Structural dynamics of the midnolin-proteasome during ubiquitin-independent substrate turnover

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

Abstract
The 26S proteasome typically degrades proteins marked by ubiquitin chains. However, a distinct, ubiquitin-independent degradation pathway for nuclear proteins exists, mediated by the adaptor protein midnolin, yet its molecular mechanism remains poorly understood. Here, we present nine cryo-electron microscopy structures of the human 26S proteasome in complex with midnolin, which collectively delineate an near-complete catalytic cycle. Our structures reveal that midnolin binds to the proteasome via the RPN1 subunit by its C-terminal helix. Unexpectedly, its ubiquitin-like domain interacts with the RPN11 deubiquitinase in a non-catalytic role. This interaction positions the adjacent Catch domain, which is responsible for substrate binding, directly above the proteasomal entrance, potentially facilitating substrate entry into the proteasome. Furthermore, we observe four consecutive spiral staircase conformations of the AAA+ ATPase hexamer during substrate translocation. These findings provide insights into the mechanisms underlying ubiquitin-independent nuclear protein degradation and may help develop strategies for targeting nuclear proteins via direct proteasomal degradation.

Introduction
The 26S proteasome, consisting of a 19S regulatory particle and a 20S catalytic core, has been established as the principal route for regulated protein degradation, traditionally relying on ubiquitin-mediated substrate recognition1,2. This 19S regulatory particle typically engages ubiquitinated proteins, facilitating their unfolding for degradation by the 20S core. Nevertheless, the identification of the midnolin (MIDN)-proteasome pathway expands this established view by revealing an alternative, ubiquitin-independent mechanism for the degradation of numerous nuclear proteins3,4. This alternative degradation system proves particularly useful for maintaining cellular homeostasis through rapid clearance of nuclear immediate early gene (IEG) products, including key transcriptional regulators such as EGR1, FosB, and IRF43,5. Functioning as molecular switches, these IEG proteins orchestrate cellular responses to stimuli by regulating growth, differentiation, stress adaptation, and inflammatory processes6–10.

Midnolin, initially characterized as a developmentally regulated gene in mouse midbrain11, employs three folded domains to execute its degradation functions: an N-terminal ubiquitin-like (Ubl) domain, a middle substrate-capturing Catch domain, and a C-terminal α-helix (hereafter referred to as αHelix-C) proteasome-binding module3. The Catch domain specifically recognizes a β strand degron of IEG proteins such as EGR1, FosB, and IRF43. Notably, αHelix-C of midnolin has been proven to be both necessary and sufficient for proteasome binding, but how the midnolin αHelix-C functions is unclear. Paradoxically, the Ubl domain, which is essential for degradation activity, is dispensable for substrate and proteasome binding. This is unexpected because shuttle factors, such as Rad23 and ubiquilins, use Ubl domains for binding to the proteasome to deliver certain ubiquitinated substrates1. Despite recent structural insights into the midnolin-proteasome12,13, key mechanistic questions remain unanswered. In particular, the binding kinetics of midnolin to the proteasome and the functional role of the Catch domain beyond substrate capture are still poorly understood. It remains unclear whether the Catch domain solely functions in substrate binding or also plays an active role in facilitating efficient degradation, such as positioning the substrate optimally toward the proteolytic entry site. Meanwhile, a key unresolved question is whether midnolin binds its substrates prior to proteasome association, or instead constitutively engages the proteasome before recruiting substrates. These knowledge gaps fundamentally limit our understanding of how midnolin achieves its substrate delivery and degradation efficiency.

Here, we show the structural and molecular basis of midnolin-mediated substrate degradation through single-particle cryo-electron microscopy (cryo-EM) and biochemical analysis. Our findings demonstrate that midnolin’s αHelix-C anchors to the 19S regulatory particle by binding RPN1 (PSMD2), while the coordinated Ubl-Catch domains position substrates at the substrate entrance oligonucleotide- or oligosaccharide-binding (OB) ring of the AAA+ (ATPase associated with diverse cellular activities) ATPase motor. ATP hydrolysis-driven and potential Mg²⁺-release-driven mechanical forces generated by the AAA+ ATPase ring then mediates substrate unfolding and translocation into the 20S catalytic core. Furthermore, our work demonstrates that midnolin achieves optimal activity by co-expressing with substrates. This work advances our perspective on proteasome substrate processing by revealing an evolutionarily optimized degradation pathway that bypasses ubiquitination constraints to meet the urgent temporal demands of nuclear signaling.

Results
Cryo-EM structures of substrate-engaged human midnolin-26S proteasome
To understand how the midnolin-proteasome degrade substrates without ubiquitylation, we expressed midnolin in HEK293F and treated cells with proteasome inhibitor MG132 to prevent substrate or midnolin degradation and with phorbol 12-myristate 13-acetate (PMA), a protein kinase C agonist, to increase the level of the EGR1 protein as previously reported3. We purified midnolin by two-step tandem affinity chromatography followed by size-exclusion chromatography (Supplementary Fig. 1a). Endogenous 26S proteasome was co-purified with midnolin (Supplementary Fig. 1b, c), and proteasome-like particles were revealed by negative staining (Supplementary Fig. 1d). Using single-particle cryo-EM, we determined structures of midnolin-26S proteasome. As previously reported, 20S core particle is relatively stable and 19S regulatory particle is highly dynamic14–16. After 3D classification, we determined four distinct structural states (designated MIDN-PS , -PS , -PS , and -PS for subsequent discussion). Among these states, the RPN1 subunit exhibited varying degrees of resolution and occupied distinct positions relative to the AAA+ motor (Supplementary Figs. 2, 3, and Supplementary Table 1).

The 3D classification yielded structural states that all represent substrate-processing conformations. This includes classes reconstructed from particles that were not assigned to a dominant state but still contributed to the overall conformational landscape. The highest resolution (2.87Å) was achieved for the MIDN-PS state (Fig. 1a). RPN1 shows extra density when compared with previous structures15. However, its high dynamic precludes its resolution. We performed local refinement on RPN1 and refined it to high resolution of 3.07Å, which indicated the αHelix-C of midnolin interacts with RPN1 (Fig. 1b, c, and Supplementary Table 1). When the map is low pass filtered to 15Å, a more distinct yet low-resolution density at the entrance of the ATPase N-ring potentially represents the Catch domain of midnolin or substrate has been translocated into the central channel (Fig. 1d). Therefore, we solved actively processing conformations of the midnolin-bound 26S proteasome, which showed substrate density in the central channel, reaching through the N-ring, the ATPase ring, and into the degradation chamber of the 20S core peptidase (Fig. 1e). Here, we have captured the structures of the natural endogenous substrate-bound 26S proteasome in action.

In these reconstructions, we resolved distinct densities corresponding to the inhibitor MG132. This enabled precise docking of the atomic model into the density map, which shows MG132 specifically binds to the catalytic β subunits (β1, β2, β5) of the core particle (CP) (Supplementary Fig. 4). We note that, as an active-site inhibitor, MG132 may allosterically influence the conformational landscape of the 19S regulatory particle17, a consideration relevant to the interpretation of the observed states.

The αHelix-C of midnolin interacts with RPN1 (PSMD2) dynamically
RPN1 is a major docking site on the proteasome for substrates and shuttle factors, particularly those bearing Ubl domains18–20. Our cryo-EM analysis revealed that different from previous studies, αHelix-C (not the Ubl domain) of midnolin binds across both the proteasome/cyclosome (PC) repeat toroid domain and the rod-like N-terminal domain of RPN1, a mode distinct from previously characterized Ubl-mediated interactions (Fig. 2a–e). This binding is primarily driven by a hydrophobic core and extensive electrostatic complementarity (Fig. 2a, b), a finding independently corroborated by concurrent structural studies12,13.

A detailed comparative analysis highlights the distinctiveness of the midnolin αHelix-C–RPN1 interface relative to canonical Ubl domains (Fig. 2b–e). Although it shares a conserved hydrophobic core (anchored by RPN1 Leu426 and Leu465) with T2-site binders like USP14 and NUB114,21, the midnolin αHelix-C establishes a binding mode that spans both the PC repeat toroid and N-terminal domains of RPN1 (Fig. 2c). The interaction interface exhibits a distinctive tripartite pattern. The central portion of the helix engages with RPN1 through an extensive hydrophobic network, where Val392, Leu395, Leu398, and Leu399 of midnolin pack against Val422, Leu426, Cys459, Pro461, Ala464, and Leu465 of RPN1, forming a hydrophobic core (Fig. 2b). This central hydrophobic interface is flanked by two polar interaction networks, especially electrostatic interactions and hydrogen bonds (Fig. 2b). These residues in αHelix-C of midnolin are all highly conserved across species (Supplementary Fig. 5), consistent with our observations that αHelix-C is important for its interaction with RPN1. Furthermore, thermal stability analysis revealed that the binding of midnolin to RPN1 increase its stability (Supplementary Table 2). This sophisticated binding mechanism represents a mode of substrate or shuttle factor recognition by RPN1, expanding our understanding of proteasome-substrate interactions.

To evaluate the binding affinity between them, we purified the C-terminal of midnolin containing αHelix-C and the proteasome subunit RPN1, and measured their binding affinity using isothermal titration calorimetry (ITC) (Fig. 2f, g and Supplementary Table 3). The results demonstrated moderate binding affinities of approximately 130 nM and 146 nM between RPN1 and the two midnolin truncations, respectively. Surface plasmon resonance (SPR) assays further corroborated these values (390 nM and 663 nM, respectively), revealing a dynamic interaction characterized by relatively rapid association and dissociation kinetics (Fig. 2h, i and Supplementary Table 4). Together, these data suggest that midnolin and RPN1 form a moderate binding complex with readily reversible binding, rather than a high-affinity, stable interaction, which may facilitate its self-degradation.

To investigate the role of αHelix‑C in stimulating proteasomal activity, we co‑expressed the MIDN-EGR1(101‑200) complex (the rationale for this construct is detailed later) and its αHelix‑C mutant MIDNK391E/L395E/R409E-EGR1(101‑200) complex. For brevity, EGR1(101–200)-mCherry was referred to as EGR1(101–200). Compared to the wild‑type complex, MIDNK391E/L395E/R409E-EGR1(101‑200) complex significantly reduced the ability to stimulate proteasome activity (Fig. 2j, k). Since αHelix‑C alone is sufficient to bind the proteasome, we further asked whether it is also sufficient to stimulate proteasomal activity. We found that the C‑terminal truncations of midnolin (encompassing αHelix‑C) exhibited no stimulatory effect (Fig. 2l, m). These results indicate that while αHelix‑C is necessary for anchoring midnolin to the proteasome, additional domains are required to drive efficient substrate degradation, a conclusion consistent with previous reports1,13.

Uniform expression of midnolin is dependent on substrate
Structural analysis of the endogenously purified midnolin-proteasome complex revealed substantial complexity, preventing resolution of the Ubl and Catch domains. To overcome this limitation and better understand the mechanism, we sought to establish an in vitro reconstitution system. A key unresolved question is whether midnolin engages its targets after expression or instead constitutively expresses with it. To address this question, we purified the Catch domain of midnolin and its substrate, EGR1(101–200), separately and performed gel filtration assays. When expressed alone, the Catch domain of midnolin exhibits poor stability and solubility, with most of the protein unable to effectively bind its substrate even with a molar excess of substrate (Supplementary Fig. 6a, b). However, under co-expression conditions, the Catch domain displays a more uniform expression profile, suggesting that co-expression facilitates proper folding and enhances the functional performance of midnolin (Supplementary Fig. 6c, d). Compared with the co-expressed MBP-Catch-EGR1(101–200) complex eluted at 14 mL in superdex 200 increase column, the individually expressed MBP-Catch eluted at a volume of 11.5 mL, which suggests MBP-Catch forms oligomer without substrates. Therefore, we co-expressed and purified the full-length midnolin-EGR1(101–200) complex for further biochemical and structural studies (Supplementary Fig. 6e). As expected, the complex shows a single, symmetric peak, whereas midnolin alone shows obvious oligomerization/aggregation, confirming that substrate binding is essential for stabilizing the full-length midnolin (Supplementary Fig. 6e). Additionally, the bioinformatic analysis (Supplementary Fig. 6f) was performed using the public transcriptomic database GEPIA22, which integrates RNA-seq data from projects such as TCGA and GTEx. We directly extracted gene expression values (TPM) for hepatocellular carcinoma and glioblastoma multiforme samples. The results showed that MIDN mRNA levels are significantly and positively correlated with those of its substrate EGR1 in real tumor tissues (p < 1e-5), consistent with the trend observed in our biochemical assays. These findings indicate that midnolin may preferentially co-express with its substrates to achieve optimal activity.

The Ubl and catch domains direct substrate to the pore entrance
To elucidate the molecular mechanism of midnolin-mediated substrate delivery to the proteasome entry port, we purified human proteasomes via an mCherry-hexahistidine-twin-strep-tagged RPN11 (Supplementary Fig. 6g) and incubated them with purified midnolin-EGR1(101–200) complex before plunge-freezing. We determined the cryo-EM structure of the 26S proteasome in complex with midnolin and EGR1, revealing midnolin bound to the proteasome with both the Ubl and Catch domains resolved (Supplementary Fig. 7). In a subset of particles, we observed additional density on the RPN11 subunit. Focused refinement of 19S proteasome enables clear visualization of the Ubl domain bound to RPN11 (Fig. 3a). The substrate, translocated into the ATPase AAA+ ring near RPT5 (Fig. 3b), suggests a state at the initial stage of substrate degradation. Adjacent to the Ubl domain, above the proteasome entry port, weaker but discernible density indicated the presence of the Catch domain. To improve this region, we collected additional data and performed 3D classification, isolating a subset of particles with improved density (Fig. 3c, d and Supplementary Fig. 7). Rigid-body docking of the predicted Catch–EGR1 model into the refined map yielded a strong fit, allowing unambiguous placement of the Catch domain (Fig. 3e and Supplementary Fig. 8). Additionally, continuous density for the interdomain linker between the Ubl and Catch domains confirmed their spatial arrangement, further validating our structural model (Fig. 3c, d). This interpretation is further corroborated by our in silico structural model of the midnolin-proteasome-substrate complex, predicted using AlphaFold3 (Supplementary Fig. 9). The high conformational dynamics of the Catch domain are consistent with its role in delivering substrates to the proteasome entry port. Although the Catch domain was resolved at relatively lower resolution, our integrated analysis combining AlphaFold3 predictions with the cryo-EM density map demonstrates that the substrate’s C-terminus might be positioned directly above the proteasome entry port, an optimal configuration for efficient substrate degradation (Fig. 3e).

Structural analysis demonstrated that the Ubl domain interacts with RPN11 in a manner reminiscent of ubiquitin binding (Fig. 3f). The interaction is stabilized by both hydrophobic and polar contacts (Fig. 3g). Specifically, a hydrophobic core is formed by Ala72, Leu74, Val99, Pro100, Val102 and Ala104 of Ubl together with Met54, Leu56, Met75, Pro89, Trp111 and Phe133 in RPN11. The interaction network may be further reinforced by specific salt bridges, notably between Asp77 of Ubl and Lys94 in RPN11. To validate the functional role of these key residues, we mutated the core hydrophobic residues within the Ubl domain and assessed their impact on proteasome activity stimulation. The results demonstrated that both the MIDNP100E/V102E/A104E and MIDNP100E mutant exhibited a significant reduction in their ability to stimulate proteasome activity compared to the wild-type complex (Fig. 3h, i). This finding directly confirms that these hydrophobic residues are essential for the Ubl domain’s function in stimulating proteasomal activities.

The Catch domain is required for efficient substrate engagement and degradation
The αHelix-C of midnolin anchors to the proteasome by binding RPN1, while its Ubl domain engages RPN11 to position the Catch domain near the proteasome entry port for substrate degradation. To develop targeted degraders based on this mechanism, we wondered whether the core elements (αHelix-C and the Ubl domain) are sufficient to stimulate proteasome degradation activity. To test this, we constructed and purified a midnolin variant MIDNΔ(112-336) which is deficient in the Catch domain (Fig. 4a and Supplementary Fig. 10). This midnolin variant maintained its interaction with endogenous proteasome, and we determined its high-resolution structure (Supplementary Figs. 10, 11). The Catch-deficient variant MIDNΔ(112-336) demonstrated that approximately one-third of the 26S proteasome particles (29,705 particles in 84,164 particles for 26S) were in substrate-free resting states and two third in processing states (Fig. 4b). Compared with full-length MIDN purified complexes, the MIDNΔ(112–336)-proteasome sample lacking the Catch domain exhibits a significantly higher proportion of particles in the substrate-free resting state (RS). This comparison indicates that the Catch domain, together with substrate binding, plays a role in stabilizing the MIDN-proteasome complex and maintaining it in an active, substrate-processing state. We found that the substrate in the processing state structurally resembles midnolin itself, with its N-terminal disordered region (intrinsically disordered region 1, IDR1) extending into the motor domain and its Ubl domain docked at the proteasome entry port (Fig. 4c–f). This structural observation is consistent with previous biochemical evidence demonstrating that midnolin undergoes proteasomal degradation after substrate delivery3.

To clarify the core function of the Catch domain, we engineered a "Catch-replaced midnolin" construct MBP-MIDN(1-111)-EGR1(101–200)-MIDN(337-468), where the native Catch domain is replaced by the known substrate fragment EGR1(101–200), covalently tethered via a flexible linker. This chimera retains constitutive substrate binding but lacks the native Catch interface. We then compared the stimulated proteasome activity of three proteins: wild-type full-length midnolin (MBP-MIDN+EGR1(101–200)), Catch-deleted mutant (MIDNΔ(112-336), Catch-replaced midnolin. If the role of the Catch domain is to bind substrate, the Catch-replaced midnolin should stimulate proteasome activity as efficiently as wild-type because the substrate is already effectively "provided". Conversely, if the Catch domain possesses an additional "positioning" or "optimized delivery" function, then the Catch-replaced midnolin should show markedly lower stimulated efficiency, resembling or only slightly exceeding that of Catch-deleted mutant. The results show that the Catch-replaced midnolin, despite having the substrate pre-bound, showed a significantly lower degradation efficiency than the full-length, much closer to the Catch-deleted mutant MIDNΔ(112-336) (Fig. 4g, h). These results demonstrate that the Catch domain provides a specific function beyond simple substrate binding, consistent with its proposed role in actively positioning the substrate for optimal delivery to the proteasome.

Together, these results indicate that the Catch domain is crucial for engaging substrates and facilitating their efficient degradation, although midnolin itself can still be engaged by the 26S proteasome even in the absence of the Catch domain in a less efficient manner.

The human midnolin-26S proteasomes are captured in four continuous states
Our current understanding of proteasomal translocation comes from cryo-EM studies of 26S proteasomes with substrates in processing states. These studies revealed that the AAA+ motor (RPT1–6) adopts right-handed spiral-staircase conformations, with typically 4–5 RPT subunits engaging the substrate via their pore-1 loops and 1–2 subunits (seam subunits) positioned off the substrate22. These observations led to the ‘hand-over-hand’ translocation model, wherein the sequential ATP hydrolysis cycle coordinate the movement of these seam subunit to drive stepwise substrate translocation. Our cryo-EM structures capture the midnolin-proteasome complex within this dynamic cycle, providing snapshots of specific substrate-engaged translocation intermediates14–16,21. Within this dynamic framework, our study reveals four sequential translocation states of the endogenous human midnolin-26S proteasome complex. We classify these states as MIDN-PS , -PS , -PS , and -PS , following the established nomenclature based on the highest substrate-engaged RPT subunit21 (Fig. 5a).

