NorQD AAA+ complex drives metal insertion by a twisting mechanism

Domain: biology
Published: 2026-03-27
Editorial outcome: major_revision

Abstract
ATPases associated with diverse cellular activities (AAA+-ATPases) catalyse a wide range of remodelling events in all phyla. AAA+-ATPases of the MoxR-like family typically co-operate with von Willebrand factor type A (VWA) domain containing proteins to facilitate target remodelling and metal ion insertion, but their mechanism of action is poorly understood. We studied the bacterial AAA+-ATPase NorQ in complex with its VWA domain partner protein NorD, which are essential for nitric oxide reductase (NOR) activity. Our cryo-EM structures and biochemical analyses show that NorQ and NorD engage through two key interfaces: (i) a finger-like extension protruding from the VWA domain that penetrates the central pore of the NorQ hexamer, and (ii) the NorD C-terminus, which contacts the post-sensor 1 loop of NorQ. Our data reveal that NorQ activity remodels a linker region in NorD essential for metal insertion. Together, these findings support a model in which the NorQ complex exerts a twisting and stretching force on the NorD linker, thereby enabling metal insertion into its target NOR.

Introduction
Metalloproteins constitute about 30% of all known proteins, and half of all enzymes contain metal cofactors 1. The insertion of the correct metal ion into the active site of metal containing enzymes is crucial for their functionality and often depends on metallochaperones. Nevertheless, the mechanism of metal insertion and the interaction between metalloprotein and chaperone are not yet fully understood. Denitrification, i.e. respiration of nitrate, is performed by soil-living bacteria and is dependent on a set of highly metalated enzymes, reducing nitrate into dinitrogen in four separate enzymatic steps. The third reduction is performed by the haem-copper oxidase nitric oxide reductase (NOR), reducing nitric oxide into nitrous oxide. Cytochrome c-dependent NOR (cNOR) is a membrane protein, consisting of two subunits: NorB (52 kDa), and NorC (17 kDa), holding several cofactors: three haems and one non-haem iron (FeB) 2–4. The cNOR gene cluster contains six genes: structural genes norC and norB, chaperone encoding genes norQ and norD, and genes norE and norF, encoding small membrane proteins of unknown function. We have previously shown that the insertion of Fe in cNOR from Paracoccus (P.) denitrificans is dependent on the two chaperone proteins NorQ and NorD, and that cNOR is inactive without FeB 5.

NorQ (30 kDa) belongs to the large family of AAA+ ATPases 6,7, and shares several conserved features with AAA+ protein family members. AAA+ proteins typically form ring-shaped hexamers that thread the substrate protein in an ATP dependent manner through their central pore and thereby remodel it. This is often done in a hand-over-hand mechanism 8,9, whereby the protomers are arranged in a right-handed spiral staircase. The ATP bound, topmost protomers are bound to the substrate, while the subunit at the spiral seam usually has no nucleotide or substrate bound. Upon ATP binding, the seam subunit will relocate to the top, attaching to the substrate and pulling the amino acid chain further into the pore while remodelling it 9. NorQ belongs to the Pre-Sensor 2 Insert Clade (clade 7) of AAA+ proteins, which is formed by MoxR-like proteins such as dynein, CbbQ, and RavA 10. Their defining feature is a β-hairpin insert on helix 2 (H2i) near the substrate binding loop. MoxR-like AAA+ proteins often involve a VWA domain partner protein to perform their remodelling activity 6, but very little is known about the detailed mechanism of their interaction. The 69 kDa protein NorD is the VWA domain partner of NorQ. Its VWA domain contains a conserved metal ion dependent adhesion site (MIDAS), consisting of five residues (DxSxS-T-D) coordinating a divalent metal ion, presumed to be Mg2+. As the name implies, the metal ion provides a binding site for other proteins, with the target protein contributing a sixth ligand, adhering to the metal ion and thus to the VWA domain protein 11. The MIDAS motif is suggested to provide the binding between the chaperone complex and its final substrate 12. Even though MoxR-like AAA+ ATPases are strictly prokaryotic, interaction between AAA+ ATPases and VWA domain proteins have previously been observed also in eukaryotes, for example, in Mg-chelatases, inserting magnesium into chlorophyll 13 and Rea1, involved in the remodelling of pre60S ribosomes in yeast 14.

Here, we present cryo-EM structures of different NorQD complexes from Paracoccus denitrificans and Jhaorihella thermophila which in combination with mutagenesis studies, enzyme activity data and AlphaFold predictions, shed light on the function of the NorQD complex. We show that a finger motif protruding from the VWA domain binds in the pore of NorQ, and that this interaction is needed for cNOR activation. Finally, we propose a tentative model for how structural changes in cNOR are induced by the ATPase function of the NorQD complex.

Results
Domain architecture and structure of PdNorQWB

We collected a cryo-EM dataset of the NorQWB AAA+ complex from Paracoccus denitrificans (Pd) without NorD or ATP present. The NorQ subunits of this complex carry a mutation (E109Q) in the Walker B motif (WB), leading to low ATPase activity but retained ATP binding, which stabilises complex formation 12. Since the 2D classes showed highly preferred orientation (Supplementary Fig. 1a), we combined these data with a dataset collected at a tilt angle of 30 degrees to yield a map suitable for modelling (Fig. 1a, Supplementary Fig. 1b). The map was resolved to 3.7 Å overall and up to 3.3 Å in the core of the AAA+ module enabling us to determine the nucleotide in the binding pockets unambiguously (Supplementary Fig. 1c–e, Supplementary Table 1). The PdNorQWB hexamer forms a closed, right-handed spiral, in which the topmost three subunits A, B and C are bound to ATP and the two bottom subunits D and E as well as the seam subunit F are bound to ADP (Fig. 1a, Supplementary Fig. 2a). All helix 2 inserts (H2i) are long β-hairpins which arrange to form a 15 Å pore on the N-terminal side of the hexamer (Fig. 1a). Each pre-sensor 1 (preS1) loop forms a β-hairpin that is positioned at the subunit interface to the right-hand neighbour in the ring. Two more loops, the classical pore loop and the post-sensor 1 (postS1) loop, are lining the inside of the pore through the AAA+ complex, with the postS1 loop being closer to the C-terminal end of the pore (Fig. 1a).

Binding mode of PdNorQWB to PdNorDVWA

We also wanted to investigate the structure of the entire PdNorQD complex, but cryo-EM experiments with this complex resulted to an insurmountable preferential orientation of the particles in the ice, suggesting an even stronger hydrophobic interaction of PdNorD than PdNorQ with the air-water interface. However, binding only the PdNorDVWA fragment (PdNorDVWA) to PdNorQ overcame this limitation and we solved the PdNorQWBDVWA structure without ATP in the buffer by cryo-EM (Fig. 1b, Supplementary Fig. 3, Supplementary Table 1). The nucleotide occupancy of this complex is similar to that of the PdNorQWB complex (Supplementary Fig. 4a–c), except that subunit F at the seam is lacking the ADP. We determined the PdNorQWBDVWA structure under oxidizing conditions and with dithiothreitol (DTT) under reducing conditions, achieving resolutions of 3.2 Å and 3.8 Å, respectively (Supplementary Fig. 3 and 5). The overall root mean square deviation (RMSD) between the Cα atoms of the two models derived from maps obtained under these respective conditions is 1.075 Å across all 1525 amino acids of the AAA+ ring (Supplementary Fig. 4e). Under oxidizing conditions, we identified a disulphide bridge between cysteines 46 and 68 in the subunit F (Supplementary Fig. 4d), which distorts the nucleotide binding P-loop of the ATPase and prevents ATP from binding to this subunit. Apart from the disulphide bridge, the two maps are identical at the resolution obtained. The ATPase activity of the complexes containing the disulfide bridge is very low (Supplementary Table 2), which also explains the low activities observed previously 12. We conclude that the formation of the disulphide bridge under the oxidizing conditions of purification without DTT, practically abolishes ATP hydrolysis and traps the complex in place.

In the PdNorQWBDVWA structure PdNorDVWA forms a globular domain located in the C-terminal, concave opening of the AAA+ ring. As previously predicted 12, a finger-like domain is stretching into the pore to contact the H2i at the N-terminal end of the pore. Its fingertip intercalates in the seam of the ring between subunits A and F, shifting the large AAA+ subdomain of subunit F by 10 Å towards subunit E and opening up the nucleotide binding site (Supplementary Fig. 6a). While the local resolution in the AAA+ domains in the oxidized PdNorQWBDVWA consensus map is around 3 Å, it drops to an average of 4 Å in the VWA domain (Supplementary Fig. 3c). Extensive 3D sorting of the data shows that NorDVWA moves slightly around two attachment points in the pore of the AAA+ ring (Supplementary Movie 1): the H2i loops and the postS1 loop of subunit E. Binding of PdNorDVWA to PdNorQWB also induces the slightly steeper spiral staircase in the AAA+ ring, leading to an increased total rise of ~7 Å between subunits A to D (Supplementary Fig. 6b, 6c). We suggest that the intercalation of the finger-like domain at the spiral seam with simultaneous binding of the VWA domain to (at least) subunit E primes the complex for remodelling action.

