Occupancy of the HbYX hydrophobic pocket is sufficient to induce gate opening in the archaeal 20S proteasomes

The proteasome is a molecular machine responsible for degrading misfolded, damaged, and unneeded proteins that are targeted via multiple mechanisms, including the conventional pathway of ubiquitination (Cohen-Kaplan et al., 2016; Bialek et al., 2023; Demasi and da Cunha, 2018; Khaminets et al., 2016; Makaros et al., 2023; Tsuchiya et al., 2020). Proteasomal degradation regulates major cellular processes and functions, such as synaptic plasticity and memory formation (Hegde, 2017; Park and Kaang, 2019; Patrick et al., 2023; Lip et al., 2017; Jarome and Devulapalli, 2018). Impairment of proteasome function has correlated with areas of the brain affected by neurodegenerative diseases, supporting its implication in these diseases (Keller et al., 2000; Ciechanover and Brundin, 2003; McNaught et al., 2001; Ortega et al., 2007; McKinnon and Tabrizi, 2014; McNaught et al., 2002). Furthermore, increasing proteasome activity has been shown to induce protective or rescue effects in models of neurodegenerative diseases (Anderson et al., 2022; Njomen and Tepe, 2019; VerPlank et al., 2019; VerPlank et al., 2020; Myeku et al., 2012; Wang et al., 2010), strengthening the rationale to develop small molecule proteasome activators. The objective of this study is to provide a clear mechanistic framework of how proteasome activation occurs to inform drug discovery efforts.

The eukaryotic proteasome, also referred to as the 20 S or the Core Particle (CP), consists of four stacked heteroheptameric (homoheptameric in archaea) rings arranged as α – β – β – α (Groll et al., 2000). Three of the seven β subunits carry protease sites with distinct protease activities: caspase-like (β1), trypsin-like (β2), and chymotrypsin-like (β5). The α subunits have N-termini that extend toward the central pore, forming a gate that regulates substrate entry. The gate is primarily formed by the N-termini of α2, 3, and 4 with α3’s N-termini (the longest) acting as the lynchpin (Groll et al., 2000). The interactions that stabilize the open conformation have been reported in crystal and cryo-EM structures (Groll et al., 2000; Rabl et al., 2008; Ding et al., 2019; Dong et al., 2019; Wehmer et al., 2017; Eisele et al., 2018; Förster et al., 2005; Chuah et al., 2023a). A YDR motif, found on the α N-termini, interacts with the neighboring α subunit’s N-terminal tail to stabilize the gate in the open state. Most recently, we discovered that the open and closed states of the α N-termini conformation are stabilized by two alternating conformations of the IT switch (Chuah et al., 2023a). Two α subunit residues, I12 and T13, in the archaeal Thermoplasma acidophilum proteasome (T20S), switch positions to occupy the pocket adjacent to Helix 0 and stabilize the open and closed gate conformation, respectively. This IT switch is functionally conserved in the eukaryotic 20 S (Chuah et al., 2023a). Basal kinetics of the archaeal proteasome gate, quantified using NMR (Religa et al., 2010), indicates that the gate fluctuates between open and close on a time scale of seconds; however, the gate primarily exists in the closed state of isolated 20 S.

