhibits structural variability.\nPhe335 in human (Phe333 in mouse), which stacks with the base of the second or third nucleotide, undergoes\nconformational changes. The other residues involved in the substrate binding show no significant changes. The first\nnucleotide of the bound substrate (poly(T) or poly(A)) in the PLD3_poly(T)/poly(A) occupies the same site as that in\nthe product-bound hPLD3, and their phosphate groups are well aligned with that of the mPLD3_intermediate. Taken\ntogether, the structural changes during the enzymatic cycle appear to be minor except for the Phe335-containing loop as\ndescribed above.\ncomparison between hPLD3_poly(T) and product-bound hPLD4 can reflect conformational changes associated with\nsubstrate cleavage. The 3’-phosphate group of the product remains in approximately the same position as in\nhPLD3_poly(T), whereas the 5’-OH group of the product is shifted by 8.1 Å due to ribose-ring flipping after cleavage\n(Fig. 5b left). Accompanying this conformational change, the phosphate group of the newly formed 5’-end nucleotide\nis displaced outward (~6.9 Å) to avoid steric clashes with the 5’-OH group of the product (Fig. 5b left). In addition, the\n5’-OH of the newly formed nucleotide is displaced outward (~4.9 Å) and forms a hydrogen bond with the 5’-OH group\nof the product. In contrast, the structural comparison between the substrate-bound hPLD4 and hPLD3_poly(T) reveals\nconformational changes associated with the transition toward a substrate-cleaving state. The 3’-phosphate of dT1 or dT2\nin the PLD4 complex is displaced by 4.8 or 7.9 Å, respectively, relative to the complex with PLD3, whereas the base\nmoiety of dT1 or dT2 remains almost properly aligned due to π-π stacking interaction with Phe423 or Phe348 (Fig. 5b\nright). These observations suggest that the base moiety may serve as a structural scaffold to guide the phosphate into its\nproper position. In the case of dT3, the base moiety and the phosphate group exhibit substantial displacement (4.9 and\n10.6 Å, respectively). In summary, unlike PLD3/PLD4, which remains largely unchanged in overall structure, the\nsubstrate undergoes significant conformational changes across different states of the enzymatic reaction.”\nFig. 5c could be reduced in complexity while Fig. 5b is very neat, but should be better labelled.\nWe have revised the figure (Fig. 5b) to reduce complexity.\nTable S1, glycans:\nThe glycans are only mentioned in the data processing report, although they are of potential interest. It would be neat\nto report and illustrate which glycans are observed in these structures and how to they compare to the previously\npublished crystal/cryo-EM structures.\nWe appreciate the reviewer’s comments. We have illustrated the glycans observed in our study (Fig. 1a). Additionally,\nwe have added the following sentence in the Result section (page 11, line 150~).\n“It has been reported that hPLD3 can be N-glycosylated at five sites based on mass spectrometry analysis27. In previously\ndetermined crystal structures, only two N-linked glycans (linked to Asn236 and Asn284 in hPLD3) were modeled for\nboth human and mouse PLD327,28. In contrast, four N-linked glycans, corresponding to all sites except for Asn97, were\nobserved in our cryo-EM structure (Fig. 1a). For PLD4, no experimental data are available to identify specific\nglycosylation sites. In the crystal structure of human PLD4, no glycans were modeled28, whereas in our cryo-EM\nstructure, six N-linked glycans were observed (Fig. 1a).”\nFig. S1.\nSome labels could be included in the alignment (for example, green boxes, pink boxes). Human, mouse and bovine\nsequences from each isoform are essentially identical and the few differences are not mentioned. Would it make sense\nto provide sequences from more distantly related species to identify conserved/non-conserved areas?\nwhich are distantly related mammalian species. However, the conserved residues in each isoform were distributed\nthroughout the entire structure, and no distinct or characteristic regions could be identified.\nFig. S2:\nWhat does the triangle on top of the gels indicate? Increasing protein concentrations or increasing time intervals? What\nis one supposed to see in the band labelled with ‘bound to hPLD3’? Is the intensity of this band changing in the different\nconditions? Please add some more information to the legend. In fact, has vanadate been added to the cryo-EM\nconditions? If yes, was the vanadate binding site determined? This may (or may not) reveal an interesting structural\nintermediate.\nThe triangle indicates “increasing protein concentration”. We added the label of triangle (“Amount of hPLD3”) to clarify\nthis (Supplementary Fig. 2a, b). The exonuclease activity was approximately estimated from changes in the band\nintensity of “the ssDNA bound to hPLD3”. We added the arrow indicating the shifted band in each gel (Supplementary\nFig. 2a, b).