Cryo-EM structure of the prohibitin complex in open conformation

Significance

Prohibitins are implicated in diverse cellular processes and have emerged as promising therapeutic targets for treating cancers, neurodegenerative diseases, metabolic disorders, and inflammatory conditions. This study demonstrates that PHB1 and PHB2 assemble into a bell-like cage that functions as a membrane scaffold, facilitating compartmentalization of the MIM. The intrinsic flexibility of the cage wall enables a conformation switch of the prohibitin complex between open and closed states, promoting the turnover of proteins and lipids in the MIM. These structural insights provide a mechanistic framework for understanding PHB-mediated regulation of diverse cellular processes. Furthermore, drug-like small molecules could be developed to stabilize or destabilize the conformational states of the prohibitin complex, thereby ameliorating PHB-associated pathologies.

Abstract

Prohibitin 1 (PHB1) and Prohibitin 2 (PHB2), two conserved prohibitin members, are primarily localized to the mitochondrial inner membrane (MIM) to form a nanoscale macromolecular prohibitin complex. This prohibitin complex can facilitate the spatial organization of proteins and lipids, thus maintaining cellular metabolism and homeostasis, but its architecture remains largely unknown. Here, we report the cryo-EM structure of a prohibitin complex at 2.8 Å resolution, which contains 11 PHB1–PHB2 heterodimers. This complex displays a bell-like cage, consisting of a lid and a wall, which creates an intermembrane space-facing compartment for the MIM. The lid of the cage is stably assembled, and it is responsible for the prohibitin complex formation. In contrast, the wall of the cage is flexible and exhibits lateral openings, providing a channel for intramembrane exchange of proteins and lipids. These findings provide a structural basis for understanding the scaffold role of the prohibitin complex in organizing intramembrane proteins and lipids.

Prohibitins (PHBs) are highly conserved proteins ubiquitously expressed across eukaryotes (1). As central hubs for numerous signaling pathways, PHBs regulate critical processes such as lipid metabolism, mitochondrial homeostasis, and cell survival (2–11). The PHB1 and its homolog PHB2, primarily located in the mitochondrial inner membrane (MIM), play a vital role in mtDNA maintenance, protein quality control and homeostasis, oxidative phosphorylation system assembly, cristae structure maintenance, and apoptosis (12–20). Therefore, PHBs have emerged as promising therapeutic targets for treating cancers, neurodegenerative diseases, metabolic disorders, and inflammatory conditions (21–28). Several drugs targeting PHBs have been developed and evaluated for various clinical applications (29, 30).

PHBs belong to the SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) family, sharing common properties in self-oligomerizing into large membrane complexes and exhibiting a scaffold role in organizing functional membrane microdomains (FMM) (31–35). PHB1 and PHB2 have been reported to interact with each other to form a ring-like prohibitin complex with a diameter of 20 to 25 nm (36–38). However, the assembly of the prohibitin complex is poorly understood.

In recent years, several high-order structures of SPFH family proteins have been reported, such as the bacterial HflK–HflC complex, the QmcA–YbbJ complex, and the human flotillin complex (39–42). All these structures assemble into a giant molecular cage to confine a certain membrane fraction in the cell membrane. The prohibitin complex also acts as a membrane scaffold to organize both proteins and lipids, creating FMM with various protein and lipid compositions in the MIM (3, 6, 10, 18, 43, 44). It has been reported that these cages involved in organizing the membrane fractions are structurally closed with no observed channel for membrane fraction exchange (39–42). Thus, the structural basis of the SPFH family complexes controlling the turnover of the membrane fractions remains to be explored.

Using cryoelectron microscopy (Cryo-EM), we determined the structure of the prohibitin complex and found it to consist of 11 copies of PHB1–PHB2 heterodimers. The prohibitin structure exhibits a bell-like cage, composed of a lid and a wall, which creates a laterally segregated space to facilitate the compartmentalization of lipids and proteins in the MIM. The lid of the cage is stable and it is responsible for the prohibitin complex assembly, while its wall is flexible in charge of the turnover of proteins and lipids. Unlike solved structures of other SFPH family members, the cage-like prohibitin complex is not closed structurally, with several lateral openings at its cage wall, providing channels for membrane fractions. This study reveals a mechanism by which the prohibitin complex controls the organization of the FMM in the MIM, providing a basis for insight into the structural organization of the prohibitin complex.