The 20 S proteasome is known to associate with regulatory complexes, collectively referred to as Proteasome Activators (PAs) (Stadtmueller and Hill, 2011). PA700 or 19 S, which associates with the 20 S to form the 26 S, has been identified as a critical component of the Ubiquitin-Proteasome Pathway. PA700 recognizes ubiquitinated substrates and unfolds them to be degraded by the 20 S. Like many PAs, PA700 induces 20 S gate-opening via the HbYX-dependent mechanism, as does the archaeal homolog of PA700 called PAN (Stadtmueller and Hill, 2011; Smith et al., 2007). Three of the six C-terminal tails of PA700’s ATPases carry a HbYX motif (hydrophobic-tyrosine-almost any residue), whereby the ‘X’ residue must be the C-terminal residue. These HbYX motifs interact with the inter-subunit pockets in the 20 S α ring and allosterically induce gate opening. The precise molecular mechanism of how the motif’s binding in inter-subunit pockets induces gate opening is not known, though high-resolution structures of the 26 S (Ding et al., 2019; Dong et al., 2019; Wehmer et al., 2017; Eisele et al., 2018; de la Peña et al., 2018) and the 20 S interacting with a di-peptide HbYX-mimetic Chuah et al., 2023a have generated plausible models. Structures of the 26 S reveal that the C-terminal tails bind in a particular order during activation, though the correlation between the number of C-terminal tails/HbYX-bound to induce gate opening is controversial. A study indicated that partial gate opening occurs when Rpt2, 3, and 5, which have HbYX motifs, dock in their corresponding α inter-subunit pockets, and full gate opening occurs when Rpt6, which does not have a HbYX motif, additionally binds (Ding et al., 2019). Another study indicated that the binding of Rpt2, 3, and 5 did not open the gate, but the additional binding of Rpt6 causes full gate opening (Wehmer et al., 2017). Interestingly, multiple studies suggest that Rpt1, which carries the penultimate tyrosine, but not the Hb group (partial-HbYX), must also bind in combination with Rpt2, 3, 5, and 6 for gate opening (Dong et al., 2019; Eisele et al., 2018; de la Peña et al., 2018). Collectively, these structures suggest that each inter-subunit pocket contributes to gate opening to varying extents, which correlates with the fact that each α subunit’s N-terminal tails differentially participate in the closed state, though the minimum occupancy of inter-subunit pockets required for gate opening is unresolved.

While some PAs are HbYX-dependent like PA700, the 11 S family, such as PA26 or PA28 activate the proteasome via a HbYX-independent mechanism. Despite the divergence between mechanisms, they converge on the critical molecular interactions and conformational changes (e.g. the IT switch) that stabilize the open gate state (Chuah et al., 2023a). For example, both HbYX-dependent and HbYX-independent PAs have been shown to interact with αK66 side chain and mutating αK66 to alanine impedes PAs association and activation, validated by velocity sedimentation assay and proteasomal peptidase activity (Förster et al., 2005; Smith et al., 2007). Both mechanisms also involve a displacement of αPro17 (Förster et al., 2005; Chuah et al., 2023a) and switching of the IT switch (Chuah et al., 2023a). Therefore, though the cause of the conformational changes mentioned may differ between HbYX-dependent and HbYX-independent mechanisms, they share some of the PA-20S interactions that support the open conformational state of the gate.

Our early studies showed that the HbYX motif could function even on short 6–7 residue peptides (Smith et al., 2007) recently, we showed that HbYX-dependent gate opening could be reduced to a carboxybenzyl (CBZ)-blocked YA dipeptide called ZYA—a minimal HbYX mimetic. Our ZYA-T20S structure (Chuah et al., 2023a) indicated the minimum molecular interactions necessary for optimal HbYX-dependent gate-opening by a small molecule. In our 1.9Å resolution structure, ZYA was observed to interact with residues in the α subunit (G19, K66, L81, etc.) in a similar orientation and fashion as the native HbYX motif of various PA complexes. Our cryo-EM data indicated that αV24 (and αA154) hydrophobically interacts with the carboxybenzyl of ZYA and the hydrophobic side chain of the PAs HbYX motif. Interestingly, our previous study also indicated that a single residue mutation on αV24 (i.e. αV24Y, αV24F) increased proteasome activity, though the mechanism of activation by this mutation was not determined. To assess the significance of these mutations in activating the proteasome via the HbYX-dependent pathway, we determined the cryo-EM structure of αV24Y T20S and αV24F T20S. The results of this study indicate that occupancy of a hydrophobic pocket by a bulky hydrophobic group such as αV24Y within the α inter-subunit pocket is sufficient to trigger 20 S gate opening via the HbYX-dependent mechanism, demonstrating the surprising importance of the HbYX motif’s single Hb group for allosteric activation of the 20 S. The findings reported here are expected to translate to the human proteasome as ZYA is a robust activator of the mammalian 20 S and the HbYX-dependent mechanism is conserved; thereby, uncovering a highly specific pharmaceutical target for proteasome activation.