\nAlso we added the following sentence (page 8, line 109~; Supplementary Figure 2):\n“We conducted EMSA assay with varying concentrations of hPLD3 (0, 2.5, 5.0, 10, and 20 μM) incubated with ssDNA.\nA band corresponding to ssDNA bound to hPLD3 was detected only at 20 μM at pH 6.0; however, at pH 5.5 or below,\nthe band was observed at 10 μM.”\nSodium vanadate was not added to the cryo-EM sample of hPLD3_apo, but it was added at a low concentration (0.1\nmM sodium vanadate) to the sample of hPLD3 HA bound to substrate. However, vanadate was not observed in the\ncryo-EM map, suggesting that it is unlikely to bind to hPLD3 under the cryo-EM conditions.\nWe addressed this point (page 9, line 124~; Method).\nFig. S8: Please properly label the isoforms in the figure!\nWe apologize for the confusion, likely caused by the incorrect figure title. We corrected the figure title.\nFig. S9a/c: I do not understand from the figure how the tetramer assembles, please simply the presentation!\nBeyond the dimers, the tetrameric assembles does likely not bear any functional relevance? Could the tetramer form if PLD4\nstill contained a transmembrane region, is there actually a proper two-fold symmetry between the two dimers?\nPlease add some discussion.\nWe simplified the figures of Fig. S9a and S9c according to the suggestion.\nEither the A–B dimer or the C–D dimer is flipped across the two-fold axis. This leads to a misalignment with the opposite\ndimer (C–D or A–B, respectively), indicating that strict two-fold symmetry is absent between the two dimers\n(Supplementary Fig. 9c).\nWe addressed it (page 11, line 146~).\ntransmembrane domain are oriented toward each other. It appears unlikely that full-length PLD4 adopts this tetrameric\narrangement on the membrane, although approximately 40 residues between the TM region and the luminal domain are\nmissing in the cryo-EM structure. The crystal structure of hPLD4 has, in fact, been reported to form a homodimer\n(Structure, 2024). The functional relevance of the tetrameric assembly remains unclear.\nWe addressed these points in the Discussion section regarding the tetrameric assembly (page 28, line 391~).\nFig. S10: The difference in running behavior is small and it is unclear whether it may be related to different\nassemblies or slightly different protein concentrations applied to the column. A parallel RALS/MALS analysis would\nbe helpful to decide between these scenarios.\nAs the reviewer suggested, the elution profile in size exclusion chromatography depends on complex factors such as\noligomeric state, protein concentration and molecular shape etc. As responded to the previous comment, it remains\nunclear how the tetrameric assembly bears any functional relevance. Therefore, we did not pursue the molecular\nassembly. We addressed the protein concentration in cryoEM analysis (page 10, line 140~).\n“The cryo-EM samples were adjusted to approximately 1 mg/mL (~18 μM), at which concentration hPLD3 was\npredominantly observed as a dimer, whereas hPLD4 was mainly observed as a tetramer in the cryo-EM analysis.”\nIntroduction\nThe previously published structural data on PLD3/PLD4 should be shortly summarized that one can do a meaningful\ncomparison to the new structures. For example, shortly describe the dimer interface, the phospho-intermediate, the\nstructure of the product-bound PLD3. In particular, the phospho-intermediate structure of PLD4 bound to an uncleaved\nsubstrate (pdb: 8v08) should be described in some more detail.\nWe have revised the introduction to provide a more detailed description of the previous structural studies as follows\n(page 6, line 75~);\n“Recently, crystal structures of PLD3and PLD4 have been reported27,28, revealing the overall architecture of its luminal\ndomain and its dimeric organization mediated by inter-helical interactions. The structure of PLD3 co-crystallized with\noligonucleotides (ODNs) represents the product-bound structure, as the ODN was degraded within the crystal27. PLD3\nand PLD4 can function as a 5'-phosphatase for 5'-phosphorylated ssDNA (5'-Pi ssDNA), although their catalytic rate is\nmore than 10,000-fold slower than that of exonucleases acting on 5'-OH ssDNA. The crystal structures of PLD3 and\nPLD4 co-crystallized with 5'-Pi ssDNA have also been reported28. In PLD3 structures, due to differences in\ncrystallization times, distinct structures were obtained where the catalytic residue His was either phosphorylated via\nphosphate transfer from 5’-Pi ssDNA or dephosphorylated. In PLD4 structure, the catalytic residue His was\nphosphorylated, and the dephosphorylated ssDNA was trapped within the catalytic cavity.”\nLine 132: As a result of the EMSA assay, the bound substrate was likely degraded due to the residual exonuclease\nactivity of PLD3 HA, hindering identification of the bound substrate sequence. To unambiguously assign the nucleotide,\nwe determined hPLD3 structure bound to poly(T) or poly(A) substrate.