Results

Overall Structure of the Prohibitin Complex.

To obtain a prohibitin complex sample, human PHB1 and PHB2 were coexpressed in human embryonic kidney (HEK) 293-F cells, with a Flag tag fused to the C-terminus of PHB1. Following mitochondrial isolation, the prohibitin complex was subjected to affinity purification and further purification via size-exclusion chromatography (SI Appendix, Fig. S1A). The elution profile and blue native PAGE (BN-PAGE) jointly confirm the formation of a prohibitin complex of over 880 kDa (SI Appendix, Fig. S1 A and B), which is consistent with previous reports (18). The peak fractions containing a prohibitin complex were pooled, concentrated, and applied to cryo-EM grids for data collection. The Cryo-EM micrographs show that the prohibitin particles exhibit an obvious bell-like structure, with the maximum diameter exceeding 20 nm (SI Appendix, Fig. S1C). Subsequent 3D classification analyses identified a predominant conformational class with a cage-like structure, enabling reconstruction of a final cryo-EM density map at a 2.8-Å resolution (SI Appendix, Fig. S2). Atomic models could be built in this cryo-EM map for the sequences of PHB1 and PHB2 (SI Appendix, Fig. S3).

The overall structure of the prohibitin complex displays an obvious “bell-like cage” architecture, enclosing a central chamber approximately 200 Å in diameter (Fig. 1 A and B). This structural arrangement suggests that the cage-like prohibitin complex creates a laterally segregated space for the MIM, potentially facilitating the compartmentalization of proteins and lipids. The prohibitin cage is composed of 11 subunits for each of PHB1 and PHB2, with the total of 22 subunits assembled in a PHB1-to-PHB2 alternating arrangement (Fig. 1 A and B). The PHB1 and PHB2 are conserved across eukaryotes (SI Appendix, Fig. S4), exhibiting a similar structure with a shared N-terminal single transmembrane domain (TM), a conserved SPFH domain, and two coiled-coil domains (CC1 and CC2), as well as a different C-terminal domain (CTD) (Fig. 1 C and D and SI Appendix, Fig. S5). The CTD of PHB1 contains β9 and β10, whereas that of PHB2 consists of β9, α7, and α8. Additionally, the TM domains of both PHB1 and PHB2 are invisible, potentially due to their flexibility. The SPFH domain and the CC1 domain form the wall of the cage around the central chamber, and the CC2 domain and the CTD form the lid of the cage above the central chamber (Fig. 1). To validate the above-mentioned structure, we integrated previously reported in vivo cross-linking mass spectrometry data (19). Notably, all lysine–lysine cross-linking pairs identified in the human PHBs are similar to corresponding lysine pairs observed in our structural model in interlysine spatial proximity (SI Appendix, Fig. S6), indicating that our structure probably represents a native prohibitin structure.

Fig. 1.

Overall structure of the prohibitin complex. (A and B) The cryo-EM map (Left) and atomic model (Right) of the prohibitin complex in a Bottom view (A) and Side view (B) of the cryo-EM map (Left) and atomic model (Right) of the PHB complex. Subunits of PHB1 and PHB2 are colored medium aquamarine and light salmon, respectively. (C) Schematic illustration of the domain organization of PHB1 and PHB2, with individual domains separately colored. (D) The atomic models of the PHB1 and PHB2. Secondary structural elements are colored as in (C).

Organization of the Lid Region of the Prohibitin Complex.

The lid of the prohibitin cage is an ~85-Å diameter disk, composed of 11 alternately tightly arranged subunits of each of PHB1 and PHB2 (Fig. 2 A and B). All the protein segments in the lid region show good densities, suggesting assembly stability of lid region of the prohibitin complex (SI Appendix, Fig. S2). At the center of the lid, there is a hole to the cage chamber with a hole diameter of ~13 Å (Fig. 2C). Although the top hole is also observed in other SPFH complex structures, the hole sizes are different (39–42). Additionally, the lid can be divided into three concentric circles, including the outer circle contains 22 CC2 domains; the middle circle, 22 β9 s; and the inner circle, 11 β10 s of PHB1 (Fig. 2C). The α7 and α8 from PHB2 sit on the outer circle and the middle circle, creating extensive interface with three adjacent subunits (Fig. 2C). The lid region of PHB1 and PHB2 is enriched with hydrophobic residues (Fig. 2B). A hydrophobic groove, enriched with hydrophobic residues, was observed between the middle circle and the interior side of the outer circle (Fig. 2 D and E). Eleven α7-α8 plug-like motifs bind to the hydrophobic groove through hydrophobic interactions (Fig. 2F), thus stabilizing the assembly of the lid of the prohibitin complex.