Proteasome impairment and reduced proteostasis are common observations in clinical cases and animal models of neurodegenerative diseases such as Alzheimer’s and Parkinson’s, which has inspired the search for small molecule proteasome activators for years. Yet, there is a lack of robust small molecule proteasome activators to date, in part due to a lack of understanding in proteasome activation mechanisms. The α inter-subunit pockets have long been known to be the binding site of PA complexes but the exact molecular interactions between the inter-subunit pockets and the PAs that cause gate-opening have been elusive, preventing efficacious design of proteasome-activating small molecules. While no FDA-approved in vivo proteasome-activating small molecule exists as a tool for proof of concept, our previous work (Anderson et al., 2022) to express constitutively active 20 S proteasomes in C. elegans indicated that the mutant proteasomes supported an increased resistance to proteotoxicities and increased lifespan, supporting the rationale for this study. Here, we present a single specific binding site within the inter-subunit pocket that when occupied, is sufficient to induce gate opening and, therefore, a plausible and well-defined pharmaceutical target for proteasome activators.

To identify the fundamental molecular interaction(s) required for gate-opening, we focused on the archaeal proteasome as it is homoheptameric, which is particularly an advantage when desiring a high- resolution cryo-EM structure to map detailed interactions. Moreover, the archaeal proteasome still carries the conserved structures involved in gating, and both human and archaeal 20 S can be opened by the same HbYX peptides (e.g. ZYA), implying that the findings of this study could be applied to the human system with some caveats discussed below. We recently demonstrated that the αV24Y mutant archaeal proteasomes exhibited upregulated activity (Chuah et al., 2023a). Our αV24Y T20S structure here reveals inter-subunit conformational changes essentially identical to a HbYX-bound archaeal proteasome (ZYA-T20S)(Chuah et al., 2023a), demonstrating that it is in an open state, allowing us to further elucidate how this single-residue mutation opens the gate, and by extension, better understand the mechanism of gate opening by the HbYX motif itself.

Collectively, our findings in αV24Y T20S focus on a mechanism of gate-opening originating at the occupancy of a hydrophobic pocket between αL21 and αT124. The αV24Y mutation places an aromatic side chain in between αL21 and αT124 (as well as αL21 and αA154), similar to the position of the benzyl group (Z) of ZYA in the ZYA-T20S structure. The occupancy of this hydrophobic pocket by αV24Y expands the adjacent pocket where αI12 of the IT Switch is previously found to bind in open gate proteasome structures. αL21, displaced by αV24Y’s larger side chain compared to αV24 (Figure 3C), is connected to the αP17 loop, which has been commonly shown to be displaced in open gate proteasome structures. The results here also suggest that this mechanism applies to the ZYA-T20S (Figure 4G), though it may not be the only mechanism supporting ZYA’s gate-opening as ZYA is a dipeptide as opposed to a single residue mutation (αV24Y) that occupies the hydrophobic pocket. For example, the YA residues bridge the subunit from αK66 to the αP17 loop, which also likely stabilizes the open state (Chuah et al., 2023a). The interaction of ZYA’s carboxy group with αK66 may also help stabilize the open-state conformation of αK66 (Figure 5B), which we showed is essential for gate-opening here (Figure 5E). The conformational changes between the two models are consistent, although αV24Y T20S exhibits larger conformational changes likely due to cryo-EM averaging effects in the lower populated ligand-bound states (ZYA) compared to the V24Y mutant. ZYA, as a dipeptide has the ability to and perhaps, even rely on interacting with other parts of the inter-subunit pocket to supports its binding and orient the Z group in the hydrophobic pocket. Results from both αV24Y T20S and ZYA-T20S both suggest that occupying the hydrophobic pocket is a key to gate opening in the archaeal proteasome, and potentially the mammalian proteasome if placed in the right pocket(s).

The two key residues that ‘sandwiches’ αV24Y’s side chain and form the hydrophobic pocket, αL21 and αA154, are conserved in the human 20 S α inter-subunit pockets (Figure 3—figure supplement 2), indicating that a similar mechanism of occupying this hydrophobic pocket is used for gate-opening in the human system. Five out of seven H20S α subunits carry leucine in a similar position to αL21 in the T20S. The remaining two subunits carry an isoleucine (α6: PSMA1) and valine (α7: PSMA3), which are hydrophobic like leucine. Additionally, while αA154 is only conserved as alanine in α1: PSMA6, the rest of the subunits carry a methionine in this position, which is also hydrophobic. Previous analysis (Chuah et al., 2023a) also indicated that the H20S has analogs of the IT switch in selected α subunits, which suggests that targeting this hydrophobic pocket at selected α subunits with the conserved IT switch would have a greater effect on gate-opening than others. Any application of this mechanism in the human system is still complicated and should also consider the prior prediction that multiple inter-subunit pockets likely need to be occupied by the HbYX motifs for gate-opening (Chuah et al., 2023b). The conservation of activation motif, residues, and conformational changes between archaeal and human proteasomes for gate opening demonstrates potential for the findings in this study to translate to activating the human 20 S.