To verify whether the PdNorDVWA fragment retains similar PdNorQD activity to full-length PdNorD, we measured the ATPase activities of different PdNorQD complex variants. The PdNorQ complex has an ATPase activity of 13 ± 1 min-1 hexamer–1, which in complex with full length PdNorD under reducing conditions increases to 18 ± 3 min–1 complex–1, and with PdNorDVWA is slightly lower at 10 ± 0.7 min-1 complex-1. Note that these values differ from previously published activity data 12, see above. In the complexes with the PdNorQ Walker B mutation, these ATPase activities are severely inhibited but measurable (Supplementary Table 5). ATP exchange and binding can therefore take place despite the Walker B mutation. Structurally, we also observe this when we incubate PdNorQWBDVWA with ATP (Supplementary Fig. 7), as we obtain mainly hexameric and pentameric PdNorQ arrangements that carry ATP in each visible nucleotide binding pocket (Supplementary Fig. 2c, 2d). The fully ATP loaded, hexameric and pentameric complexes display a significantly steeper spiral staircase with an increased total rise of ~16 Å between subunits A to E and stronger seam than seen in the PdNorQWB complex without ATP addition (Supplementary Fig. 2a, 2b, 2c, 6c). We conclude that binding of ATP to subunits E and F of the PdNorQWBDVWA complex makes the connection between subunits A and F of the seam so flexible that subunits can be lost. The steep spiral arrangement of subunits D to F impairs PdNorDVWA binding, although the PdNorQWBDVWA plus ATP dataset also contained a subset of hexamers bound to the VWA domain of NorD. In these cryo-EM structures, the central pore is filled with density that must derive from PdNorDVWA, but the local resolution of this density is insufficient to make out secondary structure elements.

The PdNorDVWA—PdNorQWB interactions and their effects on ATPase activity

The local resolution at the contact points between PdNorDVWA and PdNorQWB is ~3.5 Å in the oxidized PdNorQWBDVWA consensus map and can be interpreted in detail. The alpha helix of the finger motif of PdNorDVWA form three hydrogen bonds to the H2i of subunit A of the PdNorQWB hexamer, while the adjacent downstream loop segment forms two hydrogen bonds to subunit F (Fig. 2a, b, Supplementary Table 3). The PdNorQWBDVWA interaction is further reinforced by several direct interactions involving conserved residues in the H2i of 5 out of the 6 AAA+ domains (Figs. 2a and 2c). Most prominent residues involved in the interactions are Arg80, Tyr81, Leu83 and Thr88 in the conserved GRXLL/IXXXXT motif of the H2i (Fig. 2a, c, Supplementary Table 4). The interaction between the C-terminus of PdNorDVWA and the postS1 loop of subunit E is mediated by three hydrogen bonds, one of which is between backbone oxygen and nitrogen atoms (Fig. 2d, Supplementary Table 3). We furthermore observe a strong hydrophobic interaction between residues A593/L556/L636/V637/A638 of PdNorDVWA and I159/L160 of PdNorQ, which will be discussed in more detail below. Interestingly, the postS1 loops of the ADP loaded subunits of the PdNorDVWA-associated AAA+ hexamer are more structured than those of the AAA+ ring alone, suggesting stabilization of the otherwise flexible loops by the interaction with the VWA domain (Supplementary Fig. 4a–c).

The importance of the interaction between the C-terminus of PdNorD and the postS1 loop for complex stability was investigated by deletion of the very last (638), as well as the last two (637 and 638) residues of PdNorD. The deletion of 638 led to a lower efficacy of co-purification, and the deletion of both, 637 and 638, abolished co-purification completely (Fig. 2e, Supplementary Fig. 8). Mutation of the L83 in the PdNorQ H2i also lowered the efficacy of co-purification, as did deletion of the NorD VWA finger (residues 403 to 448 exchanged for GGSIDGGS), however, not as much as the NorD 637 and 638 deletions. This suggests that the contact mediated by the C-terminus of PdNorD is stronger than that of the finger with the H2i, indicating that the integrity of the PdNorQD complex is more dependent on this interaction. Moreover, the finger is not essential in stimulating ATPase activity, as ATP hydrolysis was increased to 28 ± 3 min–1 complex–1 in the complex without the finger (Fig. 2e, Supplementary Table 5). However, in the context of target remodelling, the PdNorD finger is essential, as no non-haem iron was inserted into the cNOR protein when expressed with NorD lacking the finger (Fig. 2e, Supplementary Table 6). We conclude that binding of the VWA domain via its C-terminus to PdNorQ might stimulate nucleotide exchange in the hexamer, while the finger motif is needed for PdNorQ to perform remodelling action on cNOR, the substrate of PdNorQD. Point-mutations in the PdNorQ postS1 loop did not affect co-purification but resulted in a somewhat lower ATPase activity (Supplementary Table 5, Supplementary Fig. 8c).

Domain architecture of JtNorQWBNorD and the role of the NorD linker

All cryo-EM experiments involving the N-terminal and linker region of PdNorD in the complex lead to a strong preferential orientation of the particles in the ice, suggesting a possible hydrophobic interaction of these regions with the air-water interface 12. Indeed, we find stretches of hydrophobic amino acids in the beginning of the linker region of PdNorD, which are less pronounced in Jhaorihella thermophila (Jt) NorD, a variant of the protein from a thermophilic bacterium. Furthermore, Blue native PAGE analysis confirms a higher stability of the JtNorQD complex in comparison with the PdNorQD complex (Supplementary Fig. 8b and 8d). ATPase activities of the JtNorQ and JtNorQD are similar to the Pd complexes (Fig. 3a, Supplementary Table 5). Due to all these considerations, to obtain a full structure of the NorQD complex including also the NorD N-terminal part, we imaged and obtained structures of the JtNorQWBD complex (Fig. 3b). These experiments allowed us to identify 4 conformational states of the JtNorQWBD complex, which were resolved with a mean resolution of 3.2 Å to 3.9 Å (Fig. 3c, Supplementary Figs. 9–11). These maps also show signs of anisotropy due to preferential orientations, but they allow localization of secondary structure elements. By far the most populated subset of the dataset (state 1) shows densities for the NorQWB hexamer and the VWA domain of NorD. A Phenix-refined model of this map can be superimposed on our model of PdNorQWBDVWA with an RMSD of 0.687 Å across 1483 pruned Cα atom pairs (Supplementary Fig. 11c), indicating a very high structural similarity of this complex between the two species (sequence identity: 81% for NorQ and 65% for NorD). Even though less well resolved overall than the PdNorQWBDVWA complex, the JtNorQWBD dataset showed three states with a density for the N-terminal domain of NorD, which is bound either to the AAA+ subunit to the left of the topmost ATP-bound subunit (state 2), called subunit B in our study, or to the lowest ATP-bound subunit in the spiral arrangement (state 3 and 4), subunit C (Fig. 3c, Supplementary Fig. 11).

The binding between the N-terminal domain of NorD and the small alpha helical subdomain of the ATPase subunit appear to be mediated by hydrophobic interactions. Interestingly, the hydrophobic region of the N-terminal domain of NorD that interacts with the C-terminal helices of NorQ, is covered by parts of the linker that connect the N-terminal domain with the VWA domain in the AlphaFold model of NorD (Supplementary Fig. 12a–c). These linker residues 242-393 of NorD are not resolved in any of our JtNorQWBD maps (Supplementary Fig. 11). The arrangement of the VWA domain finger motif is identical in all 4 states of JtNorQWBD, but the orientation of the globular VWA domain harbouring the C-terminus of NorD differs between states 1–3 and state 4 (Fig. 3c, Supplementary Fig. 12d). While the unresolved linker region in state 3 spans a distance of 67 Å between A393 VWA and V241 NT, it is 53 Å in state 4, and 45 Å in state 2 (Fig. 3d). Presumably these changes in distance spanned by the linker region are involved in the cNOR remodelling mechanism of the NorQD complex (see “Discussion”). To investigate the functional significance of the NorD linker, we expressed Pd-cNOR from constructs with different parts of the NorD linker deleted (Fig. 3e and Supplementary Fig. 13). These constructs all led to non-functional cNOR (Fig. 3e) lacking the FeB, supporting that both the length and specific regions of the linker are important for target remodelling. Expression and complex formation of the PdNorQD was verified in a construct with the entire linker deleted (Δlinker or NorD Δ236-342) (Supplementary Fig. 8c).

JtNorQWBNorD interactions and their functional significance

The two main contact points between NorQ and NorD in the NorQD complex, the finger/H2i interaction and the C-terminus/postS1 loop interaction, contribute differently to the dynamics of the complex. During ATP hydrolysis, AAA+ ATPases move downwards from the top of the spiral arrangement and are normally attached to the substrate via their pore loops as long as they are bound to the nucleotide 8. The H2i are relocated next to the substrate binding loops in clade 7 AAA+ proteins (Fig. 2a). In the structures presented here, they always bind the NorD finger motif in a way that the topmost AAA+ subunits, A, B and C, direct the finger towards the seam. ATP binding to the nucleotide-free seam AAA+ subunit F would cause it to reach upwards and rotate the finger motif anticlockwise by 60 degrees in the hexamer pore, if as a reference frame the subunit location in the hexamer is assumed to be static and seen from the N-terminal side of the complex. In the JtNorQWBD dataset, states 2 and 3 can be distinguished by a rotation of the entire VWA domain (including finger) by 60-degree and 120-degree, respectively, relative to the binding position of the N-terminal domain (Fig. 4a, Supplementary Movie 2). On the other side, the VWA domain is held in place by the interaction of its C-terminus with the postS1 loop, which is bound to the last ADP bound subunit of the AAA+ spiral, subunit E, in all structures of NorQD that show the VWA domain, except for state 4 of JtNorQWBD. Here, the globular VWA domain is rotated 90 degrees anti-clockwise against the finger motif and is located close to the postS1 loop of subunit C (Fig. 4b, c, Supplementary Fig. 12d). The interactions of the C-terminus with the postS1 loop of subunits E and C differ in distance and position of the interaction partners (Fig. 4c). Interestingly, the hydrophobic residues of the postS1 loop (I157/159, L158/160) are oriented towards the centre of the AAA+ pore in ATP bound subunits and rotated away from the pore after nucleotide hydrolysis (Fig. 4e). While ATP bound NorQ has the tendency to point these residues to the channel, because the Sensor 1 (N153) interaction with the nucleotide enforces this geometry of the postS1 loop (Fig. 4d, Supplementary Fig. 14), the loops of ADP bound subunits are usually unstructured (Supplementary Fig. 4a). However, in the presence of the VWA domain I157/L158 are tightly bound to a hydrophobic pocket of this domain (Fig. 4f), suggesting that the VWA domain enforces an ATP-like conformation of the postS1 loop of ADP bound subunits.