Fig. 2.

Structure and organization of the lid of the prohibitin complex. (A) Top view of the prohibitin complex. The lid region is highlighted with colored cartoons, while the wall region is shown in gray. (B) Amino acid sequence of the lid region of the PHB1 and PHB2. Hydrophobic residues are gray-shaded, the positively charged and negatively charged residues are colored blue and red, respectively. (C) Top view of the lid of the prohibitin complex. Only eleven subunits were shown; one of the PHB2 subunits was colored in magenta, highlighting the overall organization of the lid region. Three concentric circles indicate the circularly assembled CC2 domains, β9 s, and β10 s. (D) The hydrophobic surface of the CC2 and β9 circle. A hydrophobic groove was observed between the CC2 and β9 circles. (E) The α7-α8 plug-like motif from PHB2 CTD is shown. (F) Zoomed-in view of the boxed region in (C). Extensive hydrophobic interactions contribute to the formation of the lid region. Detailed hydrophobic interactions between the CTD of PHB2 and juxtaposed CC2 subdomains were shown. (G) The electrostatic surface of the CC2 and β9 circle. (H) The interior and exterior of the CC2 circle were shown in electrostatic surface (Left) and cartoon (Right). The charged residues were displayed as sticks.

Notably, the CC2 domains of PHB1 and PHB2 contain oppositely charged residues (Fig. 2B). Specifically, the CC2 domain of PHB1 contains mainly negatively charged residues (D and E), exhibiting negative electrostatic potential, whereas that of PHB2 possesses primarily positively charged residues (K and R), displaying positive electrostatic potential (Fig. 2 G and H). The alternating subunit arrangements of PHB1 and PHB2 may be driven by electrostatic interactions.

Essentiality of the Lid Region for Prohibitin Assembly.

Unlike the tightly assembled lid, the wall of the prohibitin complex appears more flexible, exhibiting a loose subunit organization (Fig. 1A). Thus, we hypothesize that the lid region might play a central role in facilitating the prohibitin complex assembly. To validate this hypothesis, we used a lid-region-truncation strategy to analyze the assembly of the prohibitin complex using BN-PAGE (Fig. 3A). We find that the lid-truncated PHB1 (residues 1 to 231) weakly interacts with wild-type PHB2, and the >880-kDa band representing the prohibitin complex is undetected (Fig. 3B). Additionally, deletion of the CTD (residues 1 to 251) from the lid region of PHB1 retains its ability to interact with wild-type PHB2, but the >880-kDa band is undetectable, indicating that CTD truncation might abolish the complex assembly capability (Fig. 3B). The truncations of the lid region (residues 1 to 245) or the CTD (residues 1 to 267) from PHB2 yielded similar results as we observed in PHB1 (Fig. 3C). These results suggested that both CTDs of the PHB1 and PHB2 are required for the assembly of the prohibitin complex from PHB1 and PHB2, and the CC2 domains are essential for the interaction between PHB1 and PHB2.

Fig. 3.

The lid region is the assembly core of the prohibitin complex. (A) Schematic illustration of the conserved domain organization of PHB1 and PHB2, and their lid region truncated variants. Lid truncations of PHB1 or PHB2 impair formation of the prohibitin complex. The truncated variants were coexpressed with their wild-type partner. For PHB1 truncations, the C-terminally Flag-tagged PHB1 was used as bait (B). For PHB2 truncations, the C-terminally Strep-tagged PHB2 was used as bait (C). The eluate was analyzed by BN-PAGE and corresponding antibodies.