Conformational analysis shows the progressive advancement of the spiral staircase. For instance, in MIDN-PS , RPT6 occupies the top substrate-bound position, while seam subunits RPT1 and RPT2 are disengaged (Fig. 5b, c). This pattern shifts sequentially through the subsequent states.

A notable transition occurs between MIDN-PS and MIDN-PS . In MIDN-PS , RPT3 retains a bound Mg²⁺ ion, while in MIDN-PS , this Mg²⁺ is released (Fig. 5d, e). Concomitantly, the entire AAA+ ring undergoes a ~2.4Å downward rigid-body shift (Fig. 5f). The spatiotemporal correlation between Mg²⁺ release and ring movement suggests that Mg²⁺ dissociation from RPT3 may act as a coordinated trigger, working in concert with ATP hydrolysis to facilitate the stepwise substrate translocation observed in our structural continuum (Supplementary Movie 1). Collectively, these four sequential states provide a structural visualization of the hand-over-hand cycle14–16 and indicate a potential regulatory role for Mg²⁺ release in coordinating the sequential rearrangements of the ATPase subunits.

Working mode for midnolin-mediated ubiquitin-independent substrate degradation
Based on our results, we propose a working model for midnolin (Fig. 6a). Under specific stimulation conditions, midnolin may rapidly express in conjunction with substrates. After the substrate exerts its function, midnolin utilizes its αHelix-C to target the substrate to the 26S proteasome, where it binds to the RPN1 subunit. Subsequently, its Ubl domain interacts with RPN11, positioning the Catch domain above the entrance of the 26S proteasome N-ring. This proximity allows for preferential degradation of the substrate, followed by the degradation of midnolin itself possibly. This mechanism not only facilitates efficient substrate degradation but also optimizes the utilization of the limited proteasome, thereby maximizing resource efficiency. This is important for the proteasome to degrade various substrates.

Methods
Plasmids, genes, and site-directed mutagenesis
All cDNAs (unless noted otherwise) used in this study were of Homo sapiens origin, and generated from mRNAs extracted from HEK293T cells. Gene sequences used in this study are available from UniProt. The full-length cDNA of MIDN was cloned into the pFastBac Dual CMV vector, with the C-terminal mCherry, 6*His, and twin-strep tags separated by a tobacco etch virus protease cleavage site. Mutants of MIDN were generated by site-directed mutagenesis. The primers used in this study have been listed in Supplementary Table 7.

Expression and purification of human MIDN-proteasome complex
MIDN and its truncated mutants were expressed in HEK293F cells using the BacMam system. Recombinant baculoviruses were generated in Sf9 insect cells through the Bac-to-Bac baculovirus expression system. For protein production, HEK293F cells were cultured at a density of 2.5×10⁶ cells/mL in 1% serum medium at 37°C under 5% CO2 atmosphere with orbital agitation. Following transfection with recombinant baculovirus, 10mM sodium butyrate (303410-500G, Sigma-Aldrich) was added after 12 h to enhance protein expression. To promote complex yield, we supplemented 10µM MG132 (T2154, TargetMol) and 20ng/mL PMA (HY-18739, MedChemExpress) 6 h prior to harvest. Cells were harvested by centrifugation at 3500xg for 15 min. Pellets were resuspended in ice-cold lysis buffer (50mM HEPES (pH 7.4), 10% (v/v) glycerol, 5mM MgCl2, 5mM ATP, 1mM Tris(2-carboxyethyl)phosphine (TCEP), 0.5% (v/v) NP-40) supplemented with 1mM PMSF. Cell disruption was performed via sonication (150W output) under ice-cold conditions using 4s pulses alternating with 6s intervals for 6 min. Lysates were clarified by centrifugation at 46,500×g for 40 min under 4°C. For affinity purification, clarified lysates were loaded onto a pre-equilibrated immobilized metal affinity chromatography column (Ni²⁺-IDA resin, SA052100; Smart-Lifescience) at 4°C using gravity flow. The column was washed with 10 column volumes of wash buffer (lysis buffer + 20/40mM imidazole), and His-tagged proteins were eluted with elution buffer (lysis buffer + 300mM imidazole). The eluate was subsequently loaded onto a Strep-Tactin XT column (2-5010-010; IBA Lifesciences) pre-equilibrated with lysis buffer. After washing with 30 mL of lysis buffer under gravity flow, bound proteins were competitively eluted using lysis buffer containing 50mM biotin (A84479, innochem) under gravity flow. The eluate was concentrated to 500μL using a 100kDa cutoff concentrator. Finally, a Superose 6 Increase 10/300 GL column was equilibrated using the size-exclusion buffer (30mM HEPES, 10% (v/v) glycerol, 1mM MgCl2, 0.6mM ATP, 0.5mM TCEP, 60mM NaCl, pH 7.4) and a 0.5 mL injection loop was washed thoroughly with the size-exclusion buffer. The protein sample was centrifuged at 11,200xg for 5 min at 4°C to remove any debris. We manually injected 0.5 mL of the concentrated eluted protein into the loop and then fractionated it on a Superose 6 Increase column using size-exclusion buffer. Fractions containing intact 26S proteasomes were collected, pooled, and concentrated using Amicon Ultra-15, 10kDa MWCO (6200×g, 10 min intervals) to a final volume of around 50μL at 0.3 mg/mL. The samples were flash-frozen in liquid nitrogen, and stored at −80°C. Protein concentration was determined using a Bradford assay (BCA) kit (20201ES76, Yeasen). Graphene-supported grids were used for data collection for the sample.

Expression, purification, and in vitro incubation of human 26S proteasome
To purify the human 26S proteasome, we employed the BacMam system for large-scale expression of the RPN11 protein in mammalian HEK293F cells For purification, HEK293F cells infected with the virus were cultured for 48 h, then harvested via centrifugation at 3500xg. The cell pellet was resuspended in lysis buffer (40mM HEPES, 100mM NaCl, 1% (v/v) NP-40, 1mM MgCl2, 5mM ATP, 1mM TCEP, 1× Cocktail, 1mM PMSF, pH 7.4) and subjected to ice-cold sonication (150W, 4s pulse/6s interval for 6 min). The lysate was transferred to a pre-cooled centrifuge tube and centrifuged at 46,500×g for 20 min at 4°C to collect the supernatant. Intact proteasomes were obtained by sequential Strep and His affinity purification using the C-terminal twin-Strep and 6×His tags on RPN11. The lysates were applied to a pre-equilibrated Strep column, washed, and then the bound protein was eluted and collected. Then, the eluate was loaded onto a pre-equilibrated IDA column at 4°C, where the His-tagged target protein bound to the column. Contaminants were removed using a buffer (40mM HEPES, 100mM NaCl, 1% (v/v) NP-40, 1mM MgCl2, 5mM ATP, 1mM TCEP, 1× Cocktail, pH 7.4) containing 20mM and 40mM imidazole, followed by protein elution with 300mM imidazole. The eluent was collected and concentrated to 500μL using a 100kDa cutoff concentrator. The protein sample was centrifuged at 11,200×g for 5 min at 4°C, then fractionated using a Superose 6 column with size-exclusion buffer (50mM HEPES, 50mM NaCl, 1mM MgCl2, 2.5mM ATP, 1mM TCEP, pH 7.4). Fractions containing intact 26S proteasomes were collected. Protein concentration was determined using a Bradford assay kit. The complex was concentrated to approximately 4 mg/mL in size-exclusion buffer, flash-frozen in liquid nitrogen, and stored at −80°C.

The co-expressed and purified EGR1(101–200)-mCherry complexed with MBP-midnolin were mixed with the purified 26S proteasome at a 50:1 molar ratio, incubated on ice for 30s, and immediately prepared for cryo-EM analysis. 3µL of the sample was applied onto a freshly glow-discharged (at 20mA for 70s, negative charge) Quantifoil R1.2/1.3 300-mesh Au grid. Grids were blotted for 3s under 100% humidity at 4°C using a Vitrobot (Thermo Fisher Scientific), then plunge-frozen in liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until data collection.

Negative stain electron microscopy
For negative stain electron microscopy, a regular carbon-coated grid was grasped and subjected to glow discharge using plasma (PELCO easiGlow). The protein sample (3µL, 0.05 mg/mL) was then pipetted onto the grid and allowed to sit for 2 min. Following this, 3µL of uranyl acetate stain was applied to the grid and left to stand for another 2 min. Data collection was carried out using a transmission electron microscope (JEM1400PLUS).

Cryo-EM sample preparation and data collection
The purified samples were centrifuged at 11,200×g for 5 min at 4°C before cryo-EM sample preparation. Aliquots of 3.0µL samples were applied to glow-discharged grids (Quantifoil, 300 mesh, R1.2/1.3, Au). The plasma treatment conditions were set at 20mA for 70s, followed by a 10-s holding period. Graphene-supported mesh was purchased from Shuimu Biosciences Ltd. Using a Vitrobot (Thermo Fisher Scientific), glow discharged grids were placed at 100% humidity and samples were applied and immediately blotted for 3s before plunge freezing in liquid ethane. Grids were clipped and transferred to transmission electron microscope. All cryo-EM data acquisition was performed on a 300kV Titan Krios transmission electron microscope (Thermo Fisher Scientific) equipped with a Gatan K3 direct electron detector and a GIF Quantum energy filter, operating in EPU mode. All movie stacks were fractioned into 32 frames with a total electron dose 40e-/Å2. Images were taken at a nominal magnification of 105,000 with a pixel size of 0.85Å in super resolution mode with a defocus ranging from −0.8 and −1.8µm.

Data processing of 26S Proteasome-MIDN-FL dataset
This integrated cryo-EM dataset collected from four independently frozen grids of the same biological sample. Following patch-based motion correction and contrast transfer function (CTF) estimation, micrographs exhibiting severe ice contamination or suboptimal CTF fitting were systematically excluded. An initial template for particle picking was generated using a representative subset of ~20 micrographs. Template matching from 37,413 qualified micrographs yielded 2,067,075 raw particles in the initial round. After iterative 2D classification, 461,310 high-quality particles were retained for ab initio reconstruction. From this pool, 161,296 particles with well-defined features underwent further 3D classification after global refinement (I=20 classes). Particles from Classes 0, 5, 6, and 19, which exhibited features of substrate-engaged states, were selected. To improve the resolution of the AAA+ motor, these particles underwent local refinement using a soft mask focused on the AAA+ ATPase domain, yielding two locally refined proteasome complex structures at 3.45Å (PS , PDB: 9MBP, EMD-63766, 40,572 particles) and 3.66Å (PS , PDB: 9MBQ, 23,186 particles), respectively.

To resolve the interaction between midnolin’s αHelix-C and RPN1 at high resolution, we selected particles from Classes 0 and 1, which represented substrate-engaged complexes. Notably, 63,758 particles produced a high-resolution RPN1 subunit structure (PDB: 9MBO, EMD-63775, 3.07Å) after local refinement. For Classes 1–4 and 7–18 (combined 97,538 particles), further 3D classification (20 classes) was performed. Subsequent selection of 9,022 particles from Class 11 through mask focused on 19S generated a 4.48Å map (PS , PDB: 9U4M, EMD-63850). Additionally, 25,990 particles selected from Classes 0, 1, and 10 were processed identically to obtain an improved 19S structure (PS , PDB: 9U3L, EMD-63817) at 3.72Å resolution. The other classes (Classes: 2–9 and 12–19) represented unassigned processing states. The final maps were further processed using EMReady42. Model refinement and statistical analyses were performed based on the raw (EMReady-unprocessed) density maps.

Data processing of 26S Proteasome-MIDNΔ(112-336) dataset
The workflow was similar to the processing pipeline established for the 26S Proteasome-MIDN-FL dataset. From 19,307 acquired movies, 18,890 micrographs meeting quality thresholds were selected for particle picking using Topaz Pick (v0.2.5). This procedure generated 1,646,618 initial particle extracts, which underwent iterative 2D classification to yield 261,241 structurally homogeneous particles suitable for ab initio reconstruction.

Particles from Classes 0 and 2 were subjected to 3D classification comprised 10 distinct classes, which were systematically categorized into two functional states: Classes 1–6 and 8 (total 7 classes) representing the substrate processing state containing 54,459 particles, while Classes 0 and 9 were identified as the resting state comprising 29,705 particles. Particles in class 8 were subjected to sequential non-uniform (NU) 3D refinement, ultimately yielding a consensus proteasome structure with a global resolution of 3.42Å (MIDN-PS Δ(112-336) , PDB: 22MM, EMD-68472). Particles in class 9 were subjected to NU 3D refinement, ultimately yielding a consensus proteasome structure with a global resolution of 3.17Å (Resting state). To enhance the local resolution of the 19S regulatory particle, we performed signal subtraction of the 20S core particle density followed by local refinement using a soft mask focused on 19S. This local refinement using 22,379 optimally aligned particles achieved a high-resolution 3.35Å reconstruction of the 19S regulatory complex. The final maps were further processed using EMReady42. Model refinement and statistical analyses were performed based on the raw (EMReady-unprocessed) density maps.

Data processing of MIDN-EGR1-human26S proteasome complex
Data processing of MIDN-PS . This integrated three datasets of cryo-electron microscopy images acquired from the same frozen specimen. Following patch-based motion correction and CTF estimation, micrographs exhibiting severe ice contamination or suboptimal CTF fitting were systematically excluded. Particle extraction was performed using Topaz Pick (v0.2.5), which generated three sets of initial particle extracts. These extracts underwent iterative 2D classification, resulting in three distinct particle sets. Based on the resulting 2D classifications, the particles were re-extracted.

Due to structural heterogeneity, the re-extracted particles were subjected to heterogeneous refinement, yielding three different 3D structures. A total of 305,216 particles from Class 0 and Class 1 of these three groups were selected for NU 3D refinement. Prior to symmetry expansion, the selected particles were subjected to Non-uniform refinement with C2 symmetry imposed. The resulting alignment parameters were then used to perform symmetry expansion, doubling the particle count. The expanded particles were subsequently re-extracted, re-centered, and used for downstream focused classification and refinement steps. Since the 19S regulatory particle in the resulting 26S proteasome structure was not centered, symmetry expansion was applied to generate 610,432 particles. Masking was performed to generate a map of the 19S regulatory particles, and the particles were realigned to center the 19S within the box. Subsequently, 610,019 particles were re-extracted and subjected to local refinement and masking to generate a map of the 19S regulatory particles. From this pool, particles with well-defined features underwent 3D classification after global refinement using a 3Å filter resolution (10 classes). Class 7 (68,581 particles) was selected for local refinement, and masking was applied to generate a map of the Ubl domain. This was followed by an additional round of 3D classification, from which Class 0 (24,852 particles) was selected for local refinement, ultimately, a consensus proteasome structure containing a Ubl domain was determined at a global resolution of 3.54Å (MIDN-PS , PDB: 9W39, EMD-65595).

Data processing of MIDN-PS . An additional cryo-EM dataset was collected from the same specimen. Following patch-based motion correction and CTF estimation, micrographs with significant ice contamination or suboptimal CTF fits were systematically discarded. Initial particle picking was performed using Topaz (v0.2.5), yielding a set of raw particle extracts. Subsequent iterative rounds of 2D classification and particle re-extraction resulted in a final set of 68,908 particles. Owing to structural heterogeneity, these particles were subjected to heterogeneous refinement, which produced three distinct 3D classes. Particles from Class 0 and Class 1 across these groups (totaling 52,879 particles) were pooled and processed through NU 3D refinement. The resulting consensus 26S proteasome map exhibited a decentered 19S. To address this, symmetry expansion was applied, generating 105,647 particles. A mask focused on the 19S region was then used to realign the particles, centering the 19S within the box. These processed particles (105,647) were subsequently combined with the 610,019 particles derived from earlier processing stages (as described in the preceding section). The combined pool of 715,666 particles underwent high-resolution 3D classification into 10 classes. Classes 0 and 1 from this classification were selected for local refinement, followed by an additional round of 3D classification. From this subsequent classification, Classes 0 and 2 were chosen for further 3D classification. To enrich for a specific state, particles from all classes of this refinement were merged with a reference set from UBL ("seed1"). After duplicate removal, another round of 3D classification was performed. Particles from Class 0 were selected, and any duplicates overlapping with the seed1 set were excluded, yielding 16,606 particles. These were then merged back with the original seed1 particles, creating a combined set of 41,458 particles for another round of 3D classification (10 classes). Class 1 from this classification was selected for local refinement. The entire set of 41,458 particles then underwent a final round of 3D classification facilitated by a second reference ("seed2"). Particles from Class 0 (7251 particles) of this last classification were subjected to local refinement, ultimately yielding a consensus proteasome structure containing a Catch domain at an overall resolution of 4.97Å (MIDN-PS , PDB: 9WBG, EMD-65839). The final maps were further processed using EMReady42. Model refinement and statistical analyses were performed based on the raw (EMReady-unprocessed) density maps.

Model building and refinement
The data processing procedures were carried out using the cryoSPARC software v4.3.143. The density maps were further enhanced by B-factor sharpening in cryoSPARC and additional sharpening with EMReady42. A previously published proteasome model (see Supplementary Table 1) was initially placed into the cryo-EM density map using UCSF Chimera. The AlphaFold-predicted midnolin structure44 was then rigid-body fitted into the remaining density in Chimera. The combined model was subsequently manually adjusted and rebuilt in COOT (version 0.9.4.1). The model underwent iterative optimization through real-space refinement, employing both the Phenix software suite (version 1.20)45 and Coot46 for structural refinement and building. Model refinement and statistical analyses were performed based on the raw (EMReady-unprocessed) density maps. All figures were crafted using PyMOL 2.4.1 (Schrödinger) and ChimeraX47.

AlphaFold3 prediction
AlphaFold3 predictions of midnolin-RPN1-RPN11-ATPase, or complexed with EGR1 (101–200), IRF4(131-248) and FosB(231-338) were conducted by the online AlphaFold3 server website with random seed. The UniProt sequence ID are provided: RPT1 (P35998), RPT2 (P62191), RPT6 (P62195), RPT3 (P43686), RPT4 (P62333), RPT5 (P17980), RPN11 (O00487), RPN1 (Q13200), MIDN (Q504T8), EGR1 (P18146), IRF4 (Q15306), FosB (P53539). The N-terminal 10 residues of RPN11 (MDRLLRLGGG) were omitted in all the predictions. In addition to the proteins, four ATP molecules, two ADP molecules, and one zinc ion were also added. The resulting structural models have been deposited in ModelArchive (https://www.modelarchive.org/) under accession codes ma-bn2mb, ma-xuxb0, ma-vfr8j, and ma-ue0q7, respectively.