Methods
Plasmids
Primers (sequences provided in Supplementary Data) were ordered from Eurofins genomics. Genes for expression of P. denitrificans NorQ, NorD and the NorD-VWA domain were cloned from the pNOREX vector 23. J. thermophila NorQ and NorD were cloned from the J. thermophila strain CC-MHSW-1 (obtained from DMSZ). Genes were amplified using PCR and cloned into pET21a (P. denitrificans NorQ) or pETDuet1 (P. denitrificans NorQ, NorD and NorD-VWA domain, J. thermophila NorQ and NorD), with the S-tag exchanged for a Strep-tag, for details, see 12. P. denitrificans cNOR was expressed from the pNOREX vector 23, with a C-terminal 6-His tag on NorB 5. Point mutations, insertions and deletions were done using PCR mutagenesis. PdNorD linker deletions were constructed using primers flanking the deleted region with small overlaps allowing for in vivo recombination of the plasmid, as described in 24. All cloning was performed in E. coli DH5ɑ.

Protein expression and purification
His-tagged PdNorQ. Expression and purification of 6-His-tagged P. denitrificans NorQ was done as in 12. In short, the pET21a vector containing the His-tagged NorQ was transformed to E. coli BL21 DE3. Cells were grown at 37°C, 180 rpm shaking until OD600 = 0.5. The temperature was then lowered to 30°C and the cells were left to grow for another 16 h. Harvested cells were resuspended in 50 mM TRIS/HCl, pH 8, 50 mM NaCl, supplemented with one tablet of cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and DNase I, crushed and ultracentrifuged. The supernatant was mixed with imidazole to a final concentration of 20 mM and applied on a 5 mL HisTrap (Cytiva) column using an ÄKTA purification system (GE Healthcare). Protein was eluted in 20 mM TRIS/HCl, pH 8, 300 mM NaCl, 10% (v/v) glycerol with an imidazole gradient (50 mM - 500 mM). Eluted protein was dialysed against 10 mM TRIS/HCl, pH 8, 150 mM NaCl, and 10% (v/v) glycerol overnight. Protein concentration was determined using the Bradford assay.

Double-tagged NorQD and NorQDVWA. pETDuet1-strep constructs with NorQ and NorD or NorD-VWA domain genes were transformed to E. coli BL21 DE3, grown at 37°C, 180 rpm, in LB medium to OD 0.5. The temperature was then lowered to 20°C. Cells were harvested after 16 h and resuspended in 50 mM TRIS/HCl, pH 8, 50 mM NaCl, supplemented with one tablet of cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche) and DNase I, with or without the addition of 10 mM DTT and 2 mM ADP, followed by cell disruption at 22 kpsi (Emulsiflex C3 Homogenizer) and ultra-centrifugation. The supernatant was applied to a 7 mL Strep-Tactin column and washed with buffer (20 mM TRIS/HCl, pH 8, 300 mM NaCl, 10% (v/v) glycerol, with or without 10 mM DTT and 2 mM ADP). Protein was eluted with the same buffer, supplemented with 10 mM desthiobiotin. The eluted protein was mixed with 1 mL Ni-NTA resin and imidazole was added to a concentration of 20 mM, followed by 1 h rotating incubation. The resin was washed with buffer containing 50 mM imidazole and protein was eluted with 500 mM imidazole. The eluted sample was dialysed against dialysis buffer (10 mM TRIS, pH 8, 150 mM NaCl, and 10% (v/v) glycerol, with or without 10 mM DTT) overnight. Purified protein was analysed on SDS-page gel (Supplementary Fig. 8a–c) and concentration was determined using the Bradford assay.

Strep-tagged PdNorQ and JtNorQ. Expression of Strep-tagged NorQ without NorD or the VWA domain from P. denitrificans and J. thermophila was done from the pETDuet1-strep constructs, with the first MCS left empty. Expression and purification were done similarly as for the NorQD/VWA complexes, with some exceptions: cells were grown at 30°C, all buffers were without DTT and ADP, and the Ni-NTA purification and dialysis was not performed.

His-tagged cNOR. Expression and purification of 6-His-tagged P. denitrificans cNOR was done using the pNOREX plasmid with a C-terminal 6-His tag on NorB 5. Wild-type pNOREX, pNOREX with the NorD finger deleted (residues 403 to 448 exchanged for GGSIDGGS) and pNOREX with the NorD linker deletions, was transformed to E. coli JM109, also holding the pEC86 vector, enabling haem c synthesis in E. coli. The cells were grown in TB media at 37°C, 180 rpm, until OD600 reached 0.5. Protein production was induced with 1 mM IPTG and the temperature was lowered, first to 30°C for 5 h, then to 18°C and the shaking was turned off. After an additional 5 h, the temperature was increased to 30°C and shaking 180 rpm until harvesting. Harvested cells were resuspended in 100 mM TRIS/HCl, pH 7.6, 50 mM KCl, supplemented with DNase I and 1 tablet of cOmplete EDTA-free protease inhibitor cocktail (Roche), crushed at 22 kpsi and ultracentrifuged. Pelleted membranes were solubilized in 1% n-dodecyl-β-D-maltoside (DDM) for 1 h and ultracentrifuged again. The solubilized membranes were mixed with 5 mL Ni-NTA resin and 20 mM imidazole was added. The column was washed with buffer (50 mM imidazole, 50 mM TRIS/HCl, pH 7.6, 200 mM KCl, 0.05% DDM) and protein was eluted in the same buffer with 250 mM imidazole. Eluted protein was concentrated, and concentration was estimated using ε410,ox = 311 cm–1 mM–1.

Enzymatic assays
ATPase Activity measurements. ATP hydrolysis activity measurements were done using the malachite green end-point assay 5,25. The protein was diluted to 25–60 µg/mL in 20 mM TRIS/HCl, pH 8, 300 mM NaCl, 10% (v/v) glycerol, 15 mM MgCl2, with or without 2 mM DTT. To start the reaction, ATP was added to a final concentration of 2 mM. At each time point (1–61 min after start), 27.3 µL of the reaction was mixed with 109.1 µL malachite green colour reagent (0.034% malachite green oxalate, 1% ammonium molybdate tetrahydrate, 0.04% Triton-X) followed by vortexing. After 1 min, 13.64 µL 34% citrate solution was added, and the sample was again vortexed. A620 was measured 5 minutes later, and the concentration of Pi was calculated using a standard curve.

cNOR activity measurements. NO-reduction activity was measured using an NO-sensitive electrode and activity values calculated from maximum slopes 12. cNOR was diluted to 12.5–25 nM in 50 mM HEPES, pH 7.5, 50 mM KCl, 0.05% DDM, with 30 mM glucose and 10 U/mL catalase. The chamber was closed, and glucose oxidase was added to a concentration of 1–3 U/mL to deplete the chamber of oxygen. NO was added in steps of 10 µM up to 30–50 µM. Electron donors (0.5 mM TMPD, 3 mM ascorbate, 10 µM bovine cytochrome c) were added and the NO consumption was followed over time.

Non-haem iron
The non-haem iron content in cNOR was measured using the ferene method 26,27. The cNOR protein was diluted to 7 µM in 60 µL buffer (50 mM HEPES, pH 7.5, 50 mM KCl, 0.05% DDM). Protein was precipitated by addition of 6 µL HCl (37%) and incubated at room temperature for 5 min. Upon centrifugation, the supernatant was mixed with 100 µL of 3 M sodium acetate and 10 µL 1 M ascorbic acid (pH 6) and the absorbance at 593 nm was recorded. Ferene (3 mM, 10 µL) was added, and the sample was incubated in the dark for 5 min. The A593 was measured again and the difference in A593 before and after addition of ferene was used to estimate the non-haem iron content using ε593 = 35.5 mM–1 cm–1 for the ferene-iron complex 26.

Bioinformatic analysis
Sequence conservation of NorQ and NorD was analysed by alignment of 20 sequences for each protein using Clustal Omega 28. The Uniprot accession numbers for the used sequences are: Q51664; A0A1H5TIV7; Q5LL14; B8EHY5; Q16A05; Q3J136; A0A1G5P588; V6F0A1; A0A1H8I2H0; Q7CUT7; Q51481; A4VQB0; E4U3I4; E0UUV4; Q0A6S6; Q82TA2; A6X784; B1Y7Z3; H7C804 and C6WVL4 for NorQ and Q51665; A0A1H5TKL0; Q5LL15; B8EHY6; Q16A06; Q3J137; A0A1G5P5T9; V6F0E0; A0A1H8I2C8; A9CGJ7; Q51484; A4VQ97; E4U3I3; E0UUV3; Q0A6S5; Q82TA1; A6X785; B1Y7Z1; Q89QB3 and C6WVL5 for NorD. The sequence conservation of selected regions was illustrated using WebLogo 29.

Cryo-EM sample preparation and data acquisition
In general, 3 µL protein sample (0.5–1 mg/mL) was applied to freshly glow-discharged C-flat Cu 2/2 (300 and 400 mesh) grids and then plunge-frozen on a Vitrobot Mark IV at 4°C and 100% humidity. The images were collected on a Thermo Fisher Titan Krios equipped with a Gatan K3 BioQuantum detector using the Thermo Scientific EPU 3 software. Detailed information for each data acquisition session can be found in Tables 1–3.

For the PdNorQWBNorDVWA samples in presence of ATP, 4 mM ATP and 10 mM MgCl2 was added immediately prior to grid preparation.