To further investigate the role of the lid region in the assembly of the prohibitin complex, we employed the AlphaFold3-based structure prediction method to visualize structures with and without the lid region (45). The structure prediction using 11 subunits of each of PHB1 and PHB2 with their lid regions exhibited the bell-shaped cage architecture composed of a tightly assembled lid and a wall, as observed in the cryo-EM (SI Appendix, Fig. S7 A and B). The lid regions of the PHB1 and PHB2 in the predicted structure can be highly consistent with those presented in the cryo-EM structure (SI Appendix, Fig. S7C), namely, PHB1 and PHB2 were arranged in a similar conformation and strict alternating pattern. In contrast, when the lid regions were omitted from PHB1 and PHB2, the predicted structure lost this strict PHB1-to-PHB2 alternating arrangement (SI Appendix, Fig. S7D). These observations indicate that the lid regions may drive the alternating assembly of PHB1 and PHB2, which confirms the previous inference that the charge complementarity between the CC2 domains facilitates the alternating assembly of PHB1 and PHB2 (Fig. 2 G and H). The above findings suggest that the lid region is essential for the orderly assembly and alternating arrangement of PHB1 and PHB2 in the prohibitin complex.

Organization of the Wall Region of the Prohibitin Complex.

The wall region of the prohibitin complex is composed of 22 SPFH domains and 22 CC1 domains. In the cryo-EM structure, all 22 CC1 domains are visible, but only 10 SPFH domains are present (Fig. 1A). Unlike the symmetrical lid structure, the wall region presents an asymmetric conformation, with 6 different clusters arranged as 5 tetramers and 1 dimer (SI Appendix, Fig. S8). From the cytosol view of the prohibitin complex, we designated the dimeric module as DiM and the 5 tetrameric modules as TetM1 to TetM5 in a clockwise direction (Fig. 4A).

Fig. 4.

Organization of the wall of the prohibitin complex. (A) Top view of the prohibitin complex. Six modules of the wall region were gray-shaded. Opposite arrows indicate the channels between two adjacent modules. (B and C) Superposition of eleven PHB1 (B) and PHB2 (C) subunits in the prohibitin complex based on their lid region, respectively. (D) Bottom view of the prohibitin complex. The C-terminus of CC1 domains is stabilized by hydrogen bonds (yellow sticks) of the lid region. (E) Zoomed-in view of the boxed region in (D).

Superposition of the 11 PHB1 or 11 PHB2 subunits based on their lid regions reveals poor alignment of the SPFH domain and the portion (PHB1 CC1: G176–I211; PHB2 CC1: S190–A217) proximate to the SPFH domain, potentially suggesting a dynamic conformation of the wall of the prohibitin cage (Fig. 4 B and C). Notably, another portion of CC1 domain near the lid (far away from the SPFH domain) is stable, which might be attributed to stabilizing effect by CC2 domain (in the lid) and CC1–CC2 hydrogen bonds (Fig. 4 D and E). Interestingly, two hydrogen bonds are formed between the CC1 and CC2 domains (Q227–R255 and E231–R239) in PHB2, whereas only one hydrogen bond (D217–Y248) is formed in PHB1 (Fig. 4E), implying that the CC1 domain in PHB2 may adopt a more stable conformation than that in PHB1. Consistently, superposition of the 11 PHB1 subunits shows that PHB1 exhibits a broader conformational range than PHB2, indicating that PHB1 has a larger flexible region than PHB2 (Fig. 4 B and C). Overall, the above findings highlight the intrinsic flexibility of the wall region and reveal that the interaction between lid and wall further enhances the stability of the prohibitin cage wall.

Open Conformation of the Prohibitin Complex.

SPFH family proteins can oligomerize into nanoscale cage-like complexes to compartmentalize membrane microdomains (31, 39–42). So far, all solved structures of SPFH proteins have been reported to be closed, leaving no channels for the exchange of membrane components (SI Appendix, Fig. S9 B–E). Interestingly, in our cryo-EM structure, the 10 SPFH domains were present, respectively, in modules TetM1 (4 SPFH domains), TetM2 (4), and part of TetM3 (2), whereas the remaining 12 SPFH domains were not observed in TetM3, TetM4, TetM5, and DiM (SI Appendix, Fig. S8). The absence of nearly 55% SPFH domains (12/22) results in a pronounced notch in the wall of the prohibitin complex. This structural feature is in accordance with a recent report that the intracellular prohibitin complex adopts an open conformation, with approximately 25% of wall region missing (46). The presence of this notch may reflect structural dynamics of the prohibitin cage wall.