Protein expression and purification in E. coli
The bacterial-expressed proteins were produced in BL21(DE3) E. coli cells (TransGen Biotech) cultured in LB medium (comprising 5g/L yeast extract, 10g/L tryptone, and 10g/L NaCl). Protein expression was induced by adding 0.1mM IPTG (INALCO) when the OD600 reached 0.8, followed by overnight incubation at 16°C. The bacteria were resuspended in 40 mL of TBS buffer (20mM Tris-HCl, 137mM NaCl, pH 7.4) after centrifugation at 4000×g for 10 min and then subjected to rapid freezing in liquid nitrogen. After thawing by rotation, PMSF (working concentration: 1mM) was added to the sample, which was then subjected to ultrasonic disruption on ice (150W, pulse 4s/interval 6s, 10 min). The lysate was transferred to a pre-cooled centrifuge tube and centrifuged at 15,616×g for 20 min at 4°C to collect the supernatant. Clarified lysates were loaded onto a pre-equilibrated immobilized metal affinity chromatography column (Ni²⁺-IDA resin) equilibrated with buffer A (20mM Tris–HCl, 150mM NaCl, 0.5mM TCEP). The protein-containing supernatant was loaded onto the column. Following binding of the target molecules to the column, impurities were sequentially removed by washing with buffer A containing 20mM, 40mM, and 60mM, His-tagged proteins were eluted with elution buffer (buffer A + 300mM imidazole). The eluate was concentrated to 500μL using a 30 or 50kDa cutoff concentrator. To remove contaminating nucleic acids, the eluates of MBP-MIDN, MIDN(337–468)-mCherry, MIDN(337–432)-mCherry, and MBP-MIDN-EGR1(101–200)-mCherry were treated with Benzonase nuclease (Cat. No. SSNP01-50KU, Sino Biological) in the presence of 2mM MgCl₂ for 2 h at 4°C before SEC. Finally, a Superdex 200 Increase 10/300 GL column was equilibrated using the buffer B (20mM HEPES, 150mM NaCl, 0.5mM TCEP, pH 7.4) and a 0.5 mL injection loop was washed thoroughly with the buffer B. The protein sample was centrifuged at 11,200×g for 5 min at 4°C to remove any debris. The loop was then manually injected with 0.5 mL of concentrated eluted protein. Then fractionated using a Superdex 200 Increase 10/300 GL column with buffer B. Fractions were collected, pooled, and concentrated using a 30 or 50kDa cutoff concentrator (3500xg, 20 min intervals) based on chromatographic peaks and Coomassie Brilliant Blue staining results of Tris-glycine gels. The samples were flash-frozen with liquid nitrogen in aliquots of 30μL, and stored at −80°C. Protein concentration was determined using Nanodrop.

Co-expression and purification of EGR1(101–200) and Catch
Catch domain with His-MBP tag was cloned into pET-28a(+) vector. EGR1(101–200)-mCherry without any other tag was cloned into ORF2 of pETDuet-1 vector. A mixture of 200ng Catch and 200ng EGR1(101–200) plasmids was co-transformed into 10μL of E. coli BL21(DE3). E. coli BL21(DE3) were incubated on ice for 20 min, heat-shocked at 42°C for 45s in a water bath, and immediately returned to ice for 2 min. Subsequently, the transformed cells were resuspended in LB medium supplemented with 50μg/mL ampicillin and 25μg/mL kanamycin, followed by incubation at 37°C with stirring until the OD600 reached 0.8. The culture was cooled to 16°C, and protein expression was induced by adding 0.1mM IPTG overnight. Cells were harvested by centrifugation at 4000×g for 15 min at 4°C. The pellets were flash-frozen in liquid nitrogen, and stored at −80°C. The purification steps are the same as those for protein expression in E. coli mentioned above.

Size-exclusion chromatography
To validate the assembly characteristics of the substrate proteins EGR1 and midnolin, we employed two approaches: (1) in vitro reconstitution by incubating Catch with EGR1(101–200), and (2) co-expression, combined with size-exclusion chromatography (SEC) to assess the complex formation. For in vitro reconstitution: Catch and EGR1(101–200) were individually expressed and purified using the E. coli-based method described above. A mixture containing 1mg of EGR1(101–200) and 600μg of Catch was incubated on ice for 30 min. The sample was then injected into a pre-equilibrated Superdex 200 Increase 10/300 GL column with Buffer B. Elution fractions (0.5 mL per tube) were collected during SEC separation. Elution fractions were analyzed by 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. For co-expression analysis: the recombinant Catch-EGR1(101–200) complex was co-expressed in E. coli BL21(DE3) using the protocol above. The complex was purified via the same method under Buffer B conditions. Eluted fractions were similarly collected and subjected to 10% SDS-PAGE analysis with Coomassie Brilliant Blue staining.

Isothermal titration calorimetry (ITC)
All measurements were done in ITC buffer containing 20mM HEPES, pH 8.0, 150mM NaCl. Each titration consisted of 20 successive injections (the first at 0.4μL and the remaining 19 at 2μL). The titrant, 100μM MIDN(337-468) or MIDN(337-432), and the 10μM RPN1 analyte were dissolved in ITC buffer. Before measuring the protein–protein interactions, we performed blank titrations by injecting MIDN(337-468) or MIDN(337-432) into buffer to exclude non-specific binding signals. Reference Power was set to 5μcal/s; Initial Delay is 60s; The injection interval was 120s to ensure that the signal returns to baseline; All titrations were conducted at 25°C with stirring to ensure thorough mixing. The thermal signal curve was subsequently fitted using MicroCal PEAQ-ITC Analysis Software. The resulting blank heat effects were subtracted from the raw thermograms before fitting the binding isotherms. Binding isotherms were fitted to a single-site binding model to derive thermodynamic parameters: dissociation constant (KD), enthalpy change (ΔH), entropy change (ΔS), and binding stoichiometry (N). Additionally, all ITC experiments were performed in triplicate, and the data are presented as mean ± SD.

Surface plasmon resonance (SPR)
All surface plasmon resonance (SPR) experiments were performed at 25°C on an INTERBIOMI-S200F instrument using CM5 sensor chips and running buffer (10mM HEPES, 150mM NaCl, 0.05% Tween-20, pH 7.4) that had been degassed and 0.22µm-filtered. After a 5-min deionised-water rinse, the carboxylated surface was activated with a freshly mixed 1:1 solution of 0.4M EDC and 0.1M NHS for 7 min at 10µL min⁻¹. Ligand proteins diluted in 10mM sodium acetate (pH 4.5) were immobilized by continuous flow until the target response was reached; residual NHS-esters were blocked with 1M ethanolamine-HCl (pH 8.5) for 7 min. A reference flow cell treated identically but receiving no ligand served as the blank for subsequent double-referencing.

Three binary interactions were analysed in succession. (i) RPN1 (ligand, 498µM stock) was coupled to 759 RU; analyte MIDN(337-468) (170µM stock) was injected for 120s at 30µL min⁻¹ followed by 300s dissociation, using concentrations of 1.25, 0.625, 0.3125, 0.156, 0.078, and 0.039µM. (ii) The same surface was regenerated with 20mM NaOH (30s) and reused for MIDN(337-432) (464µM stock) with an identical concentration series. (iii) A second CM5 chip was prepared with 26S proteasome (161nM stock) immobilized to 1741 RU; MIDN Δ(112-336) (90µM stock) was titrated at 3, 1.5, 0.75, 0.375, 0.187, and 0.093µM with regeneration by 10mM glycine-HCl pH 1.5 (30s). Each concentration was injected once in randomized order and the complete sequence was repeated in triplicate on independent chips. Sensorgrams were processed with the instrument software, solvent-corrected, and globally fitted to a 1:1 Langmuir model to extract ka, kd and KD values; reported constants are mean ± SD of the three independent experiments.

Proteasome stimulating activity assay
Proteasome stimulating activity of MBP-MIDN+EGR1(101–200), MBP-MIDNP100E+EGR1(101–200), MBP-MIDNP100E/V102E/A104E+EGR1(101–200), MBP-MIDNK391E/L395E/R409E+EGR1(101–200), MBP-MIDNΔ(112-336), MBP-MIDN(1-111)-EGR1(101–200)-MIDN(337-468), MBP-MIDN(337-432) and MBP-MIDN(337-468) toward 26S human proteasomes were measured using fluorogenic substrate Suc-LLVY-AMC (South Bay Bio)12. Briefly, 1nM human 26S proteasomes were incubated with 100nM of substrate in buffer (50mM HEPES pH 7.5, 100mM KCl, 0.5mM MgCl2, 1mM TCEP, 0.2mM ATP, and 25ng/μL BSA) for 5 min at room temperature. 10μM Suc-LLVY-AMC was added to the reaction mixture. For time course analyses, proteasome stimulating activity was measured immediately after substrate addition and for up to 1.5 h at 37°C using a Thermo Fisher Scientific multimode plate reader (excitation, 345nm; emission, 445nm).

Multiple sequence alignment
Multiple sequence alignment was performed using ESPript (Version 3.2)48.

Thermal stability analysis of proteins using nanoDSF
To assess the thermal stability of various proteins, we employed nanoDSF (nano Differential Scanning Fluorimetry) using the Prometheus NT.48 instrument (NanoTemper Technologies GmbH, Munich, Germany). This method enables the evaluation of protein thermal stability in a label-free manner by monitoring changes in the intrinsic fluorescence of tryptophan residues. We purified and obtained different protein samples. Then, 10µL of each protein sample was loaded into high-purity glass capillaries, and the capillaries were placed into the Prometheus NT.48 instrument. The temperature gradient was set to increase from 20°C to 95°C at a rate of 1.0°C/min. The intrinsic fluorescence of tryptophan residues was monitored at excitation wavelengths of 280nm and 350nm, with emission detected at 330nm and 350nm, respectively. The ratio of fluorescence intensities at 350nm/330nm was used to track protein unfolding. The melting temperature (Tm), defined as the midpoint of the protein unfolding transition, was determined from the fluorescence ratio curve. Each measurement was performed in triplicate to ensure reproducibility and accuracy.