Cryo-EM image processing
Image processing was performed in the software packages RELION 4 30,31 and CryoSPARC 32. In brief, movie frames were aligned using MotionCor2 33 and contrast transfer functions were calculated with CTFFIND4 34. Particles were picked using Gaussian blob template-free autopicking. The resolution reported after 3D refinements were calculated using gold standard Fourier Shell Correlation (FSC) curves with the FSC = 0.143 criterion for mask-corrected FSC curves. For final 3D refinements dynamic auto-tightened masks were used.

For PdNorQWB ~3 million particles were picked from 11,678 micrographs and cleaned in several rounds of 2D classifications in CryoSPARC (Supplementary Fig. 1). However, the 2D classes showed severe preferred orientation. We therefore collected a tilted dataset (30°) with 2658 micrographs yielding ~1.4 million particles. After several rounds of 2D classification and 2D class balancing, good particles from both datasets were combined for downstream processing. After ab initio modelling, the particles were sorted by heterogeneous refinement (4 classes) and the best class with 113,836 particles was refined to an overall resolution of 3.7 Å.

For PdNorQWBDVWA (oxidized) ~3.3 million particles were picked and extracted from a total 10,893 micrographs in RELION (Supplementary Fig. 3). After multiple rounds of 2D classification and ab initio modelling the cleaned particles were subjected to 3D classification with 3 classes. One class with poor resolution was discarded, and the remaining particles were refined to an overall resolution of 3.0 Å. However, the local resolution of the NorD VWA domain was relatively low. The particles stack was transferred to CryoSPARC for further analysis. A subsequent 3D classification yielded 10 classes with subtle differences in the position of the NorD VWA domain and no variation within the NorQ hexameric arrangement revealed a slight tilting motion of the VWA. The 3D class that yielded the highest local resolution and connectivity in the NorD VWA region after homogeneous refinement (overall 3.2 Å) was used for molecular model building. The tilting motion of the VWA was visualized using multi-body refinement in Relion (Supplementary Movie 1).

For the PdNorQWBDVWA sample in presence of ATP we extracted ~4 million particles from 16,559 micrographs in CryoSPARC (Supplementary Fig. 7). After multiple rounds of 2D classification the cleaned particles were further sorted using ab initio modelling and heterogeneous refinement with 3 classes. Each of the three subsets represents a distinct oligomeric state of the complex and attempts to further separate the particles did not yield additional states or maps of higher resolution. Homogeneous refinement of the 3 subsets yielded an overall resolution of ~3 Å.

For PdNorQWBDVWA (reduced) in presence of DTT ~1.8 million particles were picked from 7273 micrographs (Supplementary Fig. 5). Relatively few particles (100,884) remained after cleaning in multiple rounds of 2D classification likely due to the dataset suffering from relatively thick ice. The particles were further cleaned by 3D classification using 4 classes which yielded only one class suitable for further processing. The particles of the best class were exported as a particle stack and used for homogeneous refinement in CryoSPARC yielding a map of 3.8 Å.

For JtNorQWBD ~3.4 million particles were picked from a total of 11,373 micrographs in RELION (Supplementary Fig. 9). After multiple runs of 2D classification and subsequent ab initio modelling, the particles were sorted by 3D classification with yielding two poorly resolved classes and two good classes. The particles of both good classes were transferred to CryoSPARC for subsequent processing. The 3D models produced to this point showed weak density for the NorD NTD bound to the NorQ hexamer. The imported particles were sorted by 3D classification using 20 classes using a focus mask including both, the NorD VWA and NTD. 13 classes (~260,000 particles) showed an identical assembly compared to PdNorQWBDVWA with no or only faint density for the NorD NTD (State 1). 5 classes (~104,000 particles) showed relatively low resolution of the NorD VWA, and these particles were not used for molecular model building. One class (~23,000 particles) contained particles similar to State 1 but in addition showed clear density for the NorD NTD (State 3). A final class (~27,000 particles) showed clear density for the NorD NTD, but the VWA domain was rotated compared to State 1 and 3 (State 4). The density of the NorQ hexameric assembly was virtually identical in all classes on the scale of considering the asymmetric placement of NorQ protomers. Up to this point the NorD NTD was observed bound to only one specific NorQ protomer in the asymmetric NorQ spiral. We tested possible alternative binding positions of the NTD at the NorQ small subdomain by extensive 3D classifications with spherical masks at the expected binding sites. The six parallel 3D classification runs (one run for each NorQ protomer with 20 classes) yielded one additional binding position of the NorD NTD in a 3D class with ~23,000 particles (State 2). Homogeneous refinement of each particle subset yielded an overall resolution of 3.3 Å (State 1), 3.8 Å (State 2), 3.9 Å (State 3) and 3.9 Å (State 4).