In addition, we also find that adjacent SPFH domains within a single module are tightly interacting (~7 Å distance), whereas SPFH domains located between neighboring modules are no longer interacting (~25 Å distance) (SI Appendix, Fig. S10). Moreover, the spacing between SPFH domains is proximate to the distances between their corresponding CC1 domains of the same subunit, when wall region is closed, while the spacing between SPFH domains is amplified, compared with the distance between CC1 domains, when wall region is open. For instance, a 6.8-Å separation between two CC1 domains results in a 6.6-Å distance between the corresponding SPFH domains, whereas a 10.9-Å separation between CC1 domains leads to a 25.9-Å gap between the SPFH domains (SI Appendix, Fig. S10 B and C). These findings suggest that the organization of SPFH domains is largely determined by the arrangement of the CC1 domains. It is especially true with the flexible assembly of the six structural modules. This structural flexibility may underlie the formation of a transient channel within the prohibitin cage wall, potentially enabling the regulation of protein and lipid turnover.

In Vivo State of the Prohibitin Complex.

To investigate the intraorganelle conformational state of the prohibitin complex, we performed a disulfide crosslinking assay to explore the subunit arrangement of the PHB cage wall in vivo. Six site-specific cysteine pairs were individually introduced into the wall region at either the 1 to 2 interface (PHB1–PHB2) or the 2 to 1 interface (PHB2–PHB1) (Fig. 5A). Mitochondria isolated from cells coexpressing the cysteine-introduced PHB1 and PHB2 variants were treated with oxidizing agent 4,4′-dipyridyl disulfide (4-DPS) to promote the formation of disulfide bonds between closely positioned cysteine residues. Compared with the wild-type PHB1 and PHB2 with a band of ~30 kDa, all cysteine pair-introduced PHB1 and PHB2 variants presented a band of ~70 kDa, indicating the formation of crosslinked PHB1–PHB2 or PHB2–PHB1 heterodimers (Fig. 5B).

Fig. 5.

Arrangement of the wall region of the prohibitin complex in vivo. (A) The PHB2–PHB1 dimer (2 to 1 dimer) and the PHB1–PHB2 dimer (1 to 2 dimer) within one module and the 2 to 1 dimer between two adjacent modules. Cystine pairs were introduced to the 1 to 2 interface (colored in blue) and the 2 to 1 interface (colored in red) at their wall region. (B) Crosslink of PHB1 and PHB2 with an individual cystine pair was introduced either at the 12-interface or at the 2-1 interface, separately. (C) A schematic diagram shows that the subunits in the opened or closed PHB complex will be crosslinked to different bands. Short black lines indicate that PHB1 and PHB2 were crosslinked by disulfide bonds. (D) Crosslink of PHB1 and PHB2 with cystine pairs was introduced to both the 1 to 2 interface and the 2 to 1 interface.

Next, we introduced cysteine pairs into the CC1 domain at both the 1 to 2 interface (PHB1^F198C and PHB2^A203C) and the 2 to 1 interface (PHB1^A189C and PHB2^F212C) to determine whether disulfide crosslinking could reveal clustered modules in the wall of the prohibitin complex. If the complex adopts the open conformation, disulfide bonds would form only within individual modules (rather than between modules), thus crosslinking heterodimers or tetramers (Fig. 5C). In contrast, if the complex adopts a closed conformation, disulfide bonds would form between all adjacent subunits, thereby forming a ring-like crosslinking product composed of 22 subunits (Fig. 5C). To exclude spontaneous crosslinking products generated before oxidant treatment, mitochondrial samples were pretreated with dithiothreitol (DTT).

Upon treatment with a low concentration of 4-DPS, we detected two predominant bands of approximately 70 kDa and 140 kDa, which were corresponding to the DiM and the TetM, respectively (Fig. 5D). The abundance of these bands increased proportionally with the increasing concentration of the oxidant. These findings indicate that DiM and TetM represent the fundamental assembly units of the prohibitin complex in vivo, which is consistent with previous reports of stable tetrameric assemblies in flotillin proteins (47). Moreover, at high concentrations of the oxidant, additional bands of far over 195 kDa emerged, which probably represent high-order assembly products comprising multiple TetMs and DiMs (Fig. 5D). This suggests that the prohibitin complex may coexist in two structural states: one structural state in which TetMs and DiM remain discrete as open conformation, and the other in which they are tightly assembled into a closed ring-like conformation. Taken together, these results support the coexistence of both open and closed conformations of the prohibitin complex in mitochondria.

Conformation Switch of the Prohibitin Complex.