\nbound structure?\nUsing DNA without a defined sequence makes it difficult or even impossible to interpret the base sequence, since the\nbound substrate is likely to become a mixture due to the poor sequence preference of PLD3 and PLD4. However, this\nhas not been experimentally confirmed. We regret that the previous wording may have caused confusion; accordingly,\nthe corresponding sentences have been removed for clarity.\nSome text revisions, including correction of typos, are recommended.\nResponded.\nReviewer Recommendation: Minor Revision\nGeneral Comments\nThank you to the authors for sharing this valuable study. The manuscript is clearly written and presents a compelling\ninvestigation into the catalytic mechanisms of PLD3 and PLD4 using a multidisciplinary approach. The cryoEM\nstructures are particularly significant in elucidating the residues involved in catalysis, and the data appear to be of high\nquality with appropriate validation. The authors have effectively combined cryoEM, biochemical assays, and\ncomparative modeling to reveal structural details relevant to substrate recognition and catalytic mechanism.\nNonetheless, some points should be addressed prior to acceptance to improve accuracy, completeness, and clarity.\nSpecific Comments\n1. Residue Numbering Error\nThe manuscript refers to a phenylalanine residue in different conformations or rearrangements in the loop as Phe355.\nHowever, based on the PDB files, the correct residue is Phe335. Please revise this throughout the main text and figures,\nand ensure that all other residue annotations are consistent with the deposited structures.\nWe apologize for typos. We have revised the residue annotations.\n2. CryoEM Model Quality and Validation\nThe cryoEM maps are of good resolution and appear to support the atomic models well. There is no evident bias in\nparticle picking or 2D classification, and the models appropriately reflect the density maps. However, the following\nmethodological details should be clarified:\no For PDB 9VBJ, please specify in the Methods section that the map was binned to a pixel size of 1.09 Å.\no For PDB 9VBK validation report, please clarify the symmetry imposed during reconstruction.\no For PDB 9VBI validation report please clarify the magnification of 150000x\nWe appreciate the reviewer’s suggestions. We have mentioned the map was binned to a pixel size of 1.09 Å for PDB\n9VBJ in the Methods section. We have also updated the validation reports as suggested. We added C1 symmetry imposed\nduring reconstitution for PDB 9VBK and the magnification of 10,5000x for PDB 9VBI.\n3. Biochemical Assay Figures\nThe gels shown in Supplementary Figure 2a are not of optimal quality. Nevertheless, they convey the necessary\ninformation to support the claims in the main text. If possible, consider improving the image clarity for publication.\nWe apologize for the low resolution of the images (Fig. S2a, S2b). We have replaced them with higher-resolution\nversions to improve clarity.\"\n4. Substrate Comparison and Structural Insights\n9\no The authors should expand the discussion on why the HA mutant retains residual enzymatic activity. Given the\nfunctional roles of H416 and H201, this is unexpected. Importantly, H416 forms a covalent intermediate with the\nsubstrate in mouse PLD3 structures (PDBs: 8V06, 8V07), which are not discussed in the current manuscript.\nIncluding these comparisons, along with an expanded discussion of possible rotamers of His201 and His416, would\nstrengthen the mechanistic interpretation, especially considering the similarity between mouse and human PLD3 active\nsites.\nAccording to the EMSA assay, the hPLD3_HA mutant likely retains residual enzymatic activity, but the EFQO assay\nrevealed that the hPLD3_HA retained only 0.2% of the specific activity of the wild type, indicating that only a minimal\nlevel of activity remains. In the EMSA assay, the substrate would be cleaved due to the excessive amount of the protein\nbeing added.\nWe addressed this point (page 16, line 230~) and modified the description of exonuclease activity in EMSA (page 8,\nline 107).\n“hPLD3_HA nearly abolishes the exonuclease activity, but still remains.”\nAs described below, no significant conformational changes are observed, thus it is difficult to explain the residual\nactivity of the HA mutant from the structural viewpoint.