Molecular model building and analysis
Individual domains of NorQ and NorD models generated with AlphaFold 3 35 were fitted as rigid body into the density using UCSF ChimeraX 36. The models were optimized manually in COOT 37 at the level of individual amino acids using ProSMART secondary structure restrains 38 derived from the AlphaFold 3 models. The models were then refined automatically using phenix.real_space_refine in Phenix 39. Iterative model adjustment in COOT and subsequent refinement in Phenix were performed until the models were completed with a final real space refinement in Phenix. UCSF ChimeraX was used to analyse the models and to generate the model figures. The atomic models of JtNorQWBD state 2–4 were refined and deposited without the NorD NTD as the local resolution and connectivity of the maps was suboptimal for model building in this part. For the figures we fitted the AlphaFold 3 model of the NorD NTD as a rigid body and the corresponding composite models are available via the Zenodo repository (see Data availability statement). Surface representations that show hydrophobic potentials are displayed according to ChimeraX standard colour code: dark goldenrod for the most hydrophobic potentials through white to dark cyan for the most hydrophilic potential. Interaction parameters for atom pairs correspond to VDW overlap of ≥ -0.4 Å for all contacts and standard geometric parameters for hydrogen bonds.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Kahle et al. determine the structure of the MoxR AAA+ ATPase NorQ and its partner VWA protein NorD using cryoEM. NorQD have been shown to facilitate iron insertion into cNOR (cytochrome C -dependent nitric oxide reductase); cNOR is involved in nitrate respiration. The authors show that a finger protruding from the VWA domain of NorD inserts into the central pore of the NorQ hexamer. The authors also show that the the C-terminus of NorD interacts with a post sensor 1 loop in NorQ. The authors obtain the cryoEM structure of NorQD from Paracoccus denitrificans and from Jhaorihella thermophila with the latter being at higher resolution. The structures are interesting. The structural data are further verified using mutational analysis, interaction studies, and ATPase assays. The authors propose a mechanism whereby NorQD ‘twists’ and stretches the NorD linker to enable metal insertion into its target cNOR.\nWhile the structures are interesting, there is no evidence for the proposed action of NorQD on cNOR. In other words, the authors do not carry out experiments to demonstrate the proposed “twisting” mechanism. In the absence of such data, the manuscript is basically showing a series of structures of NorQD.\n1. The abstract is poorly written with no explanation of the function of NorQD. The abstract concludes with a proposed function of NorQD on cNOR, which is not experimentally addressed in the manuscript.\n2. Since the JtNorQD is obtained at higher resolution than that of PdNorQD, it might be better to start the manuscript with describing the JtNorQD structure.\n3. In line 74, the authors should provide an explanation as to why the Walker B mutant was used for cryo-EM throughout the paper. They did say the mutant has much lower ATP hydrolysis, but they did not say why they did not use wild type for cryoEM. They could have used ADP.\n4. Line 81/82: ‘Bottom’ and ‘top’ are relative terms. When describing the subunits, it might be better to use the labels presented in the figures, i.e. subunits A-F. Similarly, throughout, when describing the seam subunits, refer to them by their label as well, to increase clarity.\n5. Line 109: Why was excluding ATP useful in obtaining this structure? Why would it have affected the resolution?\n6. Line 109: The structure only includes the VWA domain of PdNorD; the other domains/regions might play a role in the interaction as well, so it would be good to introduce the complex with the understanding of this limitation.\n7. On line 118, the ‘finger’ was suggested to aid in nucleotide exchange, but activity assays showed greater ATPase activity without this finger. These data conflict with the earlier statement. How may they be reconciled?\n8. Does the disulfide bridge play any biological role, and has it been observed in other homologs?\n9. Supplementary table 4 indicates assays were conducted in DTT, but that was not sufficient for full activity as in supplementary table 6. Therefore, all the other constructs and mutants also need to be purified with DTT.\n10. In Supplemental Figure 4, the subsets 2 and 3 of have low cFAR values, which can indicate some extent of orientation bias. Perhaps the authors can comment on how confident they are regarding the biological relevance/the conclusions made on the two subsets. This puts into questions some of the mechanisms they are proposing.\n11. Line 163, when reporting RMSD values, authors should mention if it is using all atoms of the relevant amino acids, or all heavy atoms, or just alpha carbons etc.\n12. Explain the dotted circle in the panels of Figure 1b.\n13. The discussion could more explicitly acknowledge the limitations of the current data, especially the lower resolution in the VWA domain region and the lack of direct visualization of remodeling, and should consider alternative models.\n14. Figure legends could be more detailed, especially regarding the number of replicates and statistical tests for the biochemical assays presented.\n15. Although there is an overall schematic (Figure 5), it could benefit from more detail or a zoomed-in feature highlighting the proposed mechanism.", "reviewer_2": "This manuscript reports cryo-EM structures of the MoxR AAA+ ATPase NorQ in complex with its VWA domain partner NorD. NorQ and NorD are essential for insertion of an Fe into the membrane bound cytochrome c-dependent NOR (cNOR). Coupled with ATPase measurements, the authors propose a structural model for how the NorQD complex induces structural changes in cNOR. The structures are compelling and shed light on an essential biological function. The manuscript should be published.", "reviewer_3": "Kahle and colleagues present multiple structures of the AAA+ ATPase, NorQ, alone or with its VWA domain partner, NorD. NorQB structures were determined of both Paracoccus denitrificans and Jhaorihella thermophila homologs, and in oxidizing or reducing conditions. The JtNorQD cryo-EM data analysis resulted in four unique states, including one with only the VWA domain of NorD shown, and three states with unique NTD conformations of NorD. The authors conclude that these states represent the rotation of the VWA and NTD, leading to ATP dependent opening of cNOR and insertion of an iron ion.\nThe cryo-EM and image analysis was done to a high standard and well documented.\nMajor points:\n1. The order in which these structures are presented seems backwards of a logical flow of information.\nIt is unclear why the authors do not include a reducing agent in their protein purifications, experiments, and cryo-EM samples in the first place. The way the manuscript is written, it seems like an oversight that is then corrected by redoing the experiment. If there is a biological reasoning behind this, as is alluded to on Lines 258-260, please introduce it earlier. You could also start with the structures containing DTT, and then introduce the disulfide bridge found in the oxidized structure after. It seems that with an RMSD of ~1A between them, your conclusions in the results section won’t change much with this reorganization, and you can say that the under oxidizing conditions the ATPase activity is reduced, likely due to this disulfide forming.\nI feel similarly about the current order of starting with describing the Pd NorQD structures then transitioning to Jt NorQD. It seems that state 1 of the JtNorQD structures is the same as PdNorQD (RMSD 0.687A). The interesting results are coming from the conformational switching between the NorD VWA/NTDs. It seems that the reason for this ordering is because there is significant anisotropy in the JtNorQD structures, limiting ability to solve the structures from those maps alone. I think there is nothing wrong with this, but the authors could do a better job creating a logical flow from one set of structures to the next.\n2. The significance of the spiral staircase change in NorQD structures is unclear.\nStarting with the title on line 90, the authors describe the staircase of NorQ as having a “steeper spiral staircase” when bound to NorD, as compared to the structure without NorD. In Line 101 authors say the ATP “complexes display a significantly steeper spiral staircase and stronger seam than seen in the ‘as isolated’ PdNorQWB complex”. What do you mean by ‘as isolated’? Is this the previous structure? Be clear here, as you use this wording a few more times.\nDefine ‘significance’ in the change in the staircase? Can it be described in a distance change? Unfortunately, the figures do not provide enough information about this change. Rather than showing the maps with slightly altered protomer E in Supplementary Figure 2, you should have the atomic structures of subunits of F, E and D overlaid so we can see their movement down relative to each other. Perhaps it would be more appropriate to show different subunits- it depends which subunit to which you are aligning the structures.\nIn Lines 102-105 the authors also describe the loss of subunits in the ring. The maps in Supplementary Figure 2 look like there is missing density in part of the subunits E and F, which likely implies flexibility and heterogeneity of particles in your data processing. Knowing this, it is hard to compare the “movement” of these subunits, as some may appear lower in the staircase compared to their counterparts that are entirely lacking that part of the subunit.\n3. The order of progression between states in JtNorQD is confusing and hard to understand from the figures and movies.\nWhen you are comparing an ensemble of models of a homohexameric complex it can be unclear the order in which they progress in the mechanism, or how to assign the ‘top’ subunit for alignment. When watching the movies, it seems that the VWA domain is rotating a ton, with little movement of the ATPase. I wonder what the progression would look like if you instead aligned the structures based on the VWA domain to see how the subunits of NorQ moved relative to it. NorQ is the ATPase and is known to go through large conformational switching while processing substrates. It seems more likely it would be moving, and the rotation of VWA would be in response to this.\nEither way, the authors need to find a better way to demonstrate these conformational changes in their figures and movies. The current angles shown in Figure 3 are confusing, and you should find a better angle than a 90-degree rotation so we can understand that swing of the VWA domain.\n4. Proposed mechanism includes the substrate, cNOR, but the manuscript does not provide direct evidence for how NorQD interacts with this substrate.\nThe structures presented in this manuscript lack the membrane-bound substrate, cNOR. In Figure 5 they include cNOR and provide a hypothesis of how these structures would interact with that substrate. Is there previous data to suggest that NorQ does not translocate a part of cNOR into the central channel? If so, would both NorD and cNOR be accommodated in the channel? What is the evidence that NorQD would sit atop of cNOR in this way? The authors need to provide better justification for this hypothesis, which currently has no evidence from the data presented.\nMinor points:\nSome of the figures could use some adjustments for clarity.\n1. In Figure 2, please show both residues involved in the bonds, including in the backbone. The way the structure is being displayed makes it hard to see the atoms involved in the interaction.\n2. In the Figure 3 legend you need to include a description of what colors associate with which domain. It is not clear here which domain is VWA or NTD of NorD.\n3. In Figure 5 you should add arrows between states. Would be helpful to have the structure/state on the figure itself, rather than just listed in the figure legend.\nThe methods section was written rather poorly, including what appear to be typos, informal writing style and incorrect shorthand. Please edit the methods section to have a similar writing style as the rest of the manuscript and correct the mistakes.\n1. Line 638 – “BL21 DE. Cells” – do you mean BL21 DE3?\n2. Line 640 – When describing protease inhibitor tablets it was called a “pill” on Line 640 and 652 but called a “tablet” to describe it on line 681. Be consistent.\n3. Line 643- Authors claim to use a “HiTrap” column for purification but use an imidazole gradient. Do you mean a “HisTrap”?\n4. Line 737 – To describe the total number of particles, the shorthand “3 mio.” was used. Do you mean million? Either write it out or find a more appropriate shorthand.