The prohibitin complex assembles into a cage-like structure, adopting two distinct conformational states (open or closed) to exert its function (46). To explore the structural basis underlying conformational switch of the prohibitin complex, we performed a structural superposition of our cryo-EM structure representing the open state and AlphaFold-predicted model reflecting the closed conformation (Fig. 6A). The superposition reveals a good alignment in the lid region of the cage, but significant conformational difference in the wall region (Fig. 6 A and D). In the open conformation, the SPFH domains adopt blade-like radial arrangements (Fig. 6B), whereas in the closed conformation, these domains exhibit an enclosed ring-like structure (Fig. 6C).

Fig. 6.

Conformation switch of the prohibitin complex. (A) Superposition of the prohibitin complex in open conformation (experimentally solved structure) and closed conformation (predicted structure). (B) The prohibitin complex structure in open conformation. (C) The prohibitin complex in closed conformation. Black curves indicate the loose modules in the wall region, and the gray circle indicates that the wall region is fully closed. (D) Superposition of the prohibitin lid region in both the cryo-EM structure and the predicted structure. (E) Structural alignment of the tetrameric modules of the cryo-EM structure and the predicted structure. (F) Proposed working model of the prohibitin complex. An open-state prohibitin complex is displayed, which shows the potential channels (shown as black arrows) for protein and lipid exchange.

Structural alignment of individual TetMs revealed that a ~23° rotation can switch a closed conformation to an open one (Fig. 6E). This rotation also creates a lateral gap between adjacent modules, contributing to the overall opening of the cage. Based on these observations, we propose a model in which the conformation switch of six modules of the prohibitin complex facilitates the turnover of lipids and proteins, thus modulating membrane microdomain organization (Fig. 6F). In the resting state, the prohibitin complex adopts a closed conformation, with all six modules tightly assembled. In response to membrane microdomain reorganization, the prohibitin complex switches to an open conformation in which the modules adopt a looser arrangement. This conformational switch results in lateral openings, hence facilitating the turnover of proteins and lipids within the MIM.

Discussion

SPFH family proteins act as membrane-associated scaffolds that compartmentalize membranes to regulate key processes related to protein and lipid organization. Similar to all known structures of SPFH family complex (39–42), the cage-like structure of the prohibitin complex laterally compartments membrane microdomains, providing platforms for specific membrane-related processes. Different types of cages show different oligomerization. For instance, the reported HflK–HflC cage is assembled into a 24-mer cage, the QmcA cage into a 26-mer complex, and the FLOT1–FLOT2 into a 44-mer cage. In our prohibitin structure, the PHB1–PHB2 cage is composed of 22 subunits.

Although several structures of the SPHF cages from bacterial and eukaryotic cells have been solved, the mechanism underlying protein and lipid reorganization by such cages is still unknown due to the lack of an observed channel for the turnover of the membrane fractions (31, 39–42). In this study, we report an open conformational structure of the prohibitin complex, with cage wall clustered into tetrameric modules and dimeric module. This prohibitin cage exhibits lateral openings, potentially providing channels for the turnover of proteins and lipids. Interestingly, analogous tetrameric modules have been identified in other SPFH protein complexes. The flotillin proteins have been reported to organize into stable tetramers in membrane microdomains (47). The cage wall of the HflK–HflC complex is also composed of several tetrameric and dimeric modules, exhibiting the conformational plasticity of HflK–HflC interfaces (SI Appendix, Fig. S11) (41, 42). These modules might be dynamically assembled with varying distances between two adjacent modules, like what we observed in the cryo-EM structure of the prohibitin complex. This dynamic module assembly would facilitate conformational plasticity of cages, thereby providing access to the spatially restricted membrane microdomains.

The structural plasticity of the prohibitin complex suggests potential regulatory mechanisms governed by metabolic and environmental factors. A recent study indicates that the PHB conformational state can be regulated by the depolarizing agents oligomycin and antimycin A (46). In addition, the functions of PHBs are regulated by an array of posttranslational modifications (PTMs), including phosphorylation, O-GlcNAcylation, N-myristoylation, and palmitoylation (25, 29, 30). These PTMs may induce conformational rearrangements of the prohibitin complex to regulate its interactions with target proteins or lipids, thereby influencing membrane dynamics and signaling cascades (48).