\nTo consider the enzymatic activity at the structural level, we conducted a comprehensive structural comparison among\nthe human PLD3 apo, PLD3_poly(T), PLD3_poly(A), mouse PLD3 phospho-histidine intermediate (PDB ID; 8V06)\nform (PLD3_intermediate), mouse PLD3 phosphate-bound form (PDB ID; 8V07) (phosphate-bound mPLD3), and\nhuman PLD3 product-bound form (PDB ID; 8Q1X) (product-bound PLD3) to gain further insights into the catalytic\nmechanism. The conformations of the catalytic residues, as well as those interacting with the substrate are well\nconserved between human and mouse, and across various catalytic states. The catalytic residue His416 in human PLD3\n(corresponding to His414 in mouse) exhibits a rotamer shift only in the PLD3_intermediate and the product-bound\nPLD3, moving toward His201 in human (His199 in mouse) (Fig. 5a). Tyr411 in human (Tyr409 in mouse) which stacks\nwith the base of the first nucleotide, exhibits structural variability. Phe335 in human (Phe333 in mouse), which stacks\nwith the base of the second or third nucleotide, undergoes conformational changes. The other residues involved in the\nsubstrate binding show no significant changes. The first nucleotide of the bound substrate (poly(T) or poly(A)) in the\nPLD3_poly(T)/poly(A) occupies the same site as that in the product-bound PLD3, and their phosphate groups are well\naligned with that of the PLD3_intermediate. Taken together, the structural changes during the enzymatic cycle appear\nto be minor except for the Phe335-containing loop as described above.\nWe addressed these points (page 21, line 307~).\nBased on the above comparison, we further discussed the catalytic mechanism (page 25, line 345~).\n“We determined for the first time the complex structure with the substrate prior to cleavage and revealed that the binding\npocket changes depending on the substrate. By successfully capturing other states as well, we were able to propose an\nintegrative model of the enzymatic cycle in which PLD3 and PLD4 actively function. During the cycle, the protein\nundergoes minimal conformational changes except in the Phe335-containing loop, and after cleavage, the bound\nsubstrate is repositioned into a conformation suitable for further cleavage through coordinated movements of the product\n10\nand the remaining substrate. We can provide new insights into the structural details relevant to substrate recognition and\ncatalysis.”\no A structural comparison between human PLD3–poly(T)/poly(A) complexes (this study), the apo structure (this study),\nand mouse PLD3 (PDBs 8V06 and 8V07) should be incorporated into Figure 5 to provide a more complete overview of\nthe catalytic mechanism. Also, residues in Figure 5 need to be label.\nWe have added structural comparisons among human PLD3_apo, PLD3_poly(T), PLD3_poly(A), mouse PLD3 (PDB\nID; 8V06 and 8V07) and human PLD3 product-bound form (PDB ID; 8Q1X) around the catalytic site (Fig. 5a).\nDetails as described in the comment#4 are added in the text (page 21, line 307~)\n5. Supplementary Figure 14\nIt would be beneficial to include the poly(A) substrate in Supplementary Figure 14 in the same representation as poly(T),\nenabling a more informative and balanced comparison between the two.\nWe have added the poly(A) substrate in Supplementary Figure 14 in the same representation as poly(T)\n6. Biological Relevance of Substrate Conformation\nThe authors should comment on whether the observed L-shaped conformation of the ssDNA substrates (polyT or polyA)\nis likely to occur under physiological conditions or whether it is influenced by structural constraints imposed by the\nbinding pocket.\nThe worm-like chain (WLC) model has been widely employed to describe the conformational flexibility of single-\nstranded nucleic acids. The application of the worm-like chain (WLC) model to poly(T) oligonucleotides has been\nsupported by previous studies employing atomic force microscopy (AFM) (reference A) and fluorescence spectroscopy\n(reference B). A structural analysis of the protein-ssDNA complex showed that ssDNA binds to a concave groove and\nadopts a highly compact folded conformation (reference C). Therefore, we think that the observed L-shaped\nconformation of the ssDNA substrate is a consequence of structural constraints imposed by the binding pocket.\nReference A: Rivetti, C. et al., Polymer chain statistics and conformational analysis of DNA molecules with bends or\nsections of different flexibility. (1998) J. Mol. Biol., 280, 41-59\nReference B: Murphy, M.C. et al., Probing single-stranded DNA conformational flexibility using fluorescence\nspectroscopy. (2004) Biophys J. 86, 2530-2537.\nReference C: Lei et al., DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA. (2003)\nNature 426, 198-203\nWe have added the following sentence in the Result section (page 12, line 167~).\n“It is widely accepted that ssDNA exhibits considerable flexibility. Therefore, the observed L-shaped conformation of\nthe ssDNA substrate is a consequence of structural constraints imposed by the binding pocket.”\npresents important insights into PLD3/PLD4 function, and I look forward to seeing the final version.\nBest regards,\nReviewer\n12"}, {"round": 2, "reviewer_comments": {"reviewer_2": "Dear Authors,\nI appreciate the clarity and thoroughness of your responses to all the reviewers’ comments."}, "author_response": "Thank you very much for your evaluation."}]

Decision letter