\n5. An example of informal writing can be found on Line 772 – “two trash classes”. What do you mean by trash classes? Find a better way to describe your process for eliminating bad classes."}, "author_response": "We thank all reviewers for their constructive criticism of our manuscript, which we believe\nhas strengthened the manuscript and made it easier to understand. As requested, we performed\nadditional functional experiments to support our proposed mechanism, specifically we have\nverified the functional importance of the NorD linker region. We believe that these\nexperiments not only support our suggested mechanism but also help to clarify it. We have\nalso made extensive revisions to the order and text to further clarify our points. Below is our\ndetailed response to each of the reviewers’ points.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Kahle et al. determine the structure of the MoxR AAA+ ATPase NorQ and\nits partner VWA protein NorD using cryoEM. NorQD have been shown to facilitate iron\ninsertion into cNOR (cytochrome C -dependent nitric oxide reductase); cNOR is involved in\nnitrate respiration. The authors show that a finger protruding from the VWA domain of NorD\ninserts into the central pore of the NorQ hexamer. The authors also show that the the C-\nterminus of NorD interacts with a post sensor 1 loop in NorQ. The authors obtain the cryoEM\nstructure of NorQD from Paracoccus denitrificans and from Jhaorihella thermophila with the\nlatter being at higher resolution. The structures are interesting. The structural data are further\nverified using mutational analysis, interaction studies, and ATPase assays. The authors\npropose a mechanism whereby NorQD ‘twists’ and stretches the NorD linker to enable metal\ninsertion into its target cNOR.\nWhile the structures are interesting, there is no evidence for the proposed action of NorQD on\ncNOR. In other words, the authors do not carry out experiments to demonstrate the proposed\n“twisting” mechanism. In the absence of such data, the manuscript is basically showing a\nseries of structures of NorQD.\nResponse: As specified by the reviewer above, our structural data are supported by functional\ndata, so we do not agree with the assessment that we are ‘basically showing a series of\nstructures’. Nevertheless, we do agree that the final model for the NorQD mechanism of\naction was partly speculative, and we have carried out a series of new experiments aimed at\nverifying the importance of the presumably unstructured linker region between the two folded\ndomains of NorD. This data, now presented in a new Figure 3e, support the importance of the\nlength and specific regions of the linker for NorQD-mediated Fe insertion into cNOR. We\nB\nhave also included a new Supporting Figure 13 of the Alphafold-modelled interaction\nbetween NorQD and cNOR, supporting the general interaction mode via NorD. We have also\nclarified which parts of the final mechanism in Fig 5 remain partly speculative when\nconcerning binding to the target protein cNOR.\n1. The abstract is poorly written with no explanation of the function of NorQD. The abstract\nconcludes with a proposed function of NorQD on cNOR, which is not experimentally\naddressed in the manuscript.\nResponse: We have rewritten the Abstract to include the function of NorQD and specify our\nfindings as well as our proposed model.\n2. Since the JtNorQD is obtained at higher resolution than that of PdNorQD, it might be better\nto start the manuscript with describing the JtNorQD structure.\nResponse: We agree with the reviewer that it makes sense to start with the structures of\nhighest resolution and that is exactly what we did, as we have now clarified in the manuscript.\nThe Pd NorQD structure is resolved to 3.2Å, while the JtNorQD is resolved to 3.3Å. We\nwanted to build the manuscript up from building blocks of increasing complexity in order to\naid understanding, starting from NorQ alone, adding the VWA domain and finally adding also\nthe N-terminal domain.\n3. In line 74, the authors should provide an explanation as to why the Walker B mutant was\nused for cryo-EM throughout the paper. They did say the mutant has much lower ATP\nhydrolysis, but they did not say why they did not use wild type for cryoEM. They could have\nused ADP.\nResponse: We have clarified in the manuscript why the Walker B mutation was used for all\nstructures. It leads to better expression, and more stable complex formation with the substrate\nof the AAA+ protein (in our case the partner protein NorD). This is also seen in other AAA+\nproteins [1, 2].\n4. Line 81/82: ‘Bottom’ and ‘top’ are relative terms. When describing the subunits, it might\nbe better to use the labels presented in the figures, i.e. subunits A-F. Similarly, throughout,\nwhen describing the seam subunits, refer to them by their label as well, to increase clarity.\nResponse: We have revised the manuscript to include the subunit labels for better clarity.\n5. Line 109: Why was excluding ATP useful in obtaining this structure? Why would it have\naffected the resolution?\nResponse: When adding ATP to the PdNorQNorD-VWA sample just before freezing grids,\nthe complex falls apart to varying degrees, presumably affecting the resolution. Excluding\nATP (purified ‘as is’) gave the best quality data.\n6. Line 109: The structure only includes the VWA domain of PdNorD; the other\ndomains/regions might play a role in the interaction as well, so it would be good to introduce\nthe complex with the understanding of this limitation.\nResponse: We have re-written this section to clarify the composition of the complex and to\nhighlight the missing N-terminal segments of NorD.\n7. On line 118, the ‘finger’ was suggested to aid in nucleotide exchange, but activity assays\nshowed greater ATPase activity without this finger. These data conflict with the earlier\nstatement. How may they be reconciled?\nResponse: We have deleted the statement that the finger might aid in nucleotide exchange.\nThis speculation did not prove to be correct as the reviewer correctly pointed out.\n8. Does the disulfide bridge play any biological role, and has it been observed in other\nhomologs?\nResponse: As stated on lines 252-260 in the discussion, such a disulfide has not been\nobserved in other AAA+ ATPases. The biological role is hitherto unknown, but we suggest it\ncould serve a redox regulation role. We hope to investigate its role in future studies.\n9. Supplementary table 4 indicates assays were conducted in DTT, but that was not sufficient\nfor full activity as in supplementary table 6. Therefore, all the other constructs and mutants\nalso need to be purified with DTT.\nResponse: We agree and for precisely this reason Supplementary Table 4 states that all\nproteins were purified in the presence of 10mM DTT. We have also added that there is 2mM\nDTT in the measurement buffer.\n10. In Supplemental Figure 4, the subsets 2 and 3 of have low cFAR values, which can\nindicate some extent of orientation bias. Perhaps the authors can comment on how confident\nthey are regarding the biological relevance/the conclusions made on the two subsets. This puts\ninto questions some of the mechanisms they are proposing.\nResponse: We have now highlighted in the manuscript that the PdNorQD complexes\ngenerally display preferential orientation in the ice, which we partly have overcome by\ncollecting tilted data. As the reviewer correctly points out, subsets 2 and 3 of the ATP\ncontaining, reduced PdNorQWBNorDVWA dataset show orientational bias. However, as can\nalready be seen at much lower resolution than 6.6Å, the AAA+ rings are displaying high\nflexibility at the spiral seam so that NorQ subunits might be lost and NorD binding is\nimpaired. No further, more detailed conclusions have been drawn from this dataset. We are\nnot aware that above mentioned subsets 2 and 3 have any connection to our proposed\nmechanism.\n11. Line 163, when reporting RMSD values, authors should mention if it is using all atoms of\nthe relevant amino acids, or all heavy atoms, or just alpha carbons etc.\nResponse: Thank you for pointing this out. We have now clarified that we are generally using\nCA Atoms for RMSD determination.\n12. Explain the dotted circle in the panels of Figure 1b.\nResponse: We thank the reviewer for spotting this mistake, we have now explained that the\ndotted circle outlines the MIDAS motif of the VWA domain.\n13. The discussion could more explicitly acknowledge the limitations of the current data,\nespecially the lower resolution in the VWA domain region and the lack of direct visualization\nof remodeling, and should consider alternative models.\nResponse: We have performed additional experiments to support our model and we have also\nclarified in the revised text which parts of our suggested model are supported by experiments.\n14. Figure legends could be more detailed, especially regarding the number of replicates and\nstatistical tests for the biochemical assays presented.\nResponse: We have added these details.\n15. Although there is an overall schematic (Figure 5), it could benefit from more detail or a\nzoomed-in feature highlighting the proposed mechanism.\nResponse: We have revised the figure, see also response to reviewer 3.\nReviewer #2 (Remarks to the Author):\nThis manuscript reports cryo-EM structures of the MoxR AAA+ ATPase NorQ in complex\nwith its VWA domain partner NorD. NorQ and NorD are essential for insertion of an Fe into\nthe membrane bound cytochrome c-dependent NOR (cNOR). Coupled with ATPase\nmeasurements, the authors propose a structural model for how the NorQD complex induces\nstructural changes in cNOR. The structures are compelling and shed light on an essential\nbiological function. The manuscript should be published.\nResponse: We thank reviewer #2 for the very positive assessment of our manuscript.\nReviewer #3 (Remarks to the Author):\nKahle and colleagues present multiple structures of the AAA+ ATPase, NorQ, alone or with\nits VWA domain partner, NorD. NorQB structures were determined of both Paracoccus\ndenitrificans and Jhaorihella thermophila homologs, and in oxidizing or reducing conditions.\nThe JtNorQD cryo-EM data analysis resulted in four unique states, including one with only\nthe VWA domain of NorD shown, and three states with unique NTD conformations of NorD.\nThe authors conclude that these states represent the rotation of the VWA and NTD, leading to\nATP dependent opening of cNOR and insertion of an iron ion.\nThe cryo-EM and image analysis was done to a high standard and well documented.\nResponse: We thank reviewer #3 for the general positive assessment of our data.\nMajor points:\n1. The order in which these structures are presented seems backwards of a logical flow of\ninformation.\nIt is unclear why the authors do not include a reducing agent in their protein purifications,\nexperiments, and cryo-EM samples in the first place. The way the manuscript is written, it\nseems like an oversight that is then corrected by redoing the experiment. If there is a\nbiological reasoning behind this, as is alluded to on Lines 258-260, please introduce it earlier.\nYou could also start with the structures containing DTT, and then introduce the disulfide\nbridge found in the oxidized structure after. It seems that with an RMSD of ~1A between\nthem, your conclusions in the results section won’t change much with this reorganization, and\nyou can say that the under oxidizing conditions the ATPase activity is reduced, likely due to\nthis disulfide forming.\nResponse: The reason we presented the oxidised structure first is indeed somewhat historical,\nsince we have used the same buffer conditions in previous works. Only when we identified\nthe disulfide bridge, we realized that the buffer conditions were influencing the ATPase\nactivity. The structure of PdNorQWBDVWA under oxidizing conditions has the highest\nresolution, and the detailed interaction analysis was made using this structure. However, we\nagree with the reviewer that the logical flow improves if we introduce the reduced structure\nearlier, and we have reordered the manuscript this way. We now also refer to the discussion\non the possible role of the disulfide bond in the Results section.\nI feel similarly about the current order of starting with describing the Pd NorQD structures\nthen transitioning to Jt NorQD. It seems that state 1 of the JtNorQD structures is the same as\nPdNorQD (RMSD 0.687A). The interesting results are coming from the conformational\nswitching between the NorD VWA/NTDs. It seems that the reason for this ordering is because\nthere is significant anisotropy in the JtNorQD structures, limiting ability to solve the\nstructures from those maps alone. I think there is nothing wrong with this, but the authors\ncould do a better job creating a logical flow from one set of structures to the next.