Statistical analysis
All statistical analyses and data visualizations were performed using GraphPad Prism (version 10.1.2). For the in vitro peptidase activity assays, data are presented as mean ± SD from three independent experiments (n=3). Statistical significance was determined by two-tailed one-way ANOVA followed by Tukey’s multiple comparison test. The exact p values and significance thresholds (***p<0.001, ****p<0.0001) are detailed in the respective figure legends. ns, not significant.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The atomic model coordinates and cryo-EM maps have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB), respectively, under the accession codes: 9MBO and EMD-63775 (RPN1-MIDN), 9MBP and EMD-63776 (MIDN-PS ), 9MBQ and EMD-63777 (MIDN-PS ), 9U3L and EMD-63817 (MIDN-PS ), 9U4M and EMD-63850 (MIDN-PS ), 9U7R and EMD-63943 (RS), 9W39 and EMD-65595 (MIDN-PS ), 9WBG and EMD-65839 (MIDN-PS ), 22MM and EMD-68472 (MIDN-PSΔ(112-336) ). Additionally, for the previously published structures used in this study, the atomic coordinate of the USP14–RPN1 complex is under the accession code 7W3H, the NUB1–RPN1 complex is under the accession code 8USB, the canonical ubiquitin with RPN11 is under the accession code 6MSG, the initial model used for building of MIDN-PS is under the accession code 6MSK, the initial model used for building of MIDN-PS is under the accession code 6MSJ, the initial model used for building of PS is under the accession code 9E8K, the TXNL1-bound proteasome state PS is under the accession code 9E8O, the TXNL1-bound proteasome state PS is under the accession code 9E8J, and the TXNL1-bound proteasome state PS is under the accession code 9E8G. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper ‘Structural dynamics of the midnolin–proteasome during ubiquitin-independent substrate turnover’, Zhu, Qin et al. investigate how midnolin (MIDN) mediates ubiquitin-independent proteasomal degradation. By combining cryo-electron microscopy (cryo-EM) with biochemical data, they reveal the mechanisms of the interaction between MIDN and the 26S proteasome, and proposing the role of the individual MIDN domains in substrate degradation. The work includes extensive structural work which offers various snapshots of the MIDN-proteasome interaction and of the substrate-protasome interaction. Overall, this paper contains valuable findings that will interest the proteasome research community. However, in light of the recently reported structures of the midnolin–proteasome complex (Nardone et al., 2025), the novelty of the work presented in the manuscript is limited. This wouln’t be per se a problem of the authors had actually solid evidence for the two mechanisms that they try to elucidate: (i) the role of the Catch domain in correctly placing the substrate for degradation and (ii) whether MIDN binds to the substrate prior to proteasome association. The experimental results are poorly described in a disordered way, jumping from one description to the next without a clear logic. At part sit is not even clear what experiments the authors have performer until one reads the methods parts. Moreover, several critical pieces of information are missing throughout the manuscript, and the work requires major revisions before it can be considered for publication.\nSome more detailed comments below:\nThe abstract should be self standing and understandable, instead it includes many words of proteins and domains not explained at all (etc. midnolin, Catch).\nThe introduction contains many grammatical mistakes that even hinder the reading at parts.\n1. In line 48, the authors stats that the identification of the MIDN pathway disrupts theestablished Ub-dependent pathway. To use the word disrupt is incorrect as the ubiquitin-independent path was already known. The new MIDN pathway adds up to the complexity of the system. We didn’t like the use of the word disrupt becasue it i san example of how language is used all along the text to boost the presented data at expenses of honest description of the scientific knowledge at the moment. Other examples of such usage are the words ‘unprecedented’ (line 77).\n2. The naming of the domains changes multiple times in the text, sometime the C-terminus of MIDN is called αHelix-C, some others C-terminal α-helix. The authors should be more consistent in the use of the words.\n3. Cryo-EM structures are presented in a very disordered way, which makes the reading quite complicated. They are overall ok, but very little has been done with postprocessing to resolve better or at least visualise better the less-resolved MIDN parts. We suggest the authors to try different sharpening tools. As they are shown now the maps appea all oversharpened, we assume that only overall B-factor sharpening ahs been applied.\n4. In line 102, the authors state: \"These structures are mainly in substrate processing states that resemble the ED2 state (Fig. 1a and Extended Data Figs. 2, 3 ...).\" However, it is unclear why Extended Data Figs. 2 and 3 are referenced here, as they do not demonstrate that the structures are primarily in the substrate processing state. A better comparison with the reported structures should be conducted to confirm the ED2 conformation of the observed complex. Additionally, the use of the term \"mainly\" is somewhat arbitrary; a more quantitative description would be beneficial. Overall, the paper would benefit from a more thorough comparison of the observed conformations of the 26S proteasome with previously published structures of different states of the complex. Citations should be always included e.g. in line 121 after the sentece ‘different from previous studies’.\n5. In line 115, the authors state, \"Here, we have successfully captured the structures of the natural endogenous substrate-bound 26S proteasome in action.\" Additionally, the discussion emphasizes that endogenous substrate-proteasome complexes used in the study, in contrast to other reported structures obtained with non-hydrolysable ATP analogs or RPN11 inhibitors, do not promote specific conformational states. However, it has to be noted that the proteasomes were halted using MG132, which could also skew the distribution of different states. It has been shown by Haselbach et al. (https://doi.org/10.1038/ncomms15578) that inhibitors acting on the 20S core particle can achieve a long-range allosteric effect that modulates the conformational landscape of the 19S particle. Authors should mention this when discussing the conformational landscape of the complex.\n6.\n7. In line 126-129 when discussing the C-terminal α-helix, the authors do not refer at all to the Nardone et al., 2025 paper where this feature is also described.\n8. The results explained in the paragraph ‘Uniform expression of midnolin is dependent on substrate’ appears not very solid to us. It is unclear whether and how proteome analysis was performed in in hepatocellular carcinoma and glioblastoma multiforme cells and by looking at the graphs in Extended data Fig.6f the correlation is not so tight. That in vitro co-expression of a protein and its substrate stabilise the expression and folding is not surprising, it is in fact a very common phenomenon.\n9. The paragraph regarding positioning of the substrate by the Ubl and Catch domains would benefit from a figure showing a cut-through of the map and snapshots of the different positions of the substrate depending on the positioning of the Catch domain. Moreover, why would a Cter substrate position of that type optimal for degradation?\n10. In line 182, the authors mention, \"Rigid-body docking of the predicted Catch–EGR1 model into the refined map yielded a strong fit, allowing unambiguous placement of the Catch domain (Fig. 3d).\" To strengthen this claim, the authors should provide a close-up view of the model in relation to the map, supported by the Q score and the correlation coefficient between the model and the map.\n11. In line 186, the authors wrote: \"Collectively, our data capture two states: a high-resolution Ubl-bound state (RPN11 engagement) and a state in which both the Ubl and Catch–substrate modules are simultaneously engaged with the 26S proteasome.\" However, due to the poor clarity of the cryoEM data processing and numerous final maps shown in the data processing pipelines that are not well annotated or described in the manuscript, it is not evident to which maps the authors refer when saying the \"two states\".\n12. The results described on pages 9-10 suggest that the MIND's Catch domain is crucial for stabilizing the complex. In the cryo-EM dataset of the proteasome reconstituted with a Catch-deficient protein, a significant fraction of the 26S particles was found in the MIND-free resting state. However, when processing the 26S-MIND FL dataset, the authors did not investigate the presence of the resting state, which weakens their conclusion.\nWere there any other states, aside from the extended state (ED), observed in the endogenous 26S-MIND FL or MIDN-EGR1-human datasets? If so, what were the populations of these different states? Looking at the data processing pipeline of the 26S-MIND FL, a large proportion of the particles was rejected during the ab initio and 3D classification jobs. The ab initio maps were not shown, making it impossible to understand why those particles were removed and what they represent (whether they were low-quality data or different conformations).\nIn the second 3D classification, some of the maps (e.g., classes 12 and 13) appear to show a good amount of detail but were still rejected by the authors. It would be interesting to investigate whether other conformational states were discarded alongside those particles.\nIn conclusion, if the authors intend to use the presence of the resting state in the MIDN∆(112-336)-proteasome dataset as evidence for the role of the Catch domain, they should conduct a more thorough analysis of the other datasets to check for the existence of the resting state in them.\n13. It is not apparent which maps from Extended Data Fig.2 are those shown in Fig. 1 and in Extended Data Fig.4. To which map are the authors referring in the figure caption when mentioning ED2-like state? The annotations should be specific and consistent across the manuscript to ensure that the reader can easily associate the presented results with the correct maps.\n14. Maps presented in Figures 1-4 appear to be slightly oversharpened. The specific sharpening parameters are not currently outlined in the Materials and Methods section. This information should be included, the degree of sharpening should be carefully verified and various sharpening methods could and should be used.\n15. Authors revealed the Helix-C of midnolin interaction with RPN1 (PSMD2). It would be interesting to see a side-by-side comparison of the Helix-C interaction interface (at the residue level) with the other RPN1 T1/T2 interactions (like Usp14 or Ub). Moreover, view that then they use AlphaFold3 to predict the interaction between MIDN and proteasome, it would be interesting to know if this interaction was predicted and to what confidence level.\n16. The use of AlphaFold and molecular dynamics is mentioned; however, the brief and vague description of the results makes it difficult to understand why these analyses were conducted in the first place and what insights they contribute to the overall narrative. The respective description in the methods section is also insufficient; Uniprot sequence IDs used for predictions should be specified along with the AlphaFold prediction confidence statistics. Moreover, in Extended Data Fig.9, the IDR2 and Ubl are described as dark orange.\n17. In line 220, it states: \"This demonstrates that the observed presence of 26S proteasomes without midnolin is not due to asymmetric binding—where midnolin engages one 19S regulatory particle of a 30S proteasome while the other remains unbound. This suggests that in the absence of substrate engagement, midnolin dissociated during size-exclusion chromatography, consistent with the dynamic binding-dissociation nature of midnolin-proteasome interactions we previously proposed.\" However, since a significant number of particles were discarded in other datasets (in which MIND contained the Catch domain) without careful examination of their conformational and compositional heterogeneity (specifically the presence or absence of MIND), it is challenging to unequivocally state whether the presence of MIND-free resting state complexes is unique to the cryo-EM sample prepared with the Catch-deficient MIND construct. Authors should verify the effect of the Catch domain deletion and the presence of the substrate on the stability of the 26S-MIND complex using biochemical and/or other biophysical assays. This could include techniques such as native PAGE with specific immunostaining or fluorescent tagging of the proteins of interest. In addition, authors should use RP2-CP and RP-CP notation to differentiate 30S from 26S complexes.\n18. In line 214, it states: ‘we found that the substrate in the processing state structurally resembles midnolin itself’ suggesting that MIDN gets degraded. Is there any biochemical evidence for it? In such case a citation of the relevant literature should be provided.\n19. On page 11, there is no description of the various states relaying completely on the reader's good knowledge of the proteasome. Moreover no comparison is give side by side to other published structures. This makes the manuscritp difficult to read. Moreover, the states described as well as the whole Figure 6b is extremely reminescent of the paper by Arkinson et al., 2004 (https://doi.org/10.1101/2024.11.08.622731) and this paper is not even cited. We also find the proposed model of movement associated to Mg2+ release not very sound considering that the movement described is of 2.3Å so way below the overall resolution of the map (3Å at best).\nThe discussion part is again missing citations. More in details:\n20. In line 307, the authors state: \" Additionally, the IDR2 region of the Catch domain guides midnolin itself into the proteasome for degradation after substrate delivery.\" Do authors have any biochemical data confirming this?\n21. In line 313, it states: ‘Our findings indicate that ATP hydrolysis plays a pivotal role in the displacement of the proteasome's AAA+ module, with Mg²⁺ dissociation also actively facilitating substrate translocation (Fig. 6b)’. This is quite misleading as it is based on observation of motion below the actual resolution limit of the data.\n22. In line 322, it states: ‘These findings suggest that asymmetric ATP hydrolysis by the proteasome ATPase is a widespread phenomenon and may not be substantially related to substrates. The underlying reasons for this asymmetric hydrolysis merit further investigation’. This is not true, there are a wealth of data and discussions about the asymmetry of the proteasome and all other AAA+ unfoldases.\nOther points (regarding the methodology):\n23. The authors state that the 26S complex concentration was measured after the purification; however, there is no information about the concentration of the complex used for grid preparation.\n24. There is currently no information provided regarding the software utilized for cryo-EM data processing and analysis. Similarly, Figure S5 seems to be prepared using ESPript. The authors should specify this and cite the relevant software utilized.\n25. In the description of some of the cryo-EM data sets, the number of collected movies and particles in the initial stacks is missing. Authors should consider rearranging the sections that describe the processing of different samples to align with the order of the figures depicting the pipelines.\n26. Authors use \"two-dimensional\" and \"3D\" interchangeably. I suggest using the simpler 2D/3D notation for clarity.\n27. On page 22, what do the authors mean by \"subjected to AAA+ ATPase domain-focused masking\"? Are they referring to local refinement focused on the AAA region? The pipeline shown in Extended Figure 4 does not clarify the steps undertaken by the authors. One of the initial steps states \"masked on AAA+ ATPase domain,\" and then four classes (#0, #5, #6, #19) from the previous (unmasked?) classification are presented. We suggest the authors redraw their data processing pipeline, showing all the maps for the same classification side by side. They should then indicate by separate outlines which classes were used for further data processing, similar to what they did in Figure S7.\n28. On the same page, the authors state, \"Notably, 63,758 particles selected through RPN1 region masking from the proteasome particle pool produced a high-resolution RPN1 subunit structure\". However, the data processing pipeline (Extended Data Fig. 2) shows that the particles from class 0 and class 1 were pulled together, not \"selected through RPN1 region masking.\"\n29. On page 23, the authors mention \"high-resolution 3D classification.\" However, the representative maps for these classes appear to be of lower resolution compared to those from other 3D classification jobs shown. How does this high-resolution classification differ from the other 3D classifications that the authors do not specify as \"high-resolution\"? Moreover, 3D classification was performer generating far two many classes based on numer of particles available (e.g. 160k particles into 20 classes -line538). It seems that the particles just got distributed equally in the various classes, which indicates that the 3D classification didn’t really work.\n30. For symmetry expansion, particles should first be refined with C2 symmetry applied. Authors should specify the symmetry of their refinement for transparency and reproducibility of the analysis.\n31. There is no description for the processing of \"MIDN-EGR1-human 26S 1068 proteasome complex for the resolved Catch domain\" shown in Extended Fig. 8.\n32. The description of the ITC and SPR experiments reads more like a general overview of the protocol and lacks the specific details necessary to assess and reproduce the results. For the ITC experiments, the authors do not specify whether a blank titration was performed to determine the nonspecific heats of mixing, dilution, and any potential buffer mismatch. Therefore, in addition to clarifying this in the methods section, the authors should provide reviewers with the raw titration curves from the actual experiments, as well as the results from the blank experiments.\n33. In the description of SPR measurements, there is no information about the concentration of the proteins used for the experiment or the amount of protein immobilized on the sensor. Were any negative controls included to test for nonspecific binding?\n34. Authors should use black mesh or another more contrasting color combination, as it is almost impossible to glean the density details from Extended Data Fig. 4 in its current form.\n35. NLS (same as all other abbreviations used) should be explained to facilitate understanding of the text\n36. For the sake of accuracy, the color scalebar in Fig.2a should include units (e.g., kT/e)\nFigures:\nFigure 2 : the circle in figure2b is barely visible. How does this arrangement compare with the published structure? The measurements in the ITC panel (d,e) should have included full length MIDN and negative controls. Moreover more than 2 measurements should have been performed.\nFigure 4b is based on image processing. How many particles were thrown away? And why not reporting these numbers for all the other maps as well?\nFigure 5f is very confusing.\nFigure 6b is almost copied from Arkinson et al., 2024 and yet the paper is not even cited.\nThe manuscript requires careful proofreading to correct numerous typos, grammatical errors and to improve the overall readability of the text.\nWe hope that these comments will help the authors to improve the manuscript.", "reviewer_2": "The manuscript under review provides interesting and novel insights into the mechanism by which midnolin targets proteins to the proteasome for degradation, and more broadly into the proteasome's mechanism of action.\nMidnolin was recently discovered as a novel mediator of ubiquitin-independent proteasomal degradation. It was found to target products of immediate early genes for degradation by binding simultaneously to target proteins and the proteasome, shuttling the former for degradation without the need for ubiquitination. Initial studies suggested that proteasome binding occurs by an N-terminal domain and the C-terminal UBL domain, with the central Catch domain binding substrates.\nThe study under review here puts this model on solid footing by revealing a series of cryo-EM structures of the tertiary complex between substrate, midnolin, and the proteasome. I cannot assess the technical quality of the cryo-EM structure determination, but taken at face value, the structures provide valuable insights. The binding mode between midnolin and the proteasome is quite novel, including the detailed insights into the interaction between the N-terminal helix and Rpn1 and that of the C-terminal UBL domain with Rpn11. Pinning the N- and C-terminal domains of midnolin to the proteasome then allows midnolin's central catch domain to position bound substrate at the entrance to the degradation channel to be engaged by the proteasome.\nThe study caught the proteasome in a series of different conformations that are likely to represent different steps in the degradation cycle. Analysis of these structures suggests that Mg++ dissociation drives (together with ATP-hydrolysis) the proteasome through the conformational cycles that lead to substrate unfolding and translocation to the proteolytic sites. As far as I'm aware, the suggested role of Mg++ in the mechanism is novel and important.\nThere are a couple of conclusions in the manuscript that are not properly supported by the data presented, interesting as they are. I recommend that these parts of the manuscript be revised to indicate the speculative nature of the conclusions. For example, I think that the fact that midnolin is difficult to purify and aggregates when not co-expressed with a substrate can have many other interpretations - most simply that some scaffolding factor is missing at the right stoichiometry in the experimental setup. Similarly, the suggestion that the proteasome can digest midnolin by engaging the protein at its IDR2 is interesting and even plausible, but I propose that the autodegradation of midnolin as a regulatory mechanism is discussed as a hypothetical to be explored in the future.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "Proteasome is the main degradation machine in eukaryotic cells. While ubiquitin-dependent degradation is well studied, the non-canonical degradation mechanism mediated by midnolin remains elusive. In this study, Zhu et al. investigates the molecular mechanisms of the midnolin in targeting substrates to the proteasome for degradation. The authors combine structural and biochemical analyses to characterize midnolin’s interactions with the proteasome, revealing how its αHelix-C and Ubl domains bind to proteasomal subunits RPN1 and RPN11 respectively, to direct substrates to the proteasome entry port via the Catch domain. The authors captured four continuous states of the midnolin-proteasome complex, revealing the functional dynamics of the proteasomal ATPase complex, and a novel role of Mg2+ dissociation in coordinated mechanochemical coupling. Overall, this study presents compelling structural evidence and valuable insights into the mechanism of midnolin in mediating non-ubiquitination-mediated degradation. However, the current study relies heavily on static structural observations, and it would be important to complement these with functional validation such as kinetic assays of substrate recognition and degradation, together with mutational analyses of key midnolin domains, as detailed below.\nQuestions:\n1. Lines 100-101: There is grammar issue of the sentence.\n2. It is interesting that co-expression of Catch domain and EGR1 stabilizes the Catch domain and facilitates their binding. However, this may reflect artifacts arising from using the isolated Catch domain. To clarify this, it would be important to perform the gel filtration assays with full-length midnolin with or without co-expression of EGR1.