Given the pivotal role of the prohibitin complex in mitochondrial quality control, pharmacological modulation of the prohibitin complex activity represents a promising therapeutic strategy for diseases linked to mitochondrial failure, such as cancer, inflammatory diseases, cardiomyopathy, and neurodegenerative diseases (21–23). Rational design of small molecules or peptide mimetics to stabilize the prohibitin complex or enhance its interaction with target proteins and lipids may provide perspectives for treating mitochondria-related diseases (29, 30). The insight into assembly and regulation mechanism of the prohibitin complex structure provides the theoretical basis for developing medications to ameliorate PHB-associated pathologies.

Materials and Methods

Transient Expression of the Human Prohibitin Complex.

The full-length coding sequences of PHB1 and PHB2 were amplified from cDNA of HEK293F cells (Invitrogen). All site-directed mutagenesis of PHB1 and PHB2 was carried out using the overlapping PCR method. For protein expression, all the gene fragments of PHB1 and PHB2 were subcloned into a modified pMlink vector with a C-terminal 3× Flag tag (DYKDHDGDYKDHDIDYKDDDDK) and a C-terminal twin-Strep tag (WSHPQFEKGGGSGGGSGGSAWSHPQFEK). HEK293F cells were routinely cultured in Union-293 medium (Union-Biotech Co. Ltd.) under the conditions: 5% CO2, 37 °C, and 110 rpm. The plasmids individually containing PHB1 and PHB2 were cotransfected into the cells with 4 kDa linear polyethylenimine (PEI) (Polysciences). The transfected cells were harvested after 48 h of culture.

Mitochondria Isolation.

The transfected cells were harvested by centrifugation at 800 g for 20 min and washed with cold PBS. Then, the washed cells were harvested and resuspended in buffer A containing 10 mM Tris-HCl pH 7.5, 70 mM sucrose, 210 mM mannitol, 1 mM EDTA, 1mg ml⁻¹ BSA, and 1 mM PMSF. Subsequently, the cells were disrupted using a Dounce homogenizer (Sigma) for 80 cycles on ice, and the resulting homogenate was centrifuged at 3,000×g for 10 min to remove nuclei and unbroken cells. The supernatant was collected and further centrifuged at 20,000×g for 20 min to obtain the crude mitochondrial pellet. The pellet was resuspended in buffer C containing 10 mM Tris-HCl, pH 7.5, 70 mM sucrose, 210 mM mannitol, and stored at −80 °C.

Purification of the Prohibitin Complex.

For the purification of PHB complex, the crude mitochondria were resuspended in a lysis buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 10% Glycerol, 1 mM EDTA, 1 mM PMSF, 4 μg ml⁻¹ pepstatin A, 4 μg ml⁻¹ aprotinin, and 4 μg ml⁻¹ leupeptin. The prohibitin complex was extracted with lysis buffer plus 1% (w/v) LMNG (Anatrace), 0.1% (w/v) CHS (Anatrace), and 0.25% (w/v) soybean lipid (Sigma) for 2 h in a 4 °C cold room. The homogenate was centrifuged at 4 °C at 20,000×g for 1 h to remove the insoluble pellets. The supernatant was incubated with 1 ml anti-Flag G1 affinity resin (GenScript) at 4 °C for 1.5 h. The resin was washed with 30 ml washing buffer W containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 10% Glycerol, 1 mM EDTA, 0.02% (w/v) GDN (Anatrace). Subsequently, the resin-bound proteins were eluted with buffer W plus 500 μg ml⁻¹ 3× Flag peptide. The protein solution was concentrated with a 100-kDa cut-off centricon (Milipore) and further purified by Superose-6 increase 10/300 column (GE Healthcare) in a buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% GDN. The peak fractions were pooled and concentrated to ~10 mg ml⁻¹ and immediately used for cryo-EM grid preparation.

Cryo-EM Grid Preparation and Data Acquisition.

For cryo-EM grid preparation, a 3.5-μl aliquot of the prohibitin complex was dropped onto glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh) followed by a 3-s blotting time with Vitrobot Mark IV (ThermoFisher Scientific) with 100% humidity at 8 °C. The blotted grids were immediately plunge-frozen in liquid ethane cooled by liquid nitrogen. All the grids were carefully transferred and stored in a liquid nitrogen environment until the data acquisition. All data collection was performed using a FEI Titan Krios G4 microscope operated at 300 kV and equipped with a Gatan K3 direct electron detector. The prohibitin complex movies were collected with the Thermo Scientific EPU software in counting mode, with 40 total frames per movie, a total dose of 50 electrons per Ų, a dose rate of 15.938 electrons pixel⁻¹ s⁻¹, an exposure time of 2.13 s, and a magnification of 105,000×g with a pixel size of 0.824 Å. All datasets were processed in CryoSPARC.