\nResponse: As stated above in response to reviewer #1, we have presented the data in the order\nof ‘simple’ to more complex to aid the understanding. The resolution of the PdNorQWBDVWA\nstructure is higher than that of the JtNorQD complex and all our mutational studies are done\non the Pd complexes. Hence, many of the functional data rely on the Pd complexes, while the\nModel of NorQD action is derived mainly from the Jt complexes, as these structures were\nunattainable using Pd complexes. We have now restructured the manuscript and tried to create\na logical flow introducing each of the experiments.\n2. The significance of the spiral staircase change in NorQD structures is unclear.\nStarting with the title on line 90, the authors describe the staircase of NorQ as having a\n“steeper spiral staircase” when bound to NorD, as compared to the structure without NorD. In\nLine 101 authors say the ATP “complexes display a significantly steeper spiral staircase and\nstronger seam than seen in the ‘as isolated’ PdNorQWB complex”. What do you mean by ‘as\nisolated’? Is this the previous structure? Be clear here, as you use this wording a few more\ntimes.\nResponse: Thank you for pointing out this lack of clarity. We now state that the as isolated\ncomplex refers to the oxidized variant of complexes and refer to any additives during\nexperiments by naming them directly (eg DTT, ATP). In Line 101 we were comparing\nPdNorQWBDVWA complexes obtained in the presence of ATP with PdNorQWB complexes\nwithout any added ATP. Both complexes were obtained in the presence of DTT.\nDefine ‘significance’ in the change in the staircase? Can it be described in a distance change?\nUnfortunately, the figures do not provide enough information about this change. Rather than\nshowing the maps with slightly altered protomer E in Supplementary Figure 2, you should\nhave the atomic structures of subunits of F, E and D overlaid so we can see their movement\ndown relative to each other. Perhaps it would be more appropriate to show different subunits-\nit depends which subunit to which you are aligning the structures.\nResponse: We have now added guidelines and distance marks to Supplementary figures S2a,\nS2c and S6c to highlight the change in the spiral staircase arrangements. In our hands, the\nchange is best seen, when comparing the maps. An overlay of the atomic structures, even\nwhen only showing Ca traces, is very overwhelming. In AAA+ proteins, the helical shift\nbetween subunits is mostly dependent on the nucleotide bound to the subunits. Hence, the\nshift in the ATP bound subunits A-C is similar, but differs for the ADP bound subunits D-F as\nthe reviewer points out correctly. It therefore makes most sense to align all spirals on the\ntopmost subunit in the spiral, which is subunit A. And this is what we have done. We hope\nthat the changes we introduced could clarify this point.\nIn Lines 102-105 the authors also describe the loss of subunits in the ring. The maps in\nSupplementary Figure 2 look like there is missing density in part of the subunits E and F,\nwhich likely implies flexibility and heterogeneity of particles in your data processing.\nKnowing this, it is hard to compare the “movement” of these subunits, as some may appear\nlower in the staircase compared to their counterparts that are entirely lacking that part of the\nsubunit.\nResponse: The reviewer correctly points out that there is flexibility in the bottom most\nsubunits of the PdNorQWBDVWA subsets (subunit E in subset 2; subunit F in subsets 1 and 3).\nBut subset 2 also lacks subunit F completely, leading us to suggest that subunits can be lost,\nwhen all subunits in the complex are bound to ATP. Since the small and large ATPase\nsubdomains of AAA+ complexes move as rigid bodies and the densities for the large subunits\nare resolved well enough to place a PDB model, the spiral staircase of complexes can be\ncompared well.\n3. The order of progression between states in JtNorQD is confusing and hard to understand\nfrom the figures and movies.\nWhen you are comparing an ensemble of models of a homohexameric complex it can be\nunclear the order in which they progress in the mechanism, or how to assign the ‘top’ subunit\nfor alignment. When watching the movies, it seems that the VWA domain is rotating a ton,\nwith little movement of the ATPase. I wonder what the progression would look like if you\ninstead aligned the structures based on the VWA domain to see how the subunits of NorQ\nmoved relative to it. NorQ is the ATPase and is known to go through large conformational\nswitching while processing substrates. It seems more likely it would be moving, and the\nrotation of VWA would be in response to this. Either way, the authors need to find a better\nway to demonstrate these conformational changes in their figures and movies. The current\nangles shown in Figure 3 are confusing, and you should find a better angle than a 90-degree\nrotation so we can understand that swing of the VWA domain.\nResponse: In our initial movie, we aligned the structures to the finger motif of the VWA\ndomain and showed how all other domains (including the AAA+ subunits) moved relative to\nit. Now, we have revised the movie comparing all JtNorQD states such that we kept the\nsubunit bound to the NorD N-terminal domain static so that all other domains move relative to\nit. We have also labeled the linker attachment points in NorD for more clarity. As can be seen\nvery well in the movie, there is no movement between state 3 and state 4 apart from the\nflipping back of the NorD VWA domain. All other structural features are identical. Dynamic\nmovements, however, can be depicted in various ways relative to each other. One could argue\nthat in the NorQD system, the NorD N-terminus is bound to a target on the membrane as well\nas the NorQ ring and the rest of the system moves relative to this attachment point. Hence, we\nkept the N-terminal domain static showing this scenario of events. To us it is clear how to\nassign the top subunit for alignment. The top subunit will always be the first ATP bound\nAAA+ domain in the spiral, next to the linker domain of NorD.\n4. Proposed mechanism includes the substrate, cNOR, but the manuscript does not provide\ndirect evidence for how NorQD interacts with this substrate.\nThe structures presented in this manuscript lack the membrane-bound substrate, cNOR. In\nFigure 5 they include cNOR and provide a hypothesis of how these structures would interact\nwith that substrate. Is there previous data to suggest that NorQ does not translocate a part of\ncNOR into the central channel? If so, would both NorD and cNOR be accommodated in the\nchannel? What is the evidence that NorQD would sit atop of cNOR in this way? The authors\nneed to provide better justification for this hypothesis, which currently has no evidence from\nthe data presented.\nResponse: It is true that there is no direct data on the interactions between NorQD and cNOR.\nAs discussed in our previous publication [3] the chaperones presumably act on an assembly\nintermediate of cNOR and direct interactions are hard to study as we do not know the state of\ncNOR involved. We do, however, have indirect data showing that FeB insertion into cNOR\ndepends on the activity and structural integrity of NorQD for several constructs (including the\nnew NorD linker deletions described in the response to reviewer #1 above). We have now\nrewritten the discussion to clarify the evidence for our model. In detail, they are:\na) The NorD finger occupies the central pore of the NorQ hexamer where other AAA+\nproteins bind to target protein, making direct interaction with the cNOR target here unlikely.\nWe have also added an Alphafold model of the entire NorQ hexamer/NorD/cNOR complex to\nthe Supporting information. We do not intend this model to give details about interactions as\nit has regions of very low confidence, but it shows a general interaction mode we propose in\nour model whereby the NorQD complex interacts with cNOR via the NorD only.\nb) We do know from our previous publication [3] that the MIDAS residues of the VWA\ndomain of NorD are not needed for interaction with NorQ, but that they are essential for target\nremodeling. This indicates that they are involved in target (cNOR) binding, as shown for\nmany other VWA domains.\nc) We have added new experimental data on the NorD linker region, which we present in the\nnew Figure 3e. They show that deleting specific regions of the linker, which we selected\nbased on their presumed roles as deduced from the alphafold model (also shown in new\nSupplemental Figure 13), yields a NorQD complex unable to perform Fe insertion into\nB\ncNOR. This supports our suggestion that the conformational changes in NorQD are leading to\ntarget remodelling that involves stretching the NorD linker. This data supports the general\nimportance of the linker.\nMinor points:\nSome of the figures could use some adjustments for clarity.\n1. In Figure 2, please show both residues involved in the bonds, including in the backbone.\nThe way the structure is being displayed makes it hard to see the atoms involved in the\ninteraction.\nResponse: Thanks for the suggestion. For more clarity in the figure and more specificity in\npresenting the data, we have opted to include the interactions between PdNorQWB and\nPdNorDVWA in form of a table in Supplementary Table 3 & 4, including the interacting atoms\nand bond length.\n2. In the Figure 3 legend you need to include a description of what colors associate with\nwhich domain. It is not clear here which domain is VWA or NTD of NorD.\nResponse: We are using the same color codes throughout the manuscript, depicted in Figure\n1. We do agree with the reviewer however that it is beneficial to repeat/describe them in each\ncaption, which we have done in the revised manuscript.\n3. In Figure 5 you should add arrows between states. Would be helpful to have the\nstructure/state on the figure itself, rather than just listed in the figure legend.\nResponse: We have revised the figure as suggested by the reviewer.\nThe methods section was written rather poorly, including what appear to be typos, informal\nwriting style and incorrect shorthand. Please edit the methods section to have a similar writing\nstyle as the rest of the manuscript and correct the mistakes.\n1. Line 638 – “BL21 DE. Cells” – do you mean BL21 DE3?\nResponse: Yes, fixed.\n2. Line 640 – When describing protease inhibitor tablets it was called a “pill” on Line 640 and\n652 but called a “tablet” to describe it on line 681. Be consistent.\nResponse: Fixed.\n3. Line 643- Authors claim to use a “HiTrap” column for purification but use an imidazole\ngradient. Do you mean a “HisTrap”?\nResponse: Yes, fixed.\n4. Line 737 – To describe the total number of particles, the shorthand “3 mio.” was used. Do\nyou mean million? Either write it out or find a more appropriate shorthand.\nResponse: Yes, fixed\n5. An example of informal writing can be found on Line 772 – “two trash classes”. What do\nyou mean by trash classes? Find a better way to describe your process for eliminating bad\nclasses.\nResponse: Yes, fixed.\n1. Puchades, C., C.R. Sandate, and G.C. Lander, The molecular principles governing the activity\nand functional diversity of AAA+ proteins. Nature Reviews Molecular Cell Biology, 2020.\n21(1): p. 43-58.\n2. Wendler, P., et al., Structure and function of the AAA+ nucleotide binding pocket. Biochimica\net Biophysica Acta (BBA)-Molecular Cell Research, 2012. 1823(1): p. 2-14.\n3. Kahle, M., et al., Insights into the structure-function relationship of the NorQ/NorD\nchaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR\nAAA+/VWA domain proteins. BMC biology, 2023. 21(1): p. 47."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors made some effort to make the suggested changes. They do additional experiments with truncations of the linker domain that essentially show inactive protein, while still being able to form the complex. This is to try to address the lack of experimental data to support their model. They explained away other issues I raised and they made all the clarity changes I wanted for the text, but not really to the model figure. For that figure, they just added directional arrows and a couple of labels.", "reviewer_2": "The authors have adequately addressed the reviewer comments.", "reviewer_3": "Reviewer Major Point (from original review):\n2. The significance of the spiral staircase change in NorQD structures is unclear\nStarting with the title on line 90, the authors describe the staircase of NorQ as having a “steeper spiral staircase” when bound to NorD, as compared to the structure without NorD. In Line 101 authors say the ATP “complexes display a significantly steeper spiral staircase and stronger seam than seen in the ‘as isolated’ PdNorQWB complex”. What do you mean by ‘as isolated’? Is this the previous structure? Be clear here, as you use this wording a few more times.