Interestingly, our αV24F T20S structure indicates that the position of this residue in the protein chain plays a role in α subunit folding. Our αV24F T20S cryo-EM map reveals that the α subunits are partially assembled, with the majority of regions proximal to the gating residues lacking densities, suggesting disorder. Yet, the β subunits appear functional, both structurally and biochemically (Chuah et al., 2023a), which suggests that the proteasome does not require perfectly formed α subunits for complete proteasome assembly. We notice that the hydroxyl group of αV24Y’s side chain is involved in polar interactions; however, we do not presume this to be involved in α subunit assembly as valine lacks the side chain to interact similarly. Rather, we speculate that αV24F may introduce too large of a hydrophobic group that, without a hydroxyl to interact with αD152 (Figure 3H) prevents proper folding interactions.

Collectively, our analysis indicates that the HbYX hydrophobic pocket expands due to αV24Y’s more bulky side chain, which likely sets of a chain of events starting with the IT switch, which are modeled and schematized in Figure 8. The wider and now more hydrophobic IT switch pocket likely promotes IT switch to switch to the open state, providing flexibility for the αP17 loop intra-subunit motions, or ‘slack.’ In addition, the widening of the hydrophobic pocket and rearrangement of the αE25 H-bonding pattern also appears to allow more flexibility for αP17 loop motions. Combined, these intra-subunit conformational changes collectively elongate the αP17 loop, increasing its flexibility. This enhanced flexibility is crucial because the αP17 loop interfaces with Helix 0 of neighboring α subunits. The loop’s increased plasticity accommodates rigid-body rotations against the adjacent α subunits, which would otherwise be constrained. As a result, these rotational movements can propagate allosterically around the proteasome α ring. Though this causal chain of events is supported by these structures, the timing and order of these events cannot be elucidated with these static structures. The structures here suggest a plausible molecular mechanism of how a single residue mutation in the HbYX hydrophobic pocket can induce 20 S gate opening (Figure 8).

Figure 8

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Our findings in this study also highlight and revise an understanding of the αK66’s role in proteasomal gate-opening. Previous findings have consistently alluded to its critical role in the association of HbYX-dependent and HbYX-independent PAs, as αK66 was shown to interact with and orient the C-terminal tails of PAs for gate-opening (Förster et al., 2005; Chuah et al., 2023a; de la Peña et al., 2018; Hill et al., 2002). To our surprise, the αV24Y T20S structure demonstrates that αK66’s side chain conformation changes, even though it does not interact with a PA or HbYX mimetic such as ZYA. Our double mutant αV24Y&K66A T20S’s basal activity supports the necessity of the αK66 conformational change observed in the αV24Y model and indicates the involvement of αK66 in stabilizing the open-gate conformation through a previously unreported mechanism. The conformational change in αK66 is supported by changes in H-bonding interactions between αK66 and surrounding residues, which are absent in the closed structure (WT T20S). These findings, therefore, demonstrate that αK66’s conformational changes support other conformational changes such as the conformation of the back loop, which it is connected to, and has been shown to be involved in HbYX-dependent gate opening (Chuah et al., 2023a). Essentially, αK66 is deduced to contribute more than interacting with activators, and also appears to be necessary to stabilize the open state of the 20 S.