\nThe proposed working model that midnolin co-expresses with substrates to achieve rapid targeted degradation raises questions about substrate specificity. With elevated midnolin levels, how is selectivity maintained, and could this result in unintended degradation of non-target substrates?\nA minor point: Extended Data Fig. 6g is not cited in the text.\n3. In the introduction, the authors raise a question of whether midnolin first binds its substrates prior to proteasome association or constitutively engages the proteasome before recruiting substrates. This key question is not sufficiently addressed in the current work. The structural characterization of midnolin-substrate-26S proteasome complex and co-expression assays do not rule out the possibility that midnolin engages the proteasome before substrate loading.\n4. The functional importance of different midnolin domains in proteasome targeting and degradation requires experimental validations. Specifically, while Fig.2 shows the dynamic binding of αHelix-C of midnolin to RPN1, how this interaction could be strengthened by the binding of Ubl domain to the RPN11, or antagonized by the potential interaction between Ubl and RPN1 remains unclear. Structure-based site-directed mutagenesis of these domains followed by functional readouts such as binding kinetics and degradation rates would help disentangle their functional contributions to proteasome targeting.\n5. The Catch domain might allosterically regulate proteasome states but the current experiments and results in Fig. 4 do not separate this effect from substrate-induced activation. This issue could be addressed by perturbing the substrate recognition site of Catch domain. In addition, structural analysis of substrate-free midnolin-proteasome complexes within the existing datasets may provide useful insight.\n6. Line 209: Extended Data Fig. 11 should be changed to Fig. 10.\n7. The legend of Extended Data Fig.4 should be revised with further clarification."}, "author_response": "Point-by-point Responses\nStructural dynamics of the midnolin–proteasome during ubiquitin-independent\nsubstrate turnover\" (NCOMMS-25-69834-T)\nPoint-by-point responses to the comments by Reviewer #1:\nIn this paper ‘Structural dynamics of the midnolin–proteasome during ubiquitin-\nindependent substrate turnover’, Zhu, Qin et al. investigate how midnolin (MIDN)\nmediates ubiquitin-independent proteasomal degradation. By combining cryo-electron\nmicroscopy (cryo-EM) with biochemical data, they reveal the mechanisms of the\ninteraction between MIDN and the 26S proteasome, and proposing the role of the\nindividual MIDN domains in substrate degradation. The work includes extensive\nstructural work which offers various snapshots of the MIDN-proteasome interaction\nand of the substrate-proteasome interaction. Overall, this paper contains valuable\nfindings that will interest the proteasome research community. However, in light of the\nrecently reported structures of the midnolin–proteasome complex (Nardone et al.,\n2025), the novelty of the work presented in the manuscript is limited. This wouln’t be\nper se a problem of the authors had actually solid evidence for the two mechanisms that\nthey try to elucidate: (i) the role of the Catch domain in correctly placing the substrate\nfor degradation and (ii) whether MIDN binds to the substrate prior to proteasome\nassociation. The experimental results are poorly described in a disordered way, jumping\nfrom one description to the next without a clear logic. At part sit is not even clear what\nexperiments the authors have performed until one reads the methods parts. Moreover,\nseveral critical pieces of information are missing throughout the manuscript, and the\nwork requires major revisions before it can be considered for publication.\nSome more detailed comments below:\nThe abstract should be self-standing and understandable, instead it includes many\nwords of proteins and domains not explained at all (etc. midnolin, Catch).\nThe introduction contains many grammatical mistakes that even hinder the reading at\nparts.\n1. In line 48, the authors stats that the identification of the MIDN pathway disrupts the.\nestablished Ub-dependent pathway. To use the word disrupt is incorrect as the ubiquitin-\nindependent path was already known. The new MIDN pathway adds up to the\ncomplexity of the system. We didn’t like the use of the word disrupt because it is an\nexample of how language is used all along the text to boost the presented data at\nexpenses of honest description of the scientific knowledge at the moment. Other\nexamples of such usage are the words ‘unprecedented’ (line 77).\nAnswer: Thank you for your thorough and professional review of our manuscript and\nfor your invaluable suggestions on language usage. Your comments on the usage of\n\"disrupt\" and \"unprecedented\" were particularly insightful, and we fully agree. In the\nrevised version we have changed \"disrupts this established view” to the more neutral\nwording “expands this established view by revealing…\".\nWe have now replaced the term \"unprecedented\" with the more specific and objective\ndescription “revealed mechanistic details\".\n2. The naming of the domains changes multiple times in the text, sometime the C-\nterminus of MIDN is called αHelix-C, some others C-terminal α-helix. The authors\nshould be more consistent in the use of the words.\nAnswer: Thank you for carefully reviewing our manuscript and pointing this out. To\nmaintain clarity and consistency throughout the text, we used \"αHelix-C\" as the name\nfor this domain.\n3. Cryo-EM structures are presented in a very disordered way, which makes the reading\nquite complicated. They are overall ok, but very little has been done with\npostprocessing to resolve better or at least visualise better the less-resolved MIDN parts.\nWe suggest the authors to try different sharpening tools. As they are shown now the\nmaps appear all oversharpened, we assume that only overall B-factor sharpening has\nbeen applied.\nAnswer: Thank you for your expert review of the cryo-EM structures and for the\nconstructive suggestions. We agree that the presentation could be clearer and that the\npost-processing of the lower-resolution MIDN regions requires improvement. As\nrecommended, we have moved beyond global B-factor sharpening and tested multiple\nsharpening approaches. Specifically, we applied EMReady, a tool designed to enhance\nfeatures in lower-resolution areas while minimizing over-sharpening artifacts. These\nadjustments have improved map interpretability.\nA detailed description of all post-processing workflows has been provided in the revised\n\"Methods\" section to ensure reproducibility. We sincerely appreciate these valuable\ntechnical comments.\n4. In line 102, the authors state: \"These structures are mainly in substrate processing\nstates that resemble the ED2 state (Fig. 1a and Extended Data Figs. 2, 3 ...).\" However,\nit is unclear why Extended Data Figs. 2 and 3 are referenced here, as they do not\ndemonstrate that the structures are primarily in the substrate processing state. A better\ncomparison with the reported structures should be conducted to confirm the ED2\nconformation of the observed complex. Additionally, the use of the term \"mainly\" is\nsomewhat arbitrary; a more quantitative description would be beneficial. Overall, the\npaper would benefit from a more thorough comparison of the observed conformations\nof the 26S proteasome with previously published structures of different states of the\ncomplex. Citations should be always included e.g. in line 121 after the sentence\n‘different from previous studies’.\nWe thank the reviewer for these valuable comments on this section. We have changed\nthe paragraph as follow.\nPage5; Line 98-105\n\" After 3D classification, we determined four distinct structural states (designated\nMIDN-PS , -PS , -PS , and -PS for subsequent discussion). Among these\nRPT1 RPT5 RPT2 RPT6\nstates, the RPN1 subunit exhibited varying degrees of resolution and occupied distinct\npositions relative to the AAA+ motor (Extended Data Fig. 2, 3 and Supplementary\nTable 1). The 3D classification yielded structural states that all represent substrate-\nprocessing conformations. This includes classes reconstructed from particles that were\nnot assigned to a dominant state but still contributed to the overall conformational\nlandscape. The highest resolution (2.87 Å) was achieved for the MIDN-PS state\nRPT1\n(Fig. 1a). \"\nWe fully agree with the reviewer's recommendation to conduct a more comprehensive\ncomparison with previously reported proteasomal conformations. This is indeed crucial\nfor better contextualizing the novelty of our findings. In the revised manuscript, we\nadded a supplementary table that describes the diverse proteasome conformations\ndocumented in the literature (Structural landscape of the degrading 26S proteasome reveals\nconformation-specific binding of TXNL1, https://doi.org/10.1038/s41594-025-01695-2). This\ndetailed comparison help clarify the conformational cycle of the AAA+ motor during\nmidnolin-mediated, ubiquitin-independent degradation is in consistent with the\nestablished universal principles.\nSupplementary Table 6 | Summary of key structural features of TXNL1-bound\nproteasome states\nPS\nRPT6\nPS (9E8O) PS (9E8J) PS (9E8G)\nRPT2 RPT1 RPT5\n(9E8K)\nRPT6 RPT2 RPT1 RPT5\nRPT3 RPT6 RPT2 RPT1\nPore-loop staircase contacting\nRPT4 RPT3 RPT6 RPT2\nsubstrate (from top to bottom)\nRPT5 RPT4 RPT3 RPT6\nRPT1 RPT5 RPT4 RPT3\nRPT subunits disengaged from\nRPT2 RPT1 RPT5 RPT4\npore loop staircase\nRPT2 RPT1\nRPT4\nRPT1 RPT5 RPT4\nRPT subunits with ADP RPT3\nRPT5 RPT4 RPT3\nRPT6\nRPT4 RPT3\nExchange compared to previous RPT1\nN/A RPT2 no\nstate RPT5\nHydrolysis compared to\nN/A RPT3 no RPT6\nprevious state\n5. In line 115, the authors state, \"Here, we have successfully captured the structures of\nthe natural endogenous substrate-bound 26S proteasome in action.\" Additionally, the\ndiscussion emphasizes that endogenous substrate-proteasome complexes used in the\nstudy, in contrast to other reported structures obtained with non-hydrolysable ATP\nanalogs or RPN11 inhibitors, do not promote specific conformational states. However,\nit has to be noted that the proteasomes were halted using MG132, which could also\nskew the distribution of different states. It has been shown by Haselbach et al.\n(https://doi.org/10.1038/ncomms15578) that inhibitors acting on the 20S core particle\ncan achieve a long-range allosteric effect that modulates the conformational landscape\nof the 19S particle. Authors should mention this when discussing the conformational\nlandscape of the complex.\nAnswer: Thank you for raising this critical point. We agree that while hydrolyzable ATP\nwas used to minimize bias, the essential use of MG132 to preserve the native complex\nduring purification may have allosterically influenced the 19S conformational\nlandscape, as demonstrated by Haselbach et al. (2018, Nature Communications)\nWe have revised the manuscript in two places: In the Results, we have added a\nclarifying note regarding MG132 usage when claiming capture of the “native” complex.\nIn the Discussion, we have added a dedicated paragraph to transparently discuss this\npotential limitation and cite Haselbach et al. (2018, Nature Communications).\nPage 6；Line 116-121\n\" In these reconstructions, we clearly resolved distinct densities corresponding to the\ninhibitor MG132. This enabled precise docking of the atomic model into the density\nmap, which show MG132 specifically binds to the catalytic β subunits (β1, β2, β5) of\nthe core particle (CP) (Extended Data Fig.4). We note that, as an active-site inhibitor,\nMG132 may allosterically influence the conformational landscape of the 19S\nregulatory particle17, a consideration relevant to the interpretation of the observed\nstates.\"\nPage 15-16；Line 351-363\n\"Consistent with the well-established asymmetric mechanism of the proteasomal\nAAA+ motor14,21, our structures capture consecutive spiral-staircase conformations\n(PS , PS , PS , PS ) during midnolin-mediated substrate translocation. This\nRPT6 RPT2 RPT1 RPT5\nconfirms that the hand-over-hand translocation mechanism is operational in this\nubiquitin-independent pathway. The specific distribution and progression of these\nstates in our dataset provide a detailed view of the mechanochemical cycle in the\ncontext of midnolin engagement, although the general principle of asymmetry is\npervasively established across AAA+ ATPases. However, it has to be noted that the\nproteasomes were halted using MG132, which could also skew the distribution of\ndifferent states. It has been shown by Haselbach et al.17 that inhibitors acting on the 20S\ncore particle can achieve a long-range allosteric effect that modulates the\nconformational landscape of the 19S particle. Future studies using alternative\nstabilization methods or time-resolved approaches will be valuable to elucidate the\nnative conformational dynamics of the midnolin-proteasome complex in the absence of\ncore particle inhibition.\"\n7. In line 126-129 when discussing the C-terminal α-helix, the authors do not refer at\nall to the Nardone et al., 2025 paper where this feature is also described.\nAnswer: Thank you for pointing out this important citation oversight. In the section\nwhere we describe the interaction between the C-terminal α-helix of midnolin (αHelix-\nC) and RPN1, we should reference and discuss the relevant studies by Nardone et al.\n(Mol Cell, 2025) and Peddada et al. (PNAS, 2025), which also characterized this\nstructural feature. We have now added these references in the revised manuscript.\nPage 6；Line 128-130\n\"This binding is primarily driven by a hydrophobic core and extensive electrostatic\ncomplementarity (Fig. 2a, b), a finding independently corroborated by concurrent\nstructural studies,12,13.\"\n8. The results explained in the paragraph ‘Uniform expression of midnolin is dependent\non substrate’ appears not very solid to us. It is unclear whether and how proteome\nanalysis was performed in in hepatocellular carcinoma and glioblastoma multiforme\ncells and by looking at the graphs in Extended data Fig.6f the correlation is not so tight.\nThat in vitro co-expression of a protein and its substrate stabilise the expression and\nfolding is not surprising, it is in fact a very common phenomenon.\nAnswer:\n(1) On the comment that \"co-expression stabilizing a protein is a common\nphenomenon\":\nWe fully agree with the reviewer. In vitro, co-expressing an interaction partner (e.g.,\nsubstrate EGR1) can significantly enhance the solubility and stability of an unstable\nprotein (e.g., the midnolin Catch domain), which is indeed a widespread practice in\nmolecular biology and biochemistry. We cited this result not to claim novelty but to\ndocument a crucial experimental observation: the co-expression strategy was necessary\nto obtain a functional, stable midnolin-substrate complex suitable for subsequent\nbiochemical and structural studies. This indirectly hints at the functional coupling\nbetween midnolin and its substrate.\nAccordingly, we have revised the statement to: \"For subsequent biochemical and\nstructural studies, a co-expression strategy was required to obtain a functional, stable\nmidnolin-substrate complex suitable for in vitro assays.\"\n(2) Regarding the correlation analysis in Extended Data Fig. 6f and its data source:\nThe analysis mentioned in the figure legend was based on the public transcriptomic\ndatabase GEPIA, which integrates RNA-seq data from projects like TCGA and GTEx.\nWe used gene expression data (in TPM values) from hepatocellular carcinoma and\nglioblastoma samples within this database, not a proteomic analysis performed by\nourselves. We clarified this point more explicitly in the revised figure legend and main\ntext to prevent misunderstanding.\nWe understand and agree that the correlation shown (R ~ 0.3) is moderate, not strong.\nOur intent was not to present it as definitive causal evidence but as a supportive\nbioinformatic observation. It indicates a statistically significant positive trend (p < 1e-\n5) between MIDN and its substrate EGR1 mRNA levels in actual tumor samples, which\nis directionally consistent with our in vitro observation of \"co-expression promoting\nstability/function.\" We have adjusted the text to be more precise, changing phrases like\n\"supports our observation\" to \"is consistent with the trend observed in our biochemical\nassays\"\nPage 8-9;Line 184-190\n\" Additionally, the bioinformatic analysis (Extended Data Fig. 6f) was performed using\nthe public transcriptomic database GEPIA22, which integrates RNA-seq data from\nprojects such as TCGA and GTEx. We directly extracted gene expression values (TPM)\nfor hepatocellular carcinoma and glioblastoma multiforme samples. The results showed\nthat MIDN mRNA levels are significantly and positively correlated with those of its\nsubstrate EGR1 in real tumor tissues (p < 1e-5), consistent with the trend observed in\nour biochemical assays. These findings indicate that midnolin may preferentially co-\nexpress with its substrates to achieve optimal activity. \"\n9. The paragraph regarding positioning of the substrate by the Ubl and Catch domains\nwould benefit from a figure showing a cut-through of the map and snapshots of the\ndifferent positions of the substrate depending on the positioning of the Catch domain.\nMoreover, why would a Cter substrate position of that type optimal for degradation?\nAnswer: Regarding the suggestion to add a cut-through view and substrate snapshots,\nwe agree that this would be highly beneficial. While our current overall map shows the\nCatch domain and substrate density positioned above the entry port, it does not\nintuitively convey the substrate's precise spatial trajectory relative to the proteasomal\nentry port.\nIn the revised manuscript, we have addressed this by adding a new panel to Figure 3.\nThis panel features a cut-through view of the cryo-EM map along the central channel\naxis. This visualization clearly illustrates how the density for the Catch domain–\nsubstrate complex is positioned directly above and aligned with the N-terminal (OB\nring) entrance of the AAA+ ATPase ring, providing an unambiguous depiction of the\nsubstrate's path into the proteasome.\nWe suppose that the substrate is more susceptible to degradation from its C-terminus,\nprimarily because the Catch domain precisely positions the substrate's C-terminus at\nthe proteasome entry port. However, this model warrants further investigation.\nRevised Figure 3d. A cut-through of the cryo-EM map of the midnolin/substrate density\nin the central channel, just reaching the ATPase ring.\n10. In line 182, the authors mention, \"Rigid-body docking of the predicted Catch–EGR1\nmodel into the refined map yielded a strong fit, allowing unambiguous placement of\nthe Catch domain (Fig. 3d).\" To strengthen this claim, the authors should provide a\nclose-up view of the model in relation to the map, supported by the Q score and the\ncorrelation coefficient between the model and the map.\nAnswer: We thank the reviewer for this constructive suggestion to strengthen the\nvalidation of our model placement for the Catch–EGR1 module.\nWe have provided a new, detailed close-up view in Extended Data Fig. 8a, which clearly\nshows the fitted Catch–EGR1 model within the corresponding cryo-EM density.\nWe now report the Q-score and the correlation coefficient (CC) calculated specifically for\nthe Catch–EGR1 region within the refined map (Extended Data Fig. 8b-e). Importantly,\nbeyond the rigid-body docking metrics, our refined map reveals continuous density\nconnecting the Ubl and Catch domains. This unambiguous structural feature provides\nindependent and direct validation for the correct spatial placement and trajectory of the\nCatch domain relative to the anchored Ubl domain, which is a key pillar supporting our\nmodel.\nWe note that the density for the substrate portion bound by the Catch domain is weaker\nand partially fragmented. This is consistent with the expected higher conformational\ndynamics of a substrate in the process of being engaged and translocated, and it does\nnot detract from the unambiguous fit of the Catch domain itself to its well-defined\ndensity, as now supported by the quantitative metrics and the inter-domain connectivity\ndescribed above.\nWe believe these additions provide the requested objective validation and significantly\nstrengthen the claim regarding the unambiguous placement of the Catch domain.\nRevised Extended Data Figure 8. Local quality of model fitting to cryo-EM density\nmaps. (a) close-up view of the model in relation to the map for MIDN and EGR1. (b-c)\nFitted models were analyzed with PHENIX and their per-residue correlation\ncoefficients (CC) are plotted, b for MIDN and c for EGR1. (d-e) Q-score plot of MIDN\n(d) and EGR1 (e) chains. The expected Q-score at 4.97 Å resolution is shown as red\ndashed lines. The 5-residue average of Q-scores is plotted here.\n11. In line 186, the authors wrote: \"Collectively, our data capture two states: a high-\nresolution Ubl-bound state (RPN11 engagement) and a state in which both the Ubl and\nCatch–substrate modules are simultaneously engaged with the 26S proteasome.\"\nHowever, due to the poor clarity of the cryoEM data processing and numerous final\nmaps shown in the data processing pipelines that are not well annotated or described in\nthe manuscript, it is not evident to which maps the authors refer when saying the \"two\nstates\".\nAnswer: Thank you for this important point. We agree that the link between the two\nfunctional states and the corresponding cryo-EM maps was not sufficiently clear. To\naddress this, we have systematically reorganized and annotated the data processing\nworkflows (now presented in revised Extended Data Figure 7). This revision explicitly\nlinks each final map to its respective functional state, ensuring full traceability and\nimproved clarity of our structural assignments.\n12. The results described on pages 9-10 suggest that the MIND's Catch domain is\ncrucial for stabilizing the complex. In the cryo-EM dataset of the proteasome\nreconstituted with a Catch-deficient protein, a significant fraction of the 26S particles\nwas found in the MIND-free resting state. However, when processing the 26S-MIND\nFL dataset, the authors did not investigate the presence of the resting state, which\nweakens their conclusion.\nWere there any other states, aside from the extended state (ED), observed in the\nendogenous 26S-MIND FL or MIDN-EGR1-human datasets? If so, what were the\npopulations of these different states? Looking at the data processing pipeline of the 26S-\nMIND FL, a large proportion of the particles was rejected during the ab initio and 3D\nclassification jobs. The ab initio maps were not shown, making it impossible to\nunderstand why those particles were removed and what they represent (whether they\nwere low-quality data or different conformations).\nIn the second 3D classification, some of the maps (e.g., classes 12 and 13) appear to\nshow a good amount of detail but were still rejected by the authors. It would be\ninteresting to investigate whether other conformational states were discarded alongside\nthose particles.\nIn conclusion, if the authors intend to use the presence of the resting state in the\nMIDN∆(112-336)-proteasome dataset as evidence for the role of the Catch domain,\nthey should conduct a more thorough analysis of the other datasets to check for the\nexistence of the resting state in them.\nAnswer: Thank you for this critical comment. We agree that a comprehensive analysis\nof all datasets is essential to support the conclusion regarding the Catch domain's role.\nIn response, we have now systematically re-analyzed the conformational landscapes of\nthe full-length MIDN–proteasome datasets using the same classification strategy. The\nresults confirm that the resting state (RS) was not populated in these native complexes,\nin contrast to its significant presence in the MIDNΔCatch/MIDNΔ(112–336) dataset.\nTo ensure transparency, we have provided a detailed population analysis of all observed\nstates across the full-length MIDN–proteasome datasets in the revised Results section.\nPage11; Line 241-245\n\"Compared with full-length MIDN purified complexes, the MIDNΔ(112–336)–\nproteasome sample lacking the Catch domain exhibits a significantly higher proportion\nof particles in the substrate-free resting state (RS). This comparison indicates that the\nCatch domain, together with substrate binding, plays a pivotal role in stabilizing the\nMIDN–proteasome complex and maintaining it in an active, substrate-processing\nstate.