\nDefine ‘significance’ in the change in the staircase? Can it be described in a distance change? Unfortunately, the figures do not provide enough information about this change. Rather than showing the maps with slightly altered protomer E in Supplementary Figure 2, you should have the atomic structures of subunits of F, E and D overlaid so we can see their movement down relative to each other. Perhaps it would be more appropriate to show different subunits- it depends which subunit to which you are aligning the structures."}, "author_response": "Response To Reviewers; NCOMMS-25-24940A\nReviewer #1 (Remarks to the Author):\nThe authors made some effort to make the suggested changes. They do additional experiments with truncations\nof the linker domain that essentially show inactive protein, while still being able to form the complex. This is to try\nto address the lack of experimental data to support their model. They explained away other issues I raised and\nthey made all the clarity changes I wanted for the text, but not really to the model figure. For that figure, they just\nadded directional arrows and a couple of labels.\nFrom previous review round:\n15. Although there is an overall schematic (Figure 5), it could benefit from more detail or a\nzoomed-in feature highlighting the proposed mechanism.\nResponse: The reviewer raises concerns about the schematic Figure 5, in which we propose how our structural\nand biochemical data can be integrated into a mechanism of cNor remodelling by NorQD. In the absence of\nstructural data of the cNor/NorQD holo-complex, we hypothesise how the structures described in this manuscript\nfit to our biochemical data and previously published findings. With this in mind, we suggest that the observed\ntwisting motion of the NorD VWA domain with regard to its N-terminal domain translates into remodelling action on\ncNor and iron insertion into NorB. We revised Figure 5 again to highlight the hand-over-hand mechanism in NorQ\nthat twists the NorD VWA domain as well as the positioning of linker attachment points and MIDAS motif that\nindicate remodelling of the linker domain. We have also added a zoomed-in feature, as suggested by the reviewer,\nthat shows a view onto the NorQD complex as seen from the membrane to illustrate the distance change in the\nlinker attachment points. We furthermore indicate which states of NorQ exchange nucleotide.\nReviewer #2 (Remarks to the Author):\nThe authors have adequately addressed the reviewer comments.\nReviewer #3 (Remarks to the Author):\nThe significance of the spiral staircase change in NorQD structures is unclear\n2. Starting with the title on line 90, the authors describe the staircase of NorQ as having a “steeper spiral\nstaircase” when bound to NorD, as compared to the structure without NorD. In Line 101 authors say the ATP\n“complexes display a significantly steeper spiral staircase and stronger seam than seen in the ‘as isolated’\nPdNorQWB complex”. What do you mean by ‘as isolated’? Is this the previous structure? Be clear here, as you\nuse this wording a few more times.\nDefine ‘significance’ in the change in the staircase? Can it be described in a distance change? Unfortunately, the\nfigures do not provide enough information about this change. Rather than showing the maps with slightly altered\nprotomer E in Supplementary Figure 2, you should have the atomic structures of subunits of F, E and D overlaid\nso we can see their movement down relative to each other. Perhaps it would be more appropriate to show\ndifferent subunits- it depends which subunit to which you are aligning the structures.\nAuthor Response: We have now added guidelines and distance marks to Supplementary figures S2a,S2c and\nS6c to highlight the change in the spiral staircase arrangements. In our hands, the\nchange is best seen, when comparing the maps. An overlay of the atomic structures, even\nwhen only showing Ca traces, is very overwhelming. In AAA+ proteins, the helical shift\nbetween subunits is mostly dependent on the nucleotide bound to the subunits. Hence, the\nshift in the ATP bound subunits A-C is similar, but differs for the ADP bound subunits D-F as\nthe reviewer points out correctly. It therefore makes most sense to align all spirals on the\ntopmost subunit in the spiral, which is subunit A. And this is what we have done. We hope\nthat the changes we introduced could clarify this point.\nReviewer Response: Authors still have not defined what they deem as a ‘significant’ change in the staircase.\nPlease include comments in text about the distance change.\nI disagree that having an overlay of the atomic structures would be overwhelming. You do it in Supplementary\nfigure S2e. You could make a similar figure by showing the atomic structure of subunit E as an overlay of the \"as\nisolated” with each of the ATP structures, as they are currently aligned in figure S2 a and b.\nResponse: We have now included the increase in the total helical rise between subunits A to D for PdNorQWB -\nPdNorQWBDVWA (line 126) and subunits A to E for PdNorQWB - PdNorQWBDVWA (ATP, subset 1) (line 143) in the\nmanuscript. Based on the reviewers request to “make a similar figure by showing the atomic structure of subunit E\nas an overlay of the \"as isolated” with each of the ATP structures, as they are currently aligned in figure S2 a\nand b”, we have added a new Supplementary Figure 6c showing an overlay of all relevant Pd complexes to visualise\nthe changes in helical rise. We would like to point out that the increase in total helical rise is mainly due to shifts in\nsubunits C to F, as ATP binding only affects subunits D to F (in complexes in the presence of ATP). Furthermore,\nFigures S2a and b are not showing aligned structures, but only PdNorQWB as isolated. All models shown in\nSupplementary Figure 2a-d are already aligned to subunit A of NorQ to allow for visual comparison of the helical\nstaircase in the complexes. We have now also stated that in the legend of this figure.\n3. The order of progression between states in JtNorQD is confusing and hard to understand from the figures and\nmovies.\nWhen you are comparing an ensemble of models of a homohexameric complex it can be unclear the order in\nwhich they progress in the mechanism, or how to assign the ‘top’ subunit for alignment. When watching the\nmovies, it seems that the VWA domain is rotating a ton, with little movement of the ATPase. I wonder what the\nprogression would look like if you instead aligned the structures based on the VWA domain to see how the\nsubunits of NorQ moved relative to it. NorQ is the ATPase and is known to go through large conformational\nswitching while processing substrates. It seems more likely it would be moving, and the rotation of VWA would be\nin response to this.\nEither way, the authors need to find a better way to demonstrate these conformational changes in their figures\nand movies. The current angles shown in Figure 3 are confusing, and you should find a better angle than a 90-\ndegree rotation so we can understand that swing of the VWA domain.\nAuthor’s Response: In our initial movie, we aligned the structures to the finger motif of the VWA\ndomain and showed how all other domains (including the AAA+ subunits) moved relative to\nit. Now, we have revised the movie comparing all JtNorQD states such that we kept the\nsubunit bound to the NorD N-terminal domain static so that all other domains move relative to\nit. We have also labeled the linker attachment points in NorD for more clarity. As can be seen\nvery well in the movie, there is no movement between state 3 and state 4 apart from the\nflipping back of the NorD VWA domain. All other structural features are identical. Dynamic\nmovements, however, can be depicted in various ways relative to each other. One could argue\nthat in the NorQD system, the NorD N-terminus is bound to a target on the membrane as well\nas the NorQ ring and the rest of the system moves relative to this attachment point. Hence, we\nkept the N-terminal domain static showing this scenario of events. To us it is clear how to\nassign the top subunit for alignment. The top subunit will always be the first ATP bound\nAAA+ domain in the spiral, next to the linker domain of NorD.\nReviewer response: Authors say that it is clear to assign the top subunit as the first ATP bound AAA+ domain in\nthe spiral. I agree, and I can see that is how they have assigned it in Supplementary Figure 11. But, since the\nmovie is aligned to the NTD, this is changing how the other subunits are assigned.\nIn the Supplementary figure 11, the NTD is bound to subunit B in state 2, and subunit C in state 3. Now that you\nhave aligned them to the NTD in the movie, in the transition between state 2 and state 3 we see the NTD bound\nto subunit B in both, and therefore now we see subunit F go from being a seam subunit to the new top of the\nspiral staircase in this transition, with subunits D, E and F all moving in an upward direction as a rigid body. Have\nyou tried aligning to the NTD and morphing from state 3 to state 2? It seems like you might have it backwards,\nbased on what we know of AAA+ motor translocation mechanisms.\nEvidence that the motor moves in a hand-over-hand mechanism can be found in numerous studies. It is\nparticularly easy to see in AAA+ motor structures of the 26S proteasome, which has a heterohexameric AAA+\nthat can be aligned to the 20S core peptidase; therefore, movement of each subunit can be followed through the\nATP hydrolysis cycle. In Dong et al. Nature 2019 and de la Peña et al. Science 2018, they saw consecutive ATP\nhydrolysis and translocation states in which the ATP-bound subunits move downwards as a rigid body and the\nbottom subunit releases substrate and rebinds at the top of the spiral. Assignments of ‘top’ and seam subunits is\ndone the same way as the authors described in this study.\nI agree with authors that you need to pick something to align to, and in your study it makes sense to align all the\nmodels to the NTD, since that is likely bound to a membrane. This would require more self-consistency in the\nmanuscript. You should align your models to the NTD in Supplementary Figure 11a and b. You should also try\nmorphing from state 3 to state 2 and see if that direction of movement is more consistent with previous studies,\nin which rigid body movement of the ATP-bound subunits is downward, and the seam unit will move up to become\nthe new ‘top’ subunit. This could show an interesting result of how the movement of the NTD and VWA is related\nto a translocation event.\nResponse: We are happy to read that the reviewer agrees with how we have aligned the complexes. In fact, the\nalignments in Figures 3c, S2a-d, S4d, S6, and S11a-b are done by aligning on subunit A of NorQ (always the top\nATP bound subunit in the spiral) while Figure 4 and the movie shows the complexes being aligned on the NTD of\nNorD, highlighting the hand-over-hand mechanism of NorQ. We deliberately chose the different perspectives of\nalignment to point out the similarities between NorQ states (alignment on subunit A) or the action of NorQD relative\nto a fixed membrane interaction (alignment on NTD). To facilitate understanding of the movements and the different\nperspectives, we consistently colored the NorQ ring with shades of blue from the topmost subunit in the spiral (A,\ndark blue) to the subunit at the seam (F, light blue).\nRegarding the movie and Figure 4, we propose the sequence of events, because NorQ exactly follows a hand-\nover-hand mechanism as seen in other AAA+ clades. For clarification, we now explicitly state that NorQ is working\naccording to the hand-over-hand mechanism (line 295, legend Fig 5). As our work demonstrates, clade 7 AAA+\nproteins do not seem to use the hand-over-hand mechanism on substrate being moved through the central pore,\nbut to rotate the VWA protein partner in the pore. We do not think the reviewer’s suggestion to morph from state 3\nto state 2 would increase clarity. As shown in the figure below, a transition from state 3 to state 2 would imply that\nthe top ATP bound subunit (subunit A) in the spiral would have to lose ATP and the apo subunit (subunit F) would\nhave to bind ADP - something that has never been observed before in any other AAA+ complex.\nTo clarify the model and avoid any misunderstandings, we have revised the movie to show the change in color\ncode upon ATP binding and reaching up of the seam subunit. We also explain the individual snapshots in more\ndetail and clearly separate the view sequences now."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I had no further comments.", "reviewer_3": "The authors have adequately addressed the reviewer comments."}, "author_response": ""}]

Decision letter