Data Processing of the Prohibitin Complex.

The schematic of the data processing pipeline is shown in SI Appendix, Fig. S2. From 6,088 micrographs, 5,251 were selected for analysis. Using the cryoSPARC blob picker, 2,285,948 particles were automatically picked (49). Following 2D classification in cryoSPARC, 1,809,233 high-quality particles were retained and subjected to multiple rounds of 3D classification. The best class, comprising 268,512 particles, was selected for further processing. These particles underwent nonuniform refinement followed by local refinement, yielding a cryo-EM density map for the PHB complex with an estimated resolution of 2.7 Å, determined using gold-standard Fourier shell correlation (FSC) (50). The assessment of local resolution was conducted using cryoSPARC.

Model Building and Refinement.

Initial structural models for PHB1 and PHB2 were generated using AlphaFold3 and rigidly fitted into the cryo-EM density map using ChimeraX (51). The fitted models were iteratively refined through manual adjustments in COOT (52). 11 molecules of PHB1 and 11 molecules of PHB2 were observed. The refined coordinates were further refined in real space using PHENIX, applying secondary structure and geometric constraints (53). The quality of the model was assessed using Molprobity scores and Ramachandran plots (54) (SI Appendix, Table S1).

Pull-Down Assay.

The C-terminal truncated PHB1 and PHB2 were similarly overexpressed with wild-type PHB2 and PHB1, respectively. The PHB1 truncated prohibitin complex was purified with anti-Flag G1 affinity resin (GenScript). The PHB2 truncated prohibitin complex was purified with Strep-Tactin Sepharose (IBA). The supernatant and eluates were subjected to SDS-PAGE and BN-PAGE, followed by analysis using Western blot with corresponding antibodies against the Flag-tag, Strep-tag, PHB1, and PHB2 (anti-flag, Cat No. 66008-3-Ig, Proteintech; anti-Strep, Cat No. ABT2010, Abbkine; anti-PHB1, Cat No. 10787-1-AP, Proteintech; anti-PHB2, Cat No. 12295-1-AP, Proteintech).

Blue Native-PAGE Analysis.

Blue native PAGE technique was used to determine the assembly state of the prohibitin complex. The purified prohibitin complex was mixed with 10 × loading buffer (0.1% (w/v) Ponceau S, 50% (w/v) glycerol) and subjected to electrophoresis using a 4 to 16% blue native PAGE at 4 °C for about 5 h. Cathode buffer A contained 15 mM Bis-Tris (pH 7.0), 50 mM Tricine, and 0.02% Coomassie G-250. Cathode buffer B was similar to cathode buffer A in formula, but 0.02% Coomassie G-250 was removed. Anode buffer contained 50 mM Bis-Tris pH 7.0.

Site-Specific Crosslinking.

We generated a cystine-free form of PHB1 (C69S) as a template to introduce cystine residues to specific sites. The cysteine-introduced sequences of PHB1 were cloned into pMlink plasmid with a C-terminal 3× Flag tag. For the PHB2, which is naturally cysteine-free, the cysteine-introduced sequences were subcloned into pMlink plasmid with a C-terminal twin-Strep tag. Mitochondria were isolated and stored at -80 °Cbefore use. Mitochondria thawed on ice and were washed with SEM buffer containing 20 mM HEPES/NaOH pH 7.4, 250 mM Sucrose, 1 mM EDTA, and 1 mM PMSF. Aliquot 100 µl mitochondria were incubated with a 4-DPS (Sigma) with a gradient concentration of 200 mM, 40 mM, 8 mM, 1.6 mM, 0.32 mM for 10 min. The reacted samples were centrifuged at 20,000×g for 5 min to remove the supernatant. Then, the pellet was washed with SEM buffer and mixed with 60 ml nonreducing SDS sampling buffer, followed by a 95 °C heating for 5 min. Finally, 10 µl heated sample was loaded onto SDS-PAGE and analyzed by western blotting with corresponding antibodies.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

3D EM map, atomic structure models data have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/emdb/) with the accession code EMD-64355 (55). The corresponding coordinates have been deposited in the Protein Data Bank (https://www.rcsb.org) with the accession 9UNL (56). All other data are included in the manuscript and/or SI Appendix.