\"\n13. It is not apparent which maps from Extended Data Fig.2 are those shown in Fig. 1\nand in Extended Data Fig.4. To which map are the authors referring in the figure caption\nwhen mentioning ED2-like state? The annotations should be specific and consistent\nacross the manuscript to ensure that the reader can easily associate the presented results\nwith the correct maps.\nAnswer: We sincerely appreciate your important comments regarding figure labeling\nand terminology consistency. Your observations are accurate: the lack of clear\ncorrespondence among figures, main text, and figure legends, as well as the nonspecific\nuse of terms such as \"ED2-like state,\" can indeed confuse readers and hinder\ncomprehension of our findings. To prevent misinterpretation, we have replaced \"ED2-\nlike state\" with \"processing state.\" We fully agree that clear labeling and cross-\nreferencing are fundamental to readability and scientific rigor. Following your\nsuggestions, we have performed a comprehensive, systematic revision of all labels in\nthe figures and text.\n14. Maps presented in Figures 1-4 appear to be slightly oversharpened. The specific\nsharpening parameters are not currently outlined in the Materials and Methods section.\nThis information should be included, the degree of sharpening should be carefully\nverified and various sharpening methods could and should be used.\nAnswer: Thank you for your expert review of the cryo-EM structures and for the\nconstructive suggestions. We agree that the presentation could be clearer and that the\npost-processing of the lower-resolution MIDN regions requires improvement. As\nrecommended, we have moved beyond global B-factor sharpening and tested multiple\nsharpening approaches. Specifically, we applied EMReady, a tool designed to enhance\nfeatures in lower-resolution areas while minimizing over-sharpening artifacts. These\nadjustments have improved map interpretability. A detailed description of all post-\nprocessing workflows has been provided in the revised \"Methods\" section to ensure\nreproducibility.\n15. Authors revealed the Helix-C of midnolin interaction with RPN1 (PSMD2). It\nwould be interesting to see a side-by-side comparison of the Helix-C interaction\ninterface (at the residue level) with the other RPN1 T1/T2 interactions (like Usp14 or\nUb). Moreover, view that then they use AlphaFold3 to predict the interaction between\nMIDN and proteasome, it would be interesting to know if this interaction was predicted\nand to what confidence level.\nAnswer: We appreciate the excellent suggestion to conduct a comparative analysis of\nthe αHelix-C–RPN1 interaction interface. This comparison has effectively highlighted\nthe similarities and distinctions between our identified binding mode and canonical T2-\nsite binders such as USP14 and NUB1.\nWe have added a dedicated panel in the revised manuscript (in Fig. 2d, e). This new\npanel features a structural comparison among: (1) our resolved αHelix-C–RPN1\ncomplex, (2) the previously reported USP14–RPN1 complex (PDB: 7W3H), and (3)\nthe NUB1–RPN1 complex (PDB: 8USB). Our analysis reveals that all three complexes\nshare a common hydrophobic core anchored by RPN1 residues L426 and L465. They\nare further stabilized by peripheral salt bridges and hydrogen bonds. A key distinction,\nhowever, lies in the fact that our αHelix-C engages RPN1 with a more extensive\ninteraction interface and adopts a distinct linear conformation.\nRevised Figure 2. (b) The interaction interface exhibits a tripartite pattern between\naHelix-C of midnolin and RPN1. The circle part indicates a hydrophobic center. This\ncentral hydrophobic interface is flanked by two polar interaction networks. (c)\nSuperimposition of the atomic models depicting the interaction interface between\nUSP14(blue) and RPN1(pink) versus the interaction interface between RPN1 and\naHelix-C of midnolin (magenta). The T2 site is indicated by a red dashed circle. (d)\nSide-chain interactions between the USP14 UBL domain (blue) and the RPN1 (pink)\nT2 site, derived from the USP14–RPN1 complex (PDB: 7W3H). The T2 site is\nindicated by a red dashed circle. (e) Side-chain interactions between the NUB1 UBL\ndomain (yellow) and the RPN1 (pink) T2 site, derived from the NUB1–RPN1 complex\n(PDB: 8USB).\nFurthermore, we note that AlphaFold3 models, independently predicted the αHelix-C–\nRPN1 interface. Crucially, the predicted binding mode, including the critical\nhydrophobic and polar interaction network, is in excellent agreement with the\nexperimental structure we ultimately determined by cryo-EM.\n16. The use of AlphaFold and molecular dynamics is mentioned; however, the brief and\nvague description of the results makes it difficult to understand why these analyses were\nconducted in the first place and what insights they contribute to the overall narrative.\nThe respective description in the methods section is also insufficient; Uniprot sequence\nIDs used for predictions should be specified along with the AlphaFold prediction\nconfidence statistics. Moreover, in Extended Data Fig.9, the IDR2 and Ubl are\ndescribed as dark orange.\nAnswer: We thank the reviewer for these essential and constructive comments\nregarding the computational analyses and figure details, which have significantly\nimproved the clarity and rigor of our manuscript. We have addressed each point as\nfollows.\nWe have revised the text to explicitly state that the AlphaFold3 prediction was used as\nan independent, prior validation tool. Specifically, the model for the midnolin-RPN1-\nRPN11-RPT1-6 complex, accurately predicted the key αHelix-C–RPN1 interaction\ninterface. Considering the high accuracy of AlphaFold3 in predicting the structure of\nMIDN and proteasome, we generated the predicted midnolin-RPN1-RPN11-RPT1-6-substrate models to better visualize the potential substrate-binding mode.\nUpon careful consideration of the reviewer's implied point—and in the interest of\nmaintaining the highest rigor—we acknowledge that our MD simulations, conducted\non a partial 19S complex, may not fully represent the physiological context. Therefore,\nwe have removed the entire MD simulation section from the manuscript.\nWe have comprehensively revised the Methods section to include all requested details\nfor the AlphaFold3 prediction. The UniProt sequence ID are provided: RPT1 (P35998),\nRPT2 (P62191), RPT6 (P62195), RPT3 (P43686), RPT4 (P62333), RPT5 (P17980),\nRPN11 (O00487), RPN1 (Q13200), MIDN (Q504T8), EGR1 (P18146), IRF4\n(Q15306), FosB (P53539). Predicted structures of the complex and MIDN are colored\nby pLDDT in AlphaFold3 pattern.\nWe sincerely apologize for the error in color labeling. We have carefully corrected the\nlegend for Extended Data Fig. 9. It now accurately describes the two distinct colors\nused: dark orange for the Ubl domain and orange red for IDR2.\n17. In line 220, it states: \"This demonstrates that the observed presence of 26S\nproteasomes without midnolin is not due to asymmetric binding—where midnolin\nengages one 19S regulatory particle of a 30S proteasome while the other remains\nunbound. This suggests that in the absence of substrate engagement, midnolin\ndissociated during size-exclusion chromatography, consistent with the dynamic\nbinding-dissociation nature of midnolin-proteasome interactions we previously\nproposed.\" However, since a significant number of particles were discarded in other\ndatasets (in which MIND contained the Catch domain) without careful examination of\ntheir conformational and compositional heterogeneity (specifically the presence or\nabsence of MIND), it is challenging to unequivocally state whether the presence of\nMIND-free resting state complexes is unique to the cryo-EM sample prepared with the\nCatch-deficient MIND construct. Authors should verify the effect of the Catch domain\ndeletion and the presence of the substrate on the stability of the 26S-MIND complex\nusing biochemical and/or other biophysical assays. This could include techniques such\nas native PAGE with specific immunostaining or fluorescent tagging of the proteins of\ninterest. In addition, authors should use RP2-CP and RP-CP notation to differentiate\n30S from 26S complexes.\nAnswer: Thank you for these important comments. We have now performed a unified,\nbinding-state-focused analysis on the raw particle sets from all relevant datasets and\nstandardized the data-processing flowcharts accordingly. To directly assess complex\nstability, we also added enzymatic-activity assays comparing Wild-type full-length\nmidnolin [MBP-MIDN + EGR(101-200)] with its Catch-deletion counterpart [MIDN\nΔ(112-336)]; the full-length complex showed high activity, whereas the Catch-deletion\nmutant exhibited low activity, corroborating the particle statistics obtained by cryo-EM.\nRevised Figure 4. (g) In-vitro peptidase activity was monitored over 90 min using the\nfluorogenic substrate Suc-LLVY-AMC (10 µM) for the 26S proteasome alone (black),\nor with Catch-deleted mutant [MBP-MIDN∆(112-336)] (purple), with Catch-replaced\nmidnolin [MBP-MIDN(1-111)-EGR1(101-200)-MIDN(337-468)], with wild-type\nMBP-MIDN + EGR1(101-200) (red). The y-axis indicates relative fluorescence units\n(RFU). (h) Quantification of in-vitro peptidase activity results in (g). RFU changes\nbetween 90 min and 0 min are plotted. Bars represent means ± SD of three replicates\n(****, p <0.0001; n =3, p values were calculated using ANOVA with Tukey’s post test.).\nns means not significant.\nWe also appreciate your note on nomenclature. Throughout the revised manuscript we\nnow use \"RP₂-CP (30S)\" for the double-capped proteasome and \"RP-CP (26S) \" for the\nsingle-capped proteasome to ensure professional clarity.\n18. In line 214, it states: ‘we found that the substrate in the processing state structurally\nresembles midnolin itself’ suggesting that MIDN gets degraded. Is there any\nbiochemical evidence for it? In such case a citation of the relevant literature should be\nprovided.\nAnswer: Following your suggestion, we have revised the relevant statement and added\nappropriate citations to make this important conclusion more rigorous and credible.\nYour request for literature support is fully justified. Indeed, the seminal study that first\nreported the midnolin–proteasome pathway (Gu et al., Science 2023) already provided\nbiochemical evidence that midnolin itself is degraded. For example, cycloheximide-\nchase assays in that paper showed that midnolin protein levels decline rapidly after\nsubstrate delivery, and this degradation depends on its ahelix-C and proteasome activity.\nOur biochemical assays confirm this observation, providing independent validation.\nOur structural data, which directly captures midnolin as a proteasome substrate, not\nonly stands as independent evidence but also provides a mechanistic and visual\nexplanation for the prior biochemical observation of midnolin’s inherent instability and\nproteasome sensitivity. Together, these evidences establish a “self-sacrificing”\ndegradation mode for midnolin. We have now inserted the following citation adjacent\nto the sentence that mentions midnolin’s possible self-degradation.\nPage 11; Line 248-249\n\"This structural observation is consistent with previous biochemical evidence\ndemonstrating that midnolin undergoes proteasomal degradation after substrate\ndelivery3.\"\nTo clarify the core function of the Catch domain, we engineered a \"Catch-functional\nreplacement\" construct in which the known substrate fragment EGR1(101–200) was\nfused, via a flexible linker, between the Ubl domain and α-Helix-C of midnolin,\ncompletely replacing the native Catch domain. This chimera retains substrate-binding\ncapacity (because the substrate is covalently \"pre-bound\") yet lacks the natural Catch\ndomain and any specific spatial interface it might provide. In essence, we stripped the\n\"substrate-binding\" role from the wild-type Catch domain and supplied it in a simple,\nconstitutive manner. If the Catch domain were solely responsible for substrate binding,\nthe replacement construct should degrade the substrate as efficiently as the wild-type\nprotein, since the substrate has already been \"pre-delivered. \" Conversely, if the Catch\ndomain additionally performs a \"positioning\" or “optimized delivery” function—e.g.,\nby contacting the proteasome entry region to guide the substrate terminus into an\noptimal location—then the replacement construct, although substrate-bound, should\nexhibit markedly lower degradation efficiency, resembling or only slightly exceeding\nthat of the Catch-deletion mutant. The results of enzymatic assays revealed that the\ndegradation activity of the full-length midnolin–substrate–proteasome complex is\nsignificantly higher than that of the Catch-deletion mutant and Catch-functional\nreplacement, confirming the essential contribution of the Catch domain to the\nfunctional stability of the complex. Our enzymatic data demonstrate that the Catch\ndomain executes a specific role beyond mere substrate binding, further supporting the\nhypothesis that it facilitates delivery of the substrate to the proteasomal entrance.\nFigure 4(g) In-vitro peptidase activity was monitored over 90 min using the fluorogenic\nsubstrate Suc-LLVY-AMC (10 µM) for the 26S proteasome alone (black), or with\nCatch-deleted mutant [MBP-MIDN∆(112-336)] (purple), with Catch-replaced\nmidnolin [MBP-MIDN(1-111)-EGR1(101-200)-MIDN(337-468)], with wild-type\nMBP-MIDN + EGR1(101-200) (red). The y-axis indicates relative fluorescence units\n(RFU). (h) Quantification of in-vitro peptidase activity results in (g). RFU changes\nbetween 90 min and 0 min are plotted. Bars represent means ± SD of three replicates\n(****, p <0.0001; n =3, p values were calculated using ANOVA with Tukey’s post test.).\nns means not significant.\nPage11-12; Line 251-266\n\" To clarify the core function of the Catch domain, we engineered a \"Catch-replaced\nmidnolin\" construct MBP-MIDN(1-111)-EGR1(101-200)-MIDN(337-468),\nwhere the\nnative Catch domain is replaced by the known substrate fragment EGR1(101-200),\ncovalently tethered via a flexible linker. This chimera retains constitutive substrate\nbinding but lacks the native Catch interface. We then compared the stimulated\nproteasome activity of three proteins: wild-type full-length midnolin [MBP-MIDN +\nEGR1(101-200)], Catch-deleted mutant [MIDNΔ(112-336)], Catch-replaced midnolin.\nIf the role of the Catch domain is to bind substrate, the Catch-replaced midnolin should\nstimulate proteasome activity as efficiently as wild-type because the substrate is already\neffectively “provided”. Conversely, if the Catch domain possesses an additional\n“positioning” or “optimized delivery” function, then the Catch-replaced midnolin\nshould show markedly lower stimulated efficiency, resembling or only slightly\nexceeding that of Catch-deletion mutant. The results show that the Catch-replaced\nmidnolin, despite having the substrate pre-bound, showed a significantly lower\ndegradation efficiency than to the wild type, much closer to the Catch-deleted mutant\nMIDNΔ(112-336). These results demonstrate that the Catch domain provides a specific\nfunction beyond simple substrate binding, consistent with its proposed role in actively\npositioning the substrate for optimal delivery to the proteasome.\"\nReferences\n[3] Gu, X. et al. The midnolin-proteasome pathway catches proteins for ubiquitination-\nindependent degradation. Science 381, eadh5021, doi:10.1126/science.adh5021 (2023).\n19. On page 11, there is no description of the various states relaying completely on the\nreader's good knowledge of the proteasome. Moreover no comparison is give side by\nside to other published structures. This makes the manuscritp difficult to read. Moreover,\nthe states described as well as the whole Figure 6b is extremely reminescent of the\npaper by Arkinson et al., 2004 (https://doi.org/10.1101/2024.11.08.622731) and this\npaper is not even cited. We also find the proposed model of movement associated to\nMg2+ release not very sound considering that the movement described is of 2.3Å so\nway below the overall resolution of the map (3Å at best).\nThe discussion part is again missing citations. More in details:\nWe agree that the original description relied too heavily on the reader's specialized\nknowledge. We have added the references, including the paper by Arkinson et al., 2025\n(https://doi.org/10.1038/s41594-025-01695-2). In the revised manuscript, we have\nadded a concise introductory paragraph as follow:\nPage 12; Line 272-280\n\" Our current understanding of proteasomal translocation comes from cryo-EM studies\nof 26S proteasomes with substrates in processing states. These studies revealed that the\nAAA+ motor (RPT1–6) adopts right-handed spiral-staircase conformations, with\ntypically 4–5 RPT subunits engaging the substrate via their pore-1 loops and 1–2\nsubunits (seam subunits) positioned off the substrate22. These observations led to the\n‘hand-over-hand’ translocation model, wherein the sequential ATP hydrolysis cycle\ncoordinates the movement of the seam subunit to drive stepwise substrate translocation.\nOur cryo-EM structures capture the midnolin-proteasome complex within this dynamic\ncycle, providing snapshots of specific substrate-engaged translocation intermediates.14-\n16,23. \"\nWe have added the references, including the paper by Arkinson et al., 2025\n(https://doi.org/10.1038/s41594-025-01695-2)\nWe agree with the reviewer that attributing the subtle (~2.3 Å) movement specifically\nto Mg²⁺ release is not robust given the ~3 Å global resolution. We have revised the\nsentence \"Mg²⁺ dissociation actively drives substrate translocation through coordinated\nmechanochemical coupling\" as follow:\nPage 13; Line 293-296\n\" Collectively, these four sequential states provide a structural visualization of the hand-\nover-hand cycle14-16 and indicate a potential regulatory role for Mg²⁺ release in\ncoordinating the sequential rearrangements of the ATPase subunits.\"\n20. In line 307, the authors state: \"Additionally, the IDR2 region of the Catch domain\nguides midnolin itself into the proteasome for degradation after substrate delivery.\" Do\nauthors have any biochemical data confirming this?\nOur structural and biochemical data indicate that the presence of the N-terminal\ndisordered region (IDR1) leads to the degradation of MIDN (Fig. 4c-g). Therefore, we\ncorrected this sentence as \"Additionally, the N-terminal disordered region (IDR1) \nregion may guide midnolin itself into the proteasome for degradation after substrate\ndelivery. \"\n21. In line 313, it states: ‘Our findings indicate that ATP hydrolysis plays a pivotal role\nin the displacement of the proteasome's AAA+ module, with Mg²⁺ dissociation also\nactively facilitating substrate translocation (Fig. 6b)’. This is quite misleading as it is\nbased on observation of motion below the actual resolution limit of the data.\nWe sincerely thank the reviewer for raising this critical point regarding the\ninterpretation of our structural data. The reviewer is correct: attributing the observed\nsubtle conformational shifts (~2.3 Å) near the global resolution limit (~3 Å) specifically\nto Mg²⁺ dissociation and describing it as \"actively facilitating\" translocation is\nstatistically tenuous and may overstate the mechanistic evidence available from the\ncurrent dataset. We apologize for this overinterpretation in our original wording.\nWe have revised the manuscript to ensure our conclusions are framed with appropriate\ncaution as follows:\nPage13; Line 288-296\n\" A notable transition occurs between MIDN-PS and MIDN-PS . In MIDN-\nRPT1 RPT5\nPS , RPT3 retains a bound Mg²⁺ ion, while in MIDN-PS , this Mg²⁺ is released\nRPT1 RPT5\n(Fig. 5d, e). Concomitantly, the entire AAA+ ring undergoes a ~2.4 Å downward rigid-\nbody shift (Fig. 5f). The spatiotemporal correlation between Mg²⁺ release and ring\nmovement suggests that Mg²⁺ dissociation from RPT3 may act as a coordinated trigger,\nworking in concert with ATP hydrolysis to facilitate the stepwise substrate translocation\nobserved in our structural continuum (Movie S1). Collectively, these four sequential\nstates provide a structural visualization of the hand-over-hand cycle14-16 and indicate a\npotential regulatory role for Mg²⁺ release in coordinating the sequential rearrangements\nof the ATPase subunits.\"\n22. In line 322, it states: ‘These findings suggest that asymmetric ATP hydrolysis by\nthe proteasome ATPase is a widespread phenomenon and may not be substantially\nrelated to substrates. The underlying reasons for this asymmetric hydrolysis merit\nfurther investigation’. This is not true, there are a wealth of data and discussions about\nthe asymmetry of the proteasome and all other AAA+ unfoldases.\nAnswer: We thank the reviewer for this critical comment. You are absolutely correct\nthat \"there are a wealth of data and discussions about the asymmetry of the proteasome\nand all other AAA+ unfoldases. \" Our original line 322—\"These findings suggest that\nasymmetric ATP hydrolysis by the proteasome ATPase is a widespread\nphenomenon…\"—was inappropriate because it wrongly implied that the universality\nof this asymmetry was first or primarily \"discovered\" or \"suggested\" by our study. In\nreality, the asymmetric, spiral-staircase mode of sequential ATP hydrolysis by the\nproteasomal AAA+ motor has been definitively established and is widely accepted as\na fundamental principle (e.g., de la Peña et al., Science 2018; Dong et al., Nature 2019).\nOur work simply visualizes how this established mechanism operates within the\nspecific context of midnolin-mediated, ubiquitin-independent degradation.\nAccordingly, we have revised the offending statement and added citations to the key\nstudies that elucidated proteasomal asymmetry.\nPage15-16; Line 351-357\n\" Consistent with the well-established asymmetric mechanism of the proteasomal\nAAA+ motor14,21, our structures capture consecutive spiral-staircase conformations\n(PS , PS , PS , PS ) during midnolin-mediated substrate translocation. This\nRPT6 RPT2 RPT1 RPT5\nconfirms that the hand-over-hand translocation mechanism is operational in this\nubiquitin-independent pathway. The specific distribution and progression of these\nstates in our dataset provide a detailed view of the mechanochemical cycle in the\ncontext of midnolin engagement, although the general principle of asymmetry is\npervasively established across AAA+ ATPases. \"\nReferences\n[14] Zhang, S. et al. USP14-regulated allostery of the human proteasome by time-\nresolved cryo-EM. Nature 605, 567-574, doi:10.1038/s41586-022-04671-8 (2022).\n[30] Arkinson, C., Gee, C. L., Zhang, Z., Dong, K. C. & Martin, A. Structural landscape\nof the degrading 26S proteasome reveals conformation-specific binding of TXNL1.\nNature structural & molecular biology 32, 2403-2415, doi:10.1038/s41594-025-01695-\n2 (2025).\nOther points (regarding the methodology):\n23. The authors state that the 26S complex concentration was measured after the\npurification; however, there is no information about the concentration of the complex\nused for grid preparation.\nAnswer: We have added the concentration of the complex used for cryo-grid\npreparation. Although the sample concentration was low, we obtained data of sufficient\nquality to determine the target structure by employing a two-round sample-blot strategy\non graphene-oxide (GO) grids. For the in-vitro reconstituted complex (e.g., MIDN-\nEGR1-26S), we also added the concentration information.\nPage27; Line 607-620\n\" The complex was concentrated to approximately 0.3 mg/mL in size-exclusion buffer,\nflash-frozen in liquid nitrogen, and stored at -80°C.\nThe E. coli co-expressed MBP-EGR1(101–200) and MBP-midnolin complex were\nmixed with the purified 26S proteasome at a 2:1 molar ratio, incubated on ice for 30\nseconds , and immediately prepared for cryo-EM analysis. For cryo-EM grid\npreparation, the sample was thawed on ice and used at this concentration. The\nreconstituted complex(MIDN-EGR1-26S) was immediately used for cryo-EM grid\npreparation at a final concentration of approximately 4 mg/mL. 3 µL of the freshly\nconcentrated sample was applied onto a freshly glow-discharged (at 20 mA for 70\nseconds, negative charge) Quantifoil R1.2/1.3 300-mesh Au grid. Grids were blotted\nfor 3 seconds under 100% humidity at 4°C using a Vitrobot (Thermo Fisher Scientific),\nthen plunge-frozen in liquid ethane cooled by liquid nitrogen. The entire process from\nfinal concentration adjustment to plunge-freezing was completed within 10 minutes to\nensure complex stability and homogeneity. Frozen grids were stored in liquid nitrogen\nuntil data collection.