Our prior study showed the possible roles waters could play in proteasome activation and gating regulation. Our ZYA-T20S structure indicated that the hydroxyl of ZYA’s tyrosine side chain interacted with a water molecule that further hydrogen bonded with αL21 and αE25. To elucidate the relevance and importance of this water interaction, we mutated αE25 (Chuah et al., 2023a). αE25A proteasome mutants exhibited an upregulated basal activity that was not further activated by PAN or PA26, suggesting it plays an important role in gating. To elucidate whether a similar mechanism occurs in αV24Y T20S open gate conformation, we sought to identify where waters might be found within the inter-subunit pocket. Broadly, we observed significantly fewer waters in the inter-subunit pockets of αV24Y T20S than we did in the WT T20S and ZYA-T20S. We note that αV24Y T20S’s resolution is not as high as ZYA-T20S but is reasonable for predicting water coordinates. Because no common water was found in the two different open states, the necessity and role of specific water molecule interactions in gate-opening could not be elucidated from these structures.

Ultimately, the findings described indicate that the occupancy of the hydrophobic pocket within the α inter-subunit pocket is sufficient to induce gate-opening in archaeal proteasomes. The application of this finding in the mammalian system relies on further identification of the critical inter-subunit pockets that need to be occupied by the HbYX motif in that system to stabilize gate-opening. Our prior studies involving ZYA have indicated a minimum of two HbYX motifs binding for gate-opening in the mammalian system (Chuah et al., 2023b). However, the mammalian 20 S has 7 different pockets that can be occupied in different ways by 6 different Rpt1-6 C-termini, so the specific inter-subunit pockets that need C-termini/HbYX motifs bound to activate gate opening have not been worked out. All cryo-EM studies to date agree that occupancy of five Rpt C-termini is sufficient for gate opening, but which of those are required for gate opening is not known. Further progress on the HbYX mechanism based on the results thus far, would be to identify the specific inter-subunit pockets that need to be occupied by the HbYX motif to induce gate-opening in the eukaryotic proteasomes.

While this study provides significant insights, it is important to acknowledge certain limitations. A key limitation stems from using the homoheptameric archaeal T20S as our model. Although this simpler system allows for more reliable dissection of fundamental mechanisms, and core elements like HbYX-induced gate opening are conserved at the intersubunit pocket level, the overall T20S and eukaryotic 20 S/26 S proteasomes differ significantly in their complexity. Specifically, our engineered αV24Y mutation results in a tyrosine constitutively occupying all seven identical hydrophobic pockets. This contrasts with the eukaryotic proteasome, which possesses seven distinct α-subunit pockets that interact with various Rpt C-termini through dynamic binding. Moreover, the specific Rpt C-termini interactions—whether acting individually or cooperatively—that are essential to drive gate opening in the eukaryotic system remain incompletely understood. Therefore, while insights from our archaeal system are valuable for understanding general principles, direct comparisons and extrapolations to the intricate allostery and interaction complexities of the eukaryotic 26 S proteasome must be made with caution.

The work presented here is conducted with the intention of understanding proteasome activation mechanisms and developing the molecular framework for restoring or activating proteasome activity to combat the impairment consistently observed in neurodegenerative diseases, which is barred by our limited understanding of proteasome regulation. Here, we advance the field’s understanding of proteasomal gate-opening by extending beyond the vague idea that HbYX motif binding induces gate opening to pinpointing a sole molecular interaction that significantly and sufficiently opens the proteasome’s gate. Our findings inform proteasome-activating drug discovery efforts to focus on small molecules that occupy the HbYX hydrophobic pocket.

Cryo-EM maps are deposited in the Electron Microscopy Data Bank (EMDB) under accession codes, EMD-44914 (αV24F T20S) and EMD-44926, coordinates are available from the RCSB Protein Data Bank under accession codes, 9BUZ (αV24Y T20S), 8F7K (ZYA-T20S), 8F6A (WT T20S).

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Author details

1. Janelle JY Chuah

   Department of Biochemistry and Molecular Medicine, West Virginia University School of Medicine, Morgantown, United States

   Contribution

   Formal analysis, Supervision, Validation, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1139-694X

2. Madalena R Daugherty

   Department of Biochemistry and Molecular Medicine, West Virginia University School of Medicine, Morgantown, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0004-9274-4134

3. David M Smith

   1. Department of Biochemistry and Molecular Medicine, West Virginia University School of Medicine, Morgantown, United States
   2. Department of Neuroscience, Rockefeller Neuroscience Institute, West Virginia University, Morgantown, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   dmsmith@hsc.wvu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1502-676X

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