In Situ Architecture of the Human Prohibitin Complex

Biophysics Cell Biology Life Sciences
prohibitin complex mitochondrial inner membrane structural biology cryo-electron tomography molecular modeling

Academic Background

Mitochondria are the energy factories of cells, and the integrity of the mitochondrial inner membrane (MIM) is crucial for cellular function. Prohibitin (PHB) is a highly conserved protein family, consisting of two subtypes, PHB1 and PHB2, which play important roles in various cellular processes, including mitochondrial stress signaling, cell cycle regulation, apoptosis, and lifespan regulation. Although PHB1 and PHB2 are thought to function as scaffold proteins in the mitochondrial inner membrane, their molecular organization has long remained unclear. Previous studies using negative-stain electron microscopy (EM) in yeast suggested that PHB1/PHB2 heterodimers might form ring-like structures with a diameter of approximately 20 nm, but these speculations lacked direct experimental evidence. Therefore, elucidating the molecular structure of the human prohibitin complex is of great significance for understanding its role in maintaining the integrity and spatial organization of the mitochondrial inner membrane.

Source of the Paper

This study was conducted by a research team from the Max Planck Institute for Multidisciplinary Sciences, the University Medical Center Göttingen, Altos Labs, and other institutions. The primary authors include Felix Lange, Michael Ratz, Jan-Niklas Dohrke, and others. The paper was published in April 2025 in the journal Nature Cell Biology, titled “In situ architecture of the human prohibitin complex.”

Research Process

1. Protein Localization and Expression Regulation

The study began by using CRISPR-Cas9 gene editing technology to generate fluorescently labeled cell lines (PHB1-DK and PHB2-DK) endogenously expressing PHB1 and PHB2 in human U2OS cells. Through fluorescence microscopy and immunogold electron microscopy (immunogold EM), the researchers found that PHB1 and PHB2 are primarily located on the crista membrane (CM) of mitochondria, with their concentration being 3-5 times higher than in the inner boundary membrane (IBM). Additionally, fluorescence recovery after photobleaching (FRAP) experiments indicated that PHB1 and PHB2 exhibit low mobility on the crista membrane, further supporting their stable localization there.

2. Quantitative Analysis and Complex Abundance Estimation

Using quantitative Western blotting, the researchers determined that each U2OS cell contains approximately 3.38 × 10^6 PHB1 molecules and 3.46 × 10^6 PHB2 molecules. Based on these data, the researchers estimated that each U2OS cell may contain about 2.14 × 10^5 prohibitin ring-like complexes, with approximately 1.7 × 10^5 located on the crista membrane. Further calculations showed that each crista membrane, on average, contains about 15 prohibitin ring-like complexes, covering 0.7-1.4% of the crista membrane surface area.

3. Cryo-Electron Tomography (Cryo-ET) and Subtomogram Averaging

To directly observe the structure of the prohibitin complex, the researchers used cryo-electron tomography (cryo-ET) to image U2OS cells. Cryo-sections approximately 150 nm thick were prepared using focused ion beam (FIB) technology, and data were collected using a Titan Krios G2 microscope. The researchers observed numerous convex structures with a diameter of about 20 nm on the crista membrane, which appeared convex in side views with a height of approximately 9 nm. Through subtomogram averaging, the researchers obtained a three-dimensional structure of the prohibitin complex with a resolution of 16.3 Å. This structure revealed that the prohibitin complex is bell-shaped, composed of 11 alternating PHB1 and PHB2 molecules.

4. Molecular Modeling and Dynamic Simulation

Based on the PHB1 and PHB2 structures predicted by AlphaFold2, the researchers manually fitted 11 monomeric molecules into the cryo-EM density map, constructing a molecular model of the prohibitin complex. The model showed that PHB1 and PHB2 are embedded into the lipid bilayer through their N-terminal transmembrane domains, while their C-terminal coiled-coil domains stabilize the top of the complex through charge interactions. Furthermore, molecular dynamics (MD) simulations validated the stability of the model, revealing that the N-terminal domains are highly dynamic, whereas the C-terminal domains are more stable.

5. Crosslinking Mass Spectrometry (XL-MS) Validation

To further validate the molecular model, the researchers analyzed crosslinking mass spectrometry (XL-MS) data from mice and humans. The results showed that most crosslinked peptide pairs in the model had Cα-Cα distances of less than 30 Å, supporting the interactions of the C-terminal coiled-coil domains. Additionally, self-links were only satisfied at the PHB1-PHB1 or PHB2-PHB2 interfaces, further supporting the complex structure composed of 11 alternating PHB1 and PHB2 molecules.

Research Findings

  1. Bell-shaped Structure of the Prohibitin Complex: The study revealed, for the first time, the bell-shaped structure of the human prohibitin complex, which is composed of 11 alternating PHB1 and PHB2 molecules embedded in the mitochondrial inner membrane.
  2. Abundance and Distribution of the Complex: Each U2OS cell contains approximately 2.14 × 10^5 prohibitin complexes, with about 1.7 × 10^5 located on the crista membrane, and each crista membrane, on average, contains about 15 complexes.
  3. Stability of the Molecular Model: The stability and accuracy of the prohibitin complex model were validated through molecular dynamics simulations and XL-MS data.

Research Significance

  1. Scientific Value: This study is the first to reveal the structure of the human prohibitin complex at the atomic level, providing a structural basis for understanding its role in maintaining the integrity and spatial organization of the mitochondrial inner membrane.
  2. Application Value: The structural information of the prohibitin complex may offer new targets for developing therapeutic strategies for diseases related to mitochondrial dysfunction, such as neurodegenerative diseases and cancer.
  3. Technical Breakthrough: The study combined advanced techniques, including cryo-electron tomography, subtomogram averaging, molecular dynamics simulations, and crosslinking mass spectrometry, demonstrating the powerful advantages of interdisciplinary approaches in structural biology research.

Research Highlights

  1. In situ Structural Resolution: The study is the first to resolve the structure of the human prohibitin complex under in situ conditions, avoiding potential structural distortions from in vitro purification.
  2. Integration of Multiple Techniques: The study successfully integrated cryo-EM, molecular modeling, and XL-MS, providing a new research paradigm for resolving the structures of complex protein complexes.
  3. Cross-species Conservation: By comparing XL-MS data from mice and humans, the study revealed the structural conservation of the prohibitin complex across species, further validating its functional importance.

Other Valuable Information

The study also provided detailed data on the structure of the mitochondrial crista membrane, including the average length, spacing, and area of the crista membrane, which offer important references for future research on the relationship between mitochondrial structure and function. Additionally, the high-resolution cryo-EM techniques and molecular dynamics simulation methods used in the study provide technical references for resolving the structures of other protein complexes.