\"\n24. There is currently no information provided regarding the software utilized for cryo-\nEM data processing and analysis. Similarly, Figure S5 seems to be prepared using\nESPript. The authors should specify this and cite the relevant software utilized.\nAnswer: We have conducted a comprehensive review to ensure that every procedure\ninvolving specialized software or online tools, ranging from cryo-EM data processing\nto \"Isothermal titration calorimetry (ITC) \", and \"Surface plasmon resonance (SPR) \",\nexplicitly states the name and version of each software package or server used, and\nprovides the corresponding citations.\n\" Multiple sequence alignment was performed using ESPript (Version 3.2)49.\"\n25. In the description of some of the cryo-EM data sets, the number of collected movies\nand particles in the initial stacks is missing. Authors should consider rearranging the\nsections that describe the processing of different samples to align with the order of the\nfigures depicting the pipelines.\nAnswer: We fully agree with your assessment and undertook a systematic revision of\nthe relevant sections. Following your practical suggestion, we re-ordered the\nsubsections in “Methods” dedicated to data processing and adjust all in-text citations\naccordingly, ensuring an intuitive, linear path from narrative to figure.\n26. Authors use \"two-dimensional\" and \"3D\" interchangeably. I suggest using the\nsimpler 2D/3D notation for clarity.\nAnswer: We have now used 2D/3D notation for clarity.\n27. On page 22, what do the authors mean by \"subjected to AAA+ ATPase domain-\nfocused masking\"? Are they referring to local refinement focused on the AAA region?\nThe pipeline shown in Extended Figure 4 does not clarify the steps undertaken by the\nauthors. One of the initial steps states \"masked on AAA+ ATPase domain,\" and then\nfour classes (#0, #5, #6, #19) from the previous (unmasked?) classification are\npresented. We suggest the authors redraw their data processing pipeline, showing all\nthe maps for the same classification side by side. They should then indicate by separate\noutlines which classes were used for further data processing, similar to what they did\nin Figure S7.\nAnswer: Thank you for this specific and highly constructive comment on our data-\nprocessing workflow. We apologize for the lack of clarity and thoroughly revised both\nthe text and the figures according to your suggestions. \"Subjected to AAA+ ATPase\ndomain-focused masking\" indeed refers to local refinement focused on the AAA+\nregion. We redrew the data processing pipeline and corrected this sentence as follow.\nPage 29; Line 653-657\n\" Particles from Classes 0, 5, 6, and 19, which exhibited features of substrate-engaged\nstates, were selected. To improve the resolution of the AAA+ motor, these particles\nunderwent local refinement using a soft mask focused on the AAA+ ATPase domain,\nyielding two locally refined proteasome complex structures at 3.45Å (PS , PDB:\nRPT1\n9MBP, EMD-63766, 40,572 particles) and 3.66Å (PS , PDB: 9MBQ, 23,186\nRPT5\nparticles), respectively. \"\n28. On the same page, the authors state, \"Notably, 63,758 particles selected through\nRPN1 region masking from the proteasome particle pool produced a high-resolution\nRPN1 subunit structure\". However, the data processing pipeline (Extended Data Fig. 2)\nshows that the particles from class 0 and class 1 were pulled together, not \"selected\nthrough RPN1 region masking.\"\nAnswer: Thank you for this correction. The original description was indeed imprecise.\nWe have revised the text to clarify this procedure: particles from classes 0 and 1 were\nselected, followed by RPN1-focused masking and local refinement. The flowchart in\nExtended Data Fig. 2 has been updated with explicit labels (e.g., \"Particles selected for\nRPN1-focused analysis\") to reflect this accurately.\nPage 29;Line 658-668\n\" To resolve the interaction between midnolin’s αHelix-C and RPN1 at high resolution,\nwe selected particles from Classes 0 and 1, which represented substrate-engaged\ncomplexes. Notably, 63,758 particles selected through RPN1 region masking from the\nproteasome particle pool produced a high-resolution RPN1 subunit structure (PDB:\n9MBO, EMD-63775, 3.07Å) after local refinement. For Classes 1-4 and 7-18\n(combined 97,538 particles) underwent further 3D classification (20 classes),\nsubsequent screening of 9,022 particles from Class 11 through 19S-focused masking\ngenerated a 4.48Å map (PS , PDB: 9U4M, EMD-63850) of 19S regulatory particles.\nRPT6\nAdditionally, 25,990 particles selected from Classes 0, 1, and 10 using 19S-specific\nmasking were processed identically to obtain an improved 19S structure (PS , PDB:\nRPT2\n9U3L, EMD-63817) at 3.72Å resolution. The other classes (Classes:2-9 and 12-19)\nrepresented unassigned processing states. The final maps were further processed using\nEMReady43.\"\n29. On page 23, the authors mention \"high-resolution 3D classification.\" However, the\nrepresentative maps for these classes appear to be of lower resolution compared to those\nfrom other 3D classification jobs shown. How does this high-resolution classification\ndiffer from the other 3D classifications that the authors do not specify as \"high-\nresolution\"? Moreover, 3D classification was performer generating far two many\nclasses based on numer of particles available (e.g. 160k particles into 20 classes -line538). It seems that the particles just got distributed equally in the various classes,\nwhich indicates that the 3D classification didn’t really work.\nAnswer: Thank you for pointing out this inaccuracy. The term \"high-resolution 3D\nclassification\" referred to the use of a 3-Å filter resolution, not the resolution of the\nresulting maps. We have replaced this misleading term in the text (line 615) with the\nprecise description: \"3D classification after global refinement using a 3 Å filter\nresolution.\"(Page 29;Line 705-707)\nConcerning the particle classification, our initial strategy of generating a larger number\nof classes (e.g., 20 from ~160k particles) was motivated by the goal of maximally\nresolving the diverse conformational states of the proteasome complex. While this\napproach distributed particles across multiple classes, it served as a necessary\nexploratory step to identify all potential states.\n30. For symmetry expansion, particles should first be refined with C2 symmetry applied.\nAuthors should specify the symmetry of their refinement for transparency and\nreproducibility of the analysis.\nAnswer: Thank you for highlighting this essential detail for reproducibility. We\nconfirmed that the initial global refinement of the 26S proteasome complex was\nperformed with C2 symmetry applied, prior to symmetry expansion, as is standard\npractice. This specification has been explicitly stated in the revised \"Data processing of\nMIDN-EGR1-human 26S proteasome complex\".\nPage 31; Line699-705\n\" Prior to symmetry expansion, the selected particles were subjected to Non-uniform\nrefinement with C2 symmetry imposed. The resulting alignment parameters were then\nused to perform symmetry expansion, doubling the particle count. The expanded\nparticles were subsequently re-extracted, re-centered, and used for downstream focused\nclassification and refinement steps. Since the 19S regulatory particle in the resulting\n26S proteasome structure was not centered, symmetry expansion was applied to\ngenerate 610,432 particles. \"\n31. There is no description for the processing of \"MIDN-EGR1-human 26S 1068\nproteasome complex for the resolved Catch domain\" shown in Extended Fig. 8.\nAnswer: We apologize for the incomplete description in the “Methods\" section, where\nwe failed to provide a dedicated and comprehensive account of the data-processing\nworkflow corresponding to Extended Data Fig. 8 (Revised Extended Data Fig. 7,\n\"Cryo-EM data processing workflow of MIDN-EGR1-human 26S proteasome complex\nfor resolved Catch domain\"). This omission hindered full methodological transparency\nand reproducibility. In the revised manuscript, we therefore added a new subsection\nimmediately after the existing \"Data processing of MIDN-EGR1-human 26S\nproteasome complex,\" titled “Data processing for the resolution of the Catch domain in\nthe MIDN-EGR1-26S complex, \" which clearly detailed every step outlined in\nExtended Data Fig. 8 (Revised Extended Data Fig. 7).\n32. The description of the ITC and SPR experiments reads more like a general overview\nof the protocol and lacks the specific details necessary to assess and reproduce the\nresults. For the ITC experiments, the authors do not specify whether a blank titration\nwas performed to determine the nonspecific heats of mixing, dilution, and any potential\nbuffer mismatch. Therefore, in addition to clarifying this in the methods section, the\nauthors should provide reviewers with the raw titration curves from the actual\nexperiments, as well as the results from the blank experiments.\nAnswer: We apologize for the overly general description in the original manuscript. We\nhave revised the main text as the reviewer suggested.\nITC measurements of binding affinities between midnolin (337-468) or midnolin (337-\n432) and RPN1. The binding constants (K values) and stoichiometries (N) are\nD\nindicated. (a-b) Titration curves of MIDN(337 468) into RPN1. (c) Negative control\ntitration of MIDN(337-468) into buffer. (d-e) Titration curves of MIDN(337-432) into\nRPN1. (f) Negative control titration of MIDN(337-432) into buffer.\n33. In the description of SPR measurements, there is no information about the\nconcentration of the proteins used for the experiment or the amount of protein\nimmobilized on the sensor. Were any negative controls included to test for nonspecific\nbinding?\nAnswer: Thank you for raising these essential points. We have now fully detailed the\nSPR methodology in the revised manuscript to ensure reproducibility. We added the\nfollow information in the \"Methods\" section: the concentrations of both the analyte and\nligand used, the specific immobilization level of the ligand on the sensor chip (in\nresponse units), and the use of a blank reference flow cell as a negative control, with\nall binding signals double-referenced against it for specificity.\nTo correct for non-specific binding signals, an independent reference flow cell(FC2)\ncontaining no immobilized ligand was employed, and the response signals of the\nanalyte in all active channels were double-referenced by subtracting the signal from\nthis blank control channel before kinetic analysis.\n34. Authors should use black mesh or another more contrasting color combination, as\nit is almost impossible to glean the density details from Extended Data Fig. 4 in its\ncurrent form.\nAnswer: Thank you for your valuable feedback on the visual clarity of our figures. We\nfully agree with your observation that the low-contrast grid colors in Extended Data\nFig. 4 make it difficult to discern density details, thereby compromising the structural\ninformation the figure is intended to convey. In response, we have revised Extended\nData Fig. 4 to enhance contrast so that the key structural densities are now clearly\nvisible.\n35. NLS (same as all other abbreviations used) should be explained to facilitate\nunderstanding of the text\nAnswer: Thank you for your valuable feedback on the readability of the manuscript.\nWe have performed a thorough review of the entire text to ensure that the above rule is\napplied consistently and that no abbreviation is used without being defined first.\n36. For the sake of accuracy, the color scalebar in Fig.2a should include units (e.g., kT/e)\nAnswer: Thank you for pointing out this issue. We have added the standard unit \"kT/e”\n\"to the color scale bar of the electrostatic potential surface in Figure 2a.\nFigures:\nFigure 2 : the circle in figure2b is barely visible. How does this arrangement compare\nwith the published structure? The measurements in the ITC panel (d,e) should have\nincluded full length MIDN and negative controls. Moreover, more than 2 measurements\nshould have been performed.\nAnswer: To better visualize, we have boldened the circle in Figure 2b.\nWe compared our identified binding mode with canonical T2-site binders such as\nUSP14 and NUB1. This new panel features a structural comparison among: (1) our\nresolved αHelix-C–RPN1 complex, (2) the previously reported USP14–RPN1 complex\n(PDB: 7W3H), and (3) the NUB1–RPN1 complex (PDB: 8USB). Our analysis reveals\nthat all three complexes share a common hydrophobic core anchored by RPN1 residues\nL426 and L465. They are further stabilized by peripheral salt bridges and hydrogen\nbonds. A key distinction, however, lies in the fact that our αHelix-C engages RPN1 with\na more extensive interaction interface and adopts a distinct linear conformation.\nRevised Figure 2. (b) The interaction interface exhibits a tripartite pattern between\naHelix-C of midnolin and RPN1. The circle part indicates a hydrophobic center. This\ncentral hydrophobic interface is flanked by two polar interaction networks. (c)\nSuperimposition of the atomic models depicting the interaction interface between\nUSP14(blue) and RPN1(pink) versus the interaction interface between RPN1 and\naHelix-C of midnolin (magenta). The T2 site is indicated by a red dashed circle. (d)\nSide-chain interactions between the USP14 UBL domain (blue) and the RPN1 (pink)\nT2 site, derived from the USP14–RPN1 complex (PDB: 7W3H). The T2 site is\nindicated by a red dashed circle. (e) Side-chain interactions between the NUB1 UBL\ndomain (yellow) and the RPN1 (pink) T2 site, derived from the NUB1–RPN1 complex\n(PDB: 8USB).\nWe attempted to perform ITC experiments using purified full-length proteins. However,\ndue to the high protein requirements of ITC and the low yield of the full-length protein,\nwe were unable to obtain sufficient protein for the ITC measurements. As an alternative,\nwe conducted surface plasmon resonance (SPR) assays to measure the binding of\nMIDNΔ(112-336) to the 26S proteasome. We employed this truncated form, which\nlacks the Catch domain, to avoid potential interference from the substrate with binding\naffinity. The measured binding affinity (162 nM) was essentially consistent with that\nbetween the isolated αHelix-C and RPN1 (130 nM), indicating that the interaction of\nMIDN with the proteasome is primarily mediated by aHelix-C. On the other hand, we\nhave included negative controls and performed triplicate experiments. The binding\nconstant (K ) values are reported as \"mean ± standard deviation\" in both the main text\nD\nand the figures to provide statistical reliability.\nFigure 4b is based on image processing. How many particles were thrown away? And\nwhy not reporting these numbers for all the other maps as well?\nAnswer: Thank you for this important question regarding particle accounting and data\ntransparency. We have systematically reported particle counts for the datasets in the\nrevised manuscript.\nFor Figure 4b, the population percentages were derived from a 3D classification of the\nMIDNΔ(112-336)-26S dataset. The total particle pool for this analysis comprised\n84,995 particles. Following classification, 54,459 particles were assigned to the\nsubstrate-engaged Processing State, and 29,705 particles to the Resting State. The\nnegligible remainder (~800 particles) corresponded to poorly aligned or ambiguous\nclasses, which were excluded from high-resolution refinement.\nNotably, parallel analysis of the full-length MIDN–proteasome datasets consistently\nyielded only substrate-engaged states, with no detectable population of the Resting\nState. This contrast directly underscores the stabilizing role of the Catch domain.\nWe have updated the processing workfolw to clearly annotate particle counts at each\nkey step. We appreciate your suggestion to enhance the rigor of our data reporting.\nFigure 5f is very confusing.\nWe thank the reviewer for this critical feedback. We agree the original Figure 5f was\nvisually complex. In response, we have simplified the figure to provide a direct, side-\nby-side comparison of the two key conformational states. The revised version uses\ndistinct colors (cyan for PS and magenta for PS ) to separately depict these states,\nRPT1 RPT5\nfocusing on the RPT6 subunit to represent the AAA+ ring's motion. This new\nvisualization clearly demonstrates the coupled movement: the downward shift of the\nRpt6 pore-1 loop is precisely aligned with the concurrent downward translocation of\nthe substrate. The revised figure removes extraneous detail to deliver a clear message\nthat the ATPase cycle drives stepwise substrate translocation through coordinated pore-\nloop motions.\nRevised Figure 5f. Structural superimposition shows the movement of the pore-1 loop\nbetween PS and PS . Specifically, Lys222, Phe223 in RPT6 and their\nRPT1 RPT5\ncoordinated substrate residue shift downwards by 2.4 Å and 2.3 Å, respectively.\nFigure 6b is almost copied from Arkinson et al., 2024 and yet the paper is not even\ncited.\nAnswer: We sincerely apologize for the oversight in not citing Arkinson et al., 2024, as\nthe core model for the AAA+ motor cycle in Figure 6b is indeed adapted from their\nwork. We now place a clear and primary citation to Arkinson et al. at the very beginning\nof the paragraph in the main text that describes Figure 6b and its mechanism.\nReference\n[22]Arkinson, C., Gee, C. L., Zhang, Z., Dong, K. C. & Martin, A. Structural landscape\nof the degrading 26S proteasome reveals conformation-specific binding of TXNL1.\nNature structural & molecular biology 32, 2403-2415, doi:10.1038/s41594-025-01695-\n2 (2025).\nThe manuscript requires careful proofreading to correct numerous typos, grammatical\nerrors and to improve the overall readability of the text.\nWe hope that these comments will help the authors to improve the manuscript.\nAnswer: Thank you for your important comments on our manuscript. We fully agree\nthat the original text contains typographical errors, grammatical issues, and some\ndescriptions that are not fluent enough, which indeed affect the overall clarity and\nprofessionalism of the text. We apologize for this and have conducted a thorough\nlanguage proofreading and polishing of the entire text. Thank you again for your careful\nreading and valuable suggestions.\nReviewer #2 (Remarks to the Author):\nThe manuscript under review provides interesting and novel insights into the\nmechanism by which midnolin targets proteins to the proteasome for degradation, and\nmore broadly into the proteasome's mechanism of action.\nMidnolin was recently discovered as a novel mediator of ubiquitin-independent\nproteasomal degradation. It was found to target products of immediate early genes for\ndegradation by binding simultaneously to target proteins and the proteasome, shuttling\nthe former for degradation without the need for ubiquitination. Initial studies suggested\nthat proteasome binding occurs by an N-terminal domain and the C-terminal UBL\ndomain, with the central Catch domain binding substrates.\nThe study under review here puts this model on solid footing by revealing a series of\ncryo-EM structures of the tertiary complex between substrate, midnolin, and the\nproteasome. I cannot assess the technical quality of the cryo-EM structure\ndetermination, but taken at face value, the structures provide valuable insights. The\nbinding mode between midnolin and the proteasome is quite novel, including the\ndetailed insights into the interaction between the N-terminal helix and Rpn1 and that of\nthe C-terminal UBL domain with Rpn11. Pinning the N- and C-terminal domains of\nmidnolin to the proteasome then allows midnolin's central catch domain to position\nbound substrate at the entrance to the degradation channel to be engaged by the\nproteasome.\nThe study caught the proteasome in a series of different conformations that are likely\nto represent different steps in the degradation cycle. Analysis of these structures\nsuggests that Mg++ dissociation drives (together with ATP-hydrolysis) the proteasome\nthrough the conformational cycles that lead to substrate unfolding and translocation to\nthe proteolytic sites. As far as I'm aware, the suggested role of Mg++ in the mechanism\nis novel and important.\nThere are a couple of conclusions in the manuscript that are not properly supported by\nthe data presented, interesting as they are. I recommend that these parts of the\nmanuscript be revised to indicate the speculative nature of the conclusions. For example,\nI think that the fact that midnolin is difficult to purify and aggregates when not co-\nexpressed with a substrate can have many other interpretations - most simply that some\nscaffolding factor is missing at the right stoichiometry in the experimental setup.\nSimilarly, the suggestion that the proteasome can digest midnolin by engaging the\nprotein at its IDR2 is interesting and even plausible, but I propose that the\nautodegradation of midnolin as a regulatory mechanism is discussed as a hypothetical\nto be explored in the future.\nAnswer: We sincerely thank the reviewer for the positive assessment of our work and\nfor recognizing the novelty of the mechanistic insights. We fully agree that certain\nconclusions should be presented more cautiously to better distinguish between\nexperimental observations and mechanistic inferences.\nRegarding the observation that midnolin is difficult to purify and tends to aggregate\nwithout co-expressed substrate: We revised the text to clarify that this finding primarily\nrelates to the practical requirements for sample preparation in our experimental system.\nWe presented it as an intriguing observation whose in vivo relevance and underlying\nmechanism (including potential explanations such as the absence of a scaffold factor)\nremain subjects for future investigation, rather than a definitive conclusion.\nRegarding the hypothesis that \"the proteasome may degrade midnolin via IDR1\"\nand\nits potential regulatory role: Our structural and biochemical data indicate that the\npresence of the N-terminal disordered region (IDR1) can lead to the degradation of\nMIDN (Fig. 4c-g). Therefore, we corrected this sentence as “Additionally, the N-\nterminal disordered region (IDR1) region may guide midnolin itself into the proteasome\nfor degradation after substrate delivery.”\nWe are grateful for these insightful suggestions.\nReviewer #3 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nAnswer: Thank you for reviewing our manuscript and for your valuable comments. We\ngreatly appreciate Nature Communications' initiative to formally involve early-career\nresearchers in peer review, which strengthens the scientific community. Your\nparticipation as a co-reviewer brought an insightful perspective to this process, and we\nare sincerely grateful.\nReviewer #4 (Remarks to the Author)\nProteasome is the main degradation machine in eukaryotic cells. While ubiquitin-\ndependent degradation is well studied, the non-canonical degradation mechanism\nmediated by midnolin remains elusive. In this study, Zhu et al. investigates the\nmolecular mechanisms of the midnolin in targeting substrates to the proteasome for\ndegradation. The authors combine structural and biochemical analyses to characterize\nmidnolin’s interactions with the proteasome, revealing how its αHelix-C and Ubl\ndomains bind to proteasomal subunits RPN1 and RPN11 respectively, to direct\nsubstrates to the proteasome entry port via the Catch domain. The authors captured four\ncontinuous states of the midnolin-proteasome complex, revealing the functional\ndynamics of the proteasomal ATPase complex, and a novel role of Mg2+ dissociation\nin coordinated mechanochemical coupling. Overall, this study presents compelling\nstructural evidence and valuable insights into the mechanism of midnolin in mediating\nnon-ubiquitination-mediated degradation. However, the current study relies heavily on\nstatic structural observations, and it would be important to complement these with\nfunctional validation such as kinetic assays of substrate recognition and degradation,\ntogether with mutational analyses of key midnolin domains, as detailed below.\nQuestions:\n1. Lines 100-101: There is grammar issue of the sentence.\nAnswer: Thank you for pointing out the grammatical issue. We have restructured the\nsentence following your suggestion to ensure grammatical correctness and logical\nclarity. We appreciate your help in improving the language quality of our manuscript.\nPage 5; Line 98-105\n\" After 3D classification, we determined four distinct structural states (designated\nMIDN-PS , -PS , -PS , and -PS for subsequent discussion). Among these\nRPT1 RPT5 RPT2 RPT6\nstates, the RPN1 subunit exhibited varying degrees of resolution and occupied distinct\npositions relative to the AAA+ motor (Extended Data Fig. 2, 3 and Supplementary\nTable 1). The 3D classification yielded structural states that all represent substrate-\nprocessing conformations. This includes classes reconstructed from particles that were\nnot assigned to a dominant state but still contributed to the overall conformational\nlandscape. The highest resolution (2.87 Å) was achieved for the MIDN-PS state\nRPT1\n(Fig. 1a).\"\n2. It is interesting that co-expression of Catch domain and EGR1 stabilizes the Catch\ndomain and facilitates their binding. However, this may reflect artifacts arising from\nusing the isolated Catch domain. To clarify this, it would be important to perform the\ngel filtration assays with full-length midnolin with or without co-expression of EGR1.\nThe proposed working model that midnolin co-expresses with substrates to achieve\nrapid targeted degradation raises questions about substrate specificity. With elevated\nmidnolin levels, how is selectivity maintained, and could this result in unintended\ndegradation of non-target substrates?\nA minor point: Extended Data Fig. 6g is not cited in the text.\nAnswer: Thank you for these important comments.\nWe agree that using the isolated Catch domain may not fully represent the full-length\nprotein. To address this, we performed the suggested control experiment. We purified\nfull-length midnolin expressed alone or co-expression with EGR1. As expected, the\ncomplex shows a single, symmetric peak, whereas midnolin alone showed significant\noligomerization/aggregation, confirming that substrate binding is essential for\nstabilizing the full-length midnolin.\nWe agree that selectivity is a critical aspect of our model. In the revised Discussion,\nwe will emphasize that specificity is primarily ensured by the high-affinity, selective\nbinding of the Catch domain to specific β-strand degrons in substrates like EGR1\n(Nardone et al., Mol Cell, 2025, Gu et al., Science 2023). We proposed that\nspatiotemporal coupling, through co-expression and localization, further minimizes off-\ntarget degradation, while acknowledging that other regulatory layers may exist and\nwarrant future study.\nWe have cited Extended Data Fig. 6g in the text.\nPage 9; Line 193-196\n\" To elucidate the molecular mechanism of midnolin-mediated substrate delivery to the\nproteasome entry port, we purified human proteasomes via an mCherry-hexahistidine-\ntwin-strep-tagged RPN11 (Extended Data Fig. 6g) and incubated them with purified\nmidnolin-EGR1(101-200) complex before plunge-freezing. \"\nReferences\n[3] Gu, X. et al. The midnolin-proteasome pathway catches proteins for ubiquitination-\nindependent degradation. Science 381, eadh5021, doi:10.1126/science.adh5021 (2023).\n[13] Nardone, C. et al. Structural basis for the midnolin-proteasome pathway and its\nrole in suppressing myeloma. Mol Cell 85, 2597-2609 e2511,\ndoi:10.1016/j.molcel.2025.05.030 (2025).\n3. In the introduction, the authors raise a question of whether midnolin first binds its\nsubstrates prior to proteasome association or constitutively engages the proteasome\nbefore recruiting substrates. This key question is not sufficiently addressed in the\ncurrent work. The structural characterization of midnolin-substrate-26S proteasome\ncomplex and co-expression assays do not rule out the possibility that midnolin engages\nthe proteasome before substrate loading.\nAnswer: We thank you for raising this profound and pivotal question. Following your\nsuggestion, we expanded the “Discussion” in the revised manuscript to address the\nissue more deeply and cautiously, using our data to argue preferentially for one model\nwhile explicitly acknowledging that our current structural and biochemical results\ncannot temporally resolve which of the two pathways outlined in the Introduction\noperates first. The captured complex is a single \"snapshot\" along the degradation\ntrajectory, not a time-resolved record of assembly; consequently, we cannot provide\ndefinitive proof. Nevertheless, integrating our new findings allows us to present a\nlogically and energetically more favorable scenario: midnolin first binds its substrate\nand then is the binary complex recruited to the proteasome.\nPage 14-15; Line 319-332\n\" Our findings support a sequential “capture-before-delivery” mechanism for midnolin-\nmediated proteasomal degradation. In this model, substrate binding to the Catch domain\nis a prerequisite step that stabilizes midnolin, particularly the Catch domain, into a\nsoluble and functional conformation. This substrate-occupied complex constitutes a\nstable “ready-to-degrade” module, primed for proteasomal engagement. This capture-\nfirst mechanism, in which specificity is determined by the initial engagement of the\nsubstrate with midnolin, functionally parallels ubiquitination. This mechanism acts as\na \"midnolin tag\" that marks the substrate for subsequent proteasomal delivery and\ndegradation. On the other hand, in the absence of substrates, the Catch domain of\nmidnolin exhibits a deficiency in a central b-sheet structure, which predisposes it to\ninstability and subsequent aggregation. Although midnolin can express independently,\nits capacity to degrade substrates is probably rather limited on its own. Interestingly, it\nhas been reported that midnolin is required for the DNA-binding activity of EGR129,\nwhich is consistent with our observation that midnolin co-express with substrates.\nWhether the DNA-binding activity of other substrates is also dependent on midnolin\ndeserves further investigation.\"\nReferences\n[29] Chiba, A. et al. Midnolin, a Genetic Risk Factor for Parkinson's Disease, Promotes\nNeurite Outgrowth Accompanied by Early Growth Response 1 Activation in PC12\nCells. Mol Cell Biol 44, 516-527, doi:10.1080/10985549.2024.2399358 (2024).\n4. The functional importance of different midnolin domains in proteasome targeting\nand degradation requires experimental validations. Specifically, while Fig.2 shows the\ndynamic binding of αHelix-C of midnolin to RPN1, how this interaction could be\nstrengthened by the binding of Ubl domain to the RPN11, or antagonized by the\npotential interaction between Ubl and RPN1 remains unclear. Structure-based site-\ndirected mutagenesis of these domains followed by functional readouts such as\nbinding kinetics and degradation rates would help disentangle their functional contributions to\nproteasome targeting.\nAnswer: Thank you very much for this crucial suggestion. Although our structural work\nhas revealed how each midnolin domain engages proteasome subunits (Ubl–RPN11\nand ahelix-C–RPN1), it remains unclear whether these interactions act independently,\ncooperatively, or regulate one another. Without functional validation, our mechanistic\nunderstanding would stop at static correlations. We fully agree that structure-guided\npoint mutations coupled to functional assays are the “gold standard” for dissecting the\ncontribution of each domain. To answer your question directly and rigorously, we\ndesigned and performed point-mutation experiments that quantify how each domain\ncontributes to degradation efficiency. Our biochemical data demonstrate that mutations\nin either the Ubl or the aHelix-C domain significantly impair midnolin's activity.\nThese results indicate that the full-length, intact midnolin protein is required to achieve\nmaximal substrate degradation efficiency.\nRevised Figure 2. (j) In-vitro peptidase activity was monitored over 90 min using the\nfluorogenic substrate Suc-LLVY-AMC (10 µM) for the 26S proteasome alone (black),\nor with MBP-MIDNK391E/L395E/R409E (pink), with wild-type MBP-MIDN (red). The y-\naxis indicates relative fluorescence units (RFU). (k) Quantification of in-vitro peptidase\nactivity results in (j). RFU changes between 90 min and 0 min are plotted. (l) In-vitro\npeptidase activity was monitored over 90 min using the fluorogenic substrate Suc-\nLLVY-AMC (10 µM) for the 26S proteasome alone (black), or with MBP-MIDN(337-\n432) (orange), with MBP-MIDN(337-468) (blue), with wild-type MBP-MIDN +\nEGR1(101-200) (red). The y-axis indicates relative fluorescence units (RFU). (m)\nQuantification of in-vitro peptidase activity results in (l). RFU changes between 90 min\nand 0 min are plotted. Bars represent means ± SD of three replicates (****, p <0.0001;\n***, p <0.001;n =3, p values were calculated using ANOVA with Tukey’s post test.) (k and m). ns\nmeans not significant.\nRevised Figure 3. (h) In-vitro peptidase activity was monitored over 90 min using the\nfluorogenic substrate Suc-LLVY-AMC (10 µM) for the 26S proteasome alone (black),\nor with MBP-MIDNP100E + EGR1(101-200) (dark green), with MBP-\nMIDNP100E/V102E/A104E + EGR1(101-200) (light green), with wild-type MBP-MIDN +\nEGR1(101-200) (red). The y-axis indicates relative fluorescence units (RFU). (i)\nQuantification of in-vitro peptidase activity results in (h). RFU changes between 90 min\nand 0 min are plotted. Bars represent means ± SD of three replicates (****, p <0.0001;\n***, p <0.001;n =3, p values were calculated using ANOVA with Tukey’s post test.)\n5. The Catch domain might allosterically regulate proteasome states but the current\nexperiments and results in Fig. 4 do not separate this effect from substrate-induced\nactivation. This issue could be addressed by perturbing the substrate recognition site of\nCatch domain. In addition, structural analysis of substrate-free midnolin-proteasome\ncomplexes within the existing datasets may provide useful insight.\nAnswer: We thank the reviewer for this insightful point. We agree that the current data\ncannot distinguish whether the effect observed in Fig. 4 stems from the loss of substrate\nbinding or a specific positioning function of the Catch domain.\nTo directly address this, we performed a decisive domain-swap experiment. We\nengineered a \"Catch-replaced midnolin\" construct [MIDN-EGR1(fusion)], where the\nnative Catch domain is replaced by the known substrate fragment EGR1(101-200),\ncovalently tethered via a flexible linker. This chimera retains constitutive substrate\nbinding but lacks the native Catch interface.\nWe then compared the degradation efficiencies of three proteins: Wild-type full-length\nmidnolin [MBP-MIDN + EGR1(101-200)], Catch-deletion mutant [MIDNΔ(112-336)],\nCatch-replaced midnolin [MBP-MIDN(1-111)-EGR1(101-200)-MIDN(337-468)]. If\nthe role of the Catch domain is to bind substrate, the Catch-replaced midnolin should\ndegrade substrate as efficiently as wild-type because the substrate is already effectively\n“provided.” Conversely, if the Catch domain possesses an additional “positioning” or\n“optimized delivery” function, then the Catch-replaced midnolin should show\nmarkedly lower degradation efficiency, resembling or only slightly exceeding that of\nCatch-deletion mutant. The biochemical results show that the Catch-replaced midnolin,\ndespite having the substrate pre-bound, showed a significantly lower degradation\nefficiency than the full-length, much closer to the Catch-deleted mutant MIDNΔ(112-336).\nThese results demonstrate that the Catch domain provides a specific function\nbeyond simple substrate binding, consistent with its proposed role in actively\npositioning the substrate for optimal delivery to the proteasome.\nRevised Figure 4. (g) In-vitro peptidase activity was monitored over 90 min using the\nfluorogenic substrate Suc-LLVY-AMC (10 µM) for the 26S proteasome alone (black),\nor with Catch-deleted mutant [MBP-MIDN∆(112-336)] (purple), with Catch-replaced\nmidnolin [MBP-MIDN(1-111)-EGR1(101-200)-MIDN(337-468)], with wild-type\nMBP-MIDN + EGR1(101-200) (red). The y-axis indicates relative fluorescence units\n(RFU). (h) Quantification of in-vitro peptidase activity results in (g). RFU changes\nbetween 90 min and 0 min are plotted. Bars represent means ± SD of three replicates\n(****, p <0.0001; n =3, p values were calculated using ANOVA with Tukey’s post test.).\nns means not significant.\n6. Line 209: Extended Data Fig. 11 should be changed to Fig. 10.\nAnswer: Thank you for pointing out this detail. The “Extended Data Fig. 11” cited in\nline 209 has been changed to \"Extended Data Fig. 10\".\n7. The legend of Extended Data Fig.4 should be revised with further clarification.\nAnswer: We have revised the figure legend accordingly.\nExtended Data Fig. 4 | Cryo-EM densities of MG132 bound to the proteasome.\n(a) Overall surface representation of the proteasome (grey), shown in cross-section,\nwith MG132 (purple) bound to three β-subunits of the core particle (CP) in the MIDN-\nPS state. (b) Top view of the CP with seven subunits depicted in cartoon\nRPT5\nrepresentation (grey). In this view from the MIDN-PS state, MG132 molecules\nRPT5\nbound to the three β-subunits (β1, β2, β5) of the distal ring are colored purple, while\nthose bound to the equivalent subunits of the proximal (symmetry-related) ring are\ncolored blue. The β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits are annotated\nwith their respective catalytic activities: aspartyl protease-like, neutral protease, and\ncarboxypeptidase activities. (c) Cryo-EM density map (grey mesh) of MG132 (purple)\nbound to three b-subunits of the CP (dark goldenrod) in the MIDN-PS state.\nRPT5\n(d)\nCryo-EM density map (grey mesh) of MG132 (purple) bound to three β-subunits of the\nCP (dark goldenrod) in the MIDN-PS state.\nRPT1\nPoint-by-point Responses\nStructural dynamics of the midnolin–proteasome during ubiquitin-independent\nsubstrate turnover\" (NCOMMS-25-69834A)\nPoint-by-point responses to the comments by Reviewer #1:\nIn the revised version of the manuscript, the authors have made substantial efforts that\nhave significantly improved the work. We are also glad that our suggestions have been\npositively used in the current manuscript. Better annotations of the presented cryo-EM\nmaps and a more logical arrangement of the results and supplementary material\nimproved readability, facilitating interpretation. Additional biochemical analyses reveal\nthe roles of the domains, while point mutations confirm their importance for binding\nand activation. Only few issues still need to be addressed before the piece can be\nrecommended for publication.\n1. In the abstract and discussion, the authors present the \"capture-before-delivery\"\nmechanism and state that the results support it. In reality, as the authors correctly noted\nin the rebuttal, the presented structural data represent only snapshots along the catalytic\ncycle and cannot unambiguously confirm the temporal sequence of the binding events.\nTherefore, the mechanism is only hypothetical and should be presented as such, this is\nalso in line with the points raised by reviewer #4.\nResponse: We are very grateful to the reviewer for the positive comments on our\nrevision. We fully agree with the reviewer's point. Therefore, we have revised the\nwording of this model in both the Abstract and Discussion sections, now presenting it\nclearly as a hypothesis derived from the existing data.\nIn the Abstract, we have deleted the sentence \"Biochemically, we demonstrate that\nmidnolin captures substrates before delivering them to proteasomes through a\nsequential “capture-and-deliver” mechanism.\"\nIn the Discussion section, we revised \"Our findings support a sequential 'capture-\nbefore-delivery' mechanism...\" to \"Our findings lead us to propose a sequential\n'capture-before-delivery' model for midnolin-mediated proteasomal degradation.\".\n2. In Figure 1 and Figure 4, the authors present the EMReady treated maps. This is ok,\nbut it should be spelt out in the figures captions and the unsharpened maps should be\nthe one deposited to the PDB. Also, the statistics of the model refinements should be\nmade against the unsharpened map.\nResponse: We thank the reviewer for the professional suggestions. We have made the\nrequested clarifications and revisions accordingly.\nIn the figure legends for Figure 1 and Figure 4, we have stated that the displayed density\nmaps were post-processed using EMReady for optimal visualization. At the same time,\nwe emphasize that all maps deposited to the PDB and EMDB and used for the statistics\nof model refinements are the unsharpened maps. We have added relevant statements in\nthe Data processing section and the Model building and refinement section of the\nMethods.\n3. We also suggest that authors add a schematic diagram of the MIDN domain\ncomposition in linear form (similar to Figure 4a), with indications of the domains' start\nand end points in the sequence. This diagram would facilitate the interpretation of\nMIDN (337-468), MIDN (337-432), etc.\nResponse: This is an excellent suggestion that significantly improves readers'\nunderstanding of the different truncation constructs. We have added a linear schematic\ndiagram of the midnolin domain organization in revised Figure 1a.\nRevised Figure 1. Cryo-EM structures of the midnolin-26S proteasome complex\nin substrate-processing states.\n(a) Schematic representation of the midnolin (Full length, FL) and representative cryo-\nEM map of the midnolin-26S proteasome (a processing state, MIDN-PSRPT1),\nshowing the midnolin αHelix-C (magenta) interacting with the RPN1(PSMD2) of 19S\nregulatory particle. In the Figures, MIDN is short for midnolin. (b) Local refinement of\nRPN1 (pink) highlights the interaction with the midnolin αHelix-C (magenta). (c)\nAtomic model of midnolin αHelix-C and corresponding Cryo-EM map. (d) A low-pass\nfiltered map (15 Å) shows midnolin/substrate density (red) at the N-ring entrance of\n26S proteasome. (e) Cryo-EM map of the midnolin/substrate density in the central\nchannel, reaching through the N-ring, the ATPase ring, and into the degradation\nchamber of the 20S core particle. Density maps in a, b and e were post-processed with\nEMReady for optimal visualization.\n4. Another suggestion for figure improvement is to prepare the volume renders like in\nfig.4 d) (as well as in Figure 3b and Figure 4d), using the ChimeraX \"volume zone\"\ncommand with the \"new Map\" option, which will prevent \"hole\" artefacts in the\nextracted volume.\nResponse: Thank you for your professional review. This suggestion is particularly\nhelpful, and we had been looking for a way to generate cleaner volume renders without\nhole artifacts. Following your advice, we have regenerated Figures 1e, 3b, 3d, and 4d.\nThe revised figures now display the extracted volumes without holes, significantly\nimproving their clarity and visual quality.\n5. In some figure captions, the authors provide extensive details on the experimental\nsetup of the proteasome activity assay. Such information should be described in the\nrespective methods section, while keeping the figure caption concise and providing an\ninformative description of the results shown in the graphs.\nResponse: We agree with the reviewer's point. We have reviewed and streamlined all\nfigure legends involving proteasome activity assay experiments (Figures 2j, 2l, 3h, and\n4g). These legends now provide only brief descriptions of the experimental conditions\nand direct readers to the \"Proteasome stimulation activity assay\" section in the Methods\nfor detailed protocols.\n6. We are also bringing to the author's attention a few typos we noticed in the revised\ntext:\nOn page 2, line 38: underlie > should be underlying\nOn page 5, line 106: previously > previous\nOn page 10, line 218: Fig. 3d > Fig. 3e\nIn the caption of Figure 2: panels are described as \"top\"/\"bottom\" > should be\n\"left\"/\"right\"\nIn the caption of Figure 2, line 1099: the authors use the word \"equal\" when comparing\n300 nM and 600 nM affinities. More appropriate would be to say \"same order of\nmagnitude\" or similar\nIn the caption of Figure 3, it's unclear what the authors mean by \"representative maps\"\nAuthors should be precise and explicit on what maps they used. Instead, name the maps\nPS Ubl or PS Ubl_Catch if those are the final maps presented in Extended Data Figure\n7.\nResponse: We are very grateful to the reviewer for pointing these typos and unclear\ndescription out. We have made the corrections accordingly.\nPoint-by-point responses to the comments by Reviewer #2:\nThe authors addressed the concerns I raised fully in the revised manuscript. I have no\nfurther concerns.\nResponse: We thank the reviewer for the time and positive feedback.\nPoint-by-point responses to the comments by Reviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank the reviewer for the time and positive feedback.\nPoint-by-point responses to the comments by Reviewer #4:\nThe authors have addressed most of my concerns except several points:\n2. The authors state that “As expected, the complex shows a single, symmetric peak,\nwhereas midnolin alone showed significant oligomerization/aggregation, confirming\nthat substrate binding is essential for stabilizing the full-length midnolin.” However, I\ndidn’t find additional supporting data aside from Extended Data Fig. 6e, which only\nshowed the co-purification results.\nIn addition, I didn’t see a discussion about substrate selectivity in the revised Discussion.\nResponse: We are sorry for missing this data. We have revised Extended Data Fig. 6e,\nwhich shows obvious oligomerization/aggregation of full-length midnolin alone. We\nhave revised this manuscript as “As expected, the complex shows a single, symmetric\npeak, whereas midnolin alone shows obvious oligomerization/aggregation, confirming\nthat substrate binding is essential for stabilizing the full-length midnolin (Extended\nData Fig. 6e).”.\nRevised Extended Data Fig. 6e. Overlay of size-exclusion chromatography profiles\nfor MBP-midnolin purified alone (left, black line) and the co-expressed MBP-midnolin\nand EGR1(101-200)-mCherry complex (left, blue line). Coomassie brilliant blue\nstaining analysis of the purified samples revealed that MBP-midnolin co-expressed\nwith the substrate EGR1(101-200)-mCherry exhibited a homogeneous and\nmonodisperse state (right top), whereas MBP-midnolin expressed alone contained\nhigh-order oligomeric states (right bottom).\nThe discussion regarding substrate selectivity is indeed crucial. We have added a\nparagraph addressing substrate selectivity in the Discussion section.\n“The Catch domain of midnolin specifically recognizes a b-strand degron in IEG\nproteins by forming an FG zipper3,13. Our structural data show that the Catch domain\nis positioned directly above the proteasome entry port, suggesting that it may ensure\nefficient delivery and degradation of only its captured substrates through a spatial\nselection mechanism, thereby preventing off-target degradation of non-specific\nproteins. Furthermore, the possible co-expression mechanism of midnolin with its\nsubstrates may contribute to an additional regulation, ensuring that this pathway is\nactivated only when needed.”\n4. The functional assays are nicely performed. However, the conclusion in Line 228-\n230 is misleading. Based on the results from the “Catch-replaced midnolin” experiment\n(which is really a smart design), substrate binding does not necessarily equate to\nproteasome activation. A similar distinction may apply to the Ubl-RPN11 interaction,\nso the results in Fig. 3h only confirmed that this interaction is essential for stimulating\nproteasomal activities, but do not directly support its role in substrate delivery.\nTherefore, I recommend a minor revision.\nResponse: Thank you for your positive comments and constructive suggestions. We\nhave revised this sentence as “This finding directly confirms that these hydrophobic\nresidues are essential for the Ubl domain’s function in stimulating proteasomal\nactivities.”"}]

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