Structures of human organellar SPFH protein complexes

Abstract

Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) family proteins are found in all kingdoms of life and in multiple eukaryotic organelles. SPFH proteins assemble into homo- or hetero-oligomeric rings that form domed structures. Most SPFH assemblies also abut a cellular membrane, where they are implicated in diverse functions ranging from membrane organization to protein quality control. However, the precise architectures of different SPFH complexes remain unclear. Here, we report single-particle cryo-EM structures of the endoplasmic reticulum (ER)-resident Erlin1/2 complex and the mitochondrial prohibitin (PHB1/2) complex, revealing assemblies of 13 heterodimers of Erlin1 and Erlin2 and 11 heterodimers of PHB1 and PHB2, respectively. We also describe key interactions underlying the architecture of each complex and conformational heterogeneity of the PHB1/2 complex. Our findings elucidate the distinct stoichiometries and properties of human organellar SPFH complexes and highlight common principles of SPFH complex organization.

Fig. 1. Cryo-EM structure of the Erlin1/2 complex.

a Cryo-EM map of the Erlin1/2 complex, with alternating Erlin1 and Erlin2 subunits colored pink and blue, respectively, viewed from the ER lumen (“top”, left) and from the ER membrane (right). b Side view of the cryo-EM map (left) and model of the Erlin1/2 complex superposed in a low-pass filtered map showing the detergent micelle (right) as a proxy for the ER membrane (dashed lines). The dimensions of the complex and placement of Erlin1/2 domains are indicated.

Fig. 2. SPFH and CC1 domain interactions in the Erlin1/2 complex.

a Surface representation of the Erlin1/2 complex model, with two individual subunits colored in pink and blue, and the other subunits colored according to the indicated domain. b Surface representations of the Erlin1/2 complex model colored by molecular lipophilicity potential (mlp: teal – most hydrophilic, dark goldenrod – most lipophilic, left) or by Coulombic electrostatic potential (right). Orange and red arrowheads note hydrophobic and acidic surfaces. c Model of the N-terminal transmembrane (TM) and SPFH1 domains of Erlin1 (pink) and Erlin2 (blue) with hydrophobic residues at the endoplasmic reticulum (ER) membrane interface indicated. d Model of alternating Erlin1 and Erlin2 subunits with the SPFH2 domains colored as in (c). Residues involved in intersubunit hydrogen bonds are colored yellow and indicated. Residues that contribute to an acidic patch and N-linked glycosylation sites (N-glyc) are also indicated. e Model of alternating Erlin1 and Erlin2 CC1 helices with hydrogen bonding interactions indicated. f Model as in (d) with the positions of disease-linked mutations indicated in purple.

Fig. 3. The C-terminal region of the Erlin1/2 complex forms a four-layered structure.

a Clipped side view of the Erlin1/2 complex cryo-EM map with the elements that form four structural layers at the narrow end of the ‘cage’ colored as indicated. b Top view of the Erlin1/2 complex model with the narrow end colored as in (a). c Isolated elements contributing to the four layers are colored as in (a). d View of 3 subunits of the Erlin1/2 complex showing hydrophobic packing and hydrogen bonding interactions involving the CC2 helices that facilitate the transitions from the CC1 helices and into β9 of the CTs. Residues outlined are identical between Erlin1 and Erlin2. Arrow indicates counter-clockwise (CCW) direction, when viewed from the top of the complex. e The C-terminal organization of 3 subunits in the Erlin1/2 complex, showing the IPNMF motifs, a potential hydrogen bond between the Erlin1 and Erlin2 CTs (top), and hydrophobic packing interactions (bottom). f Positions of disease-linked mutations (purple) in the CC2 and CT domains of Erlin1 (pink) or Erlin2 (colored by domain or blue).

Fig. 4. Cryo-EM structures of the PHB1/2 complex.

a “Top” views of cryo-EM maps of the closed (left) and open (right) conformations of 11 PHB1 (mauve, left or cream, right) and PHB2 (robin blue) heterodimers. b Side views of the maps as in (a), with the dimensions of each complex indicated. IMS, intermembrane space c Cryo-EM maps as in (a), viewed from the inner mitochondrial membrane, with the distinct PHB1/2 heterodimers indicated. Orange arrowheads indicate gaps visible in the open conformation.

Fig. 5. Comparisons of the closed and open PHB1/2 complexes.

a Superposition of a PHB1/2 heterodimer in the closed (mauve and robin blue) and open (cream and gray) PHB1/2 complex conformations. b Model of the PHB1/2 heterodimer in the closed complex conformation as in (a), colored by root mean square deviation (RMSD) values compared to the open complex. Domain features are indicated. c ‘Bottom’ view (top) of the closed and open PHB1/2 complex models with one heterodimer in each complex, colored as in (a) and the others colored according to domain, and a rotated view showing only an isolated heterodimer (bottom). The rotation, relative to the centroid of the complex (top), and the translation (bottom) of the N-terminal residue of the PHB1 (K177, orange) or PHB2 (R191, purple) CC1 helix from the closed (mauve and robin blue) to open (cream and gray) conformation are indicated. d Zoomed inset of the superposition as in (a), showing the distance shifted by the indicated PHB1 (orange) or PHB2 (purple) residues in the SPFH domains between the closed and open conformations. e Superposition of the isolated SPFH1 and SPFH2 domains of PHB1 and PHB2 in the closed and open PHB1/2 complex conformations, colored as in (a).

Fig. 6. Interactions in the PHB1/2 complex.

a Surface representation of the closed (left) or open (right) PHB1/2 complex model, with two individual subunits colored as indicated, and the other subunits colored by domain. The buried area at the intersubunit interface on each side of the indicated PHB2 subunit is listed. b Model of the N-terminal transmembrane (TM) and SPFH1 domains of PHB1 (mauve) and PHB2 (robin blue) with hydrophobic residues at the inner mitochondrial membrane (IMM) interface indicated. IMS, intermembrane space. c Model of alternating PHB1 and PHB2 subunits in the closed PHB1/2 complex with the SPFH2 domains colored as in (a). Residues involved in intersubunit hydrogen bonds via their sidechain (yellow) or backbone (mauve or robin blue) are indicated. Residues involved in hydrophobic packing are orange. d As in (c), but for the open PHB1/2 complex and PHB1 colored cream. Residues outlined maintain interactions in both the closed and open PHB1/2 complex. e Model of alternating PHB1 and PHB2 CC1 helices in the closed (left) or open (right) complex, with hydrogen bonding interactions indicated as in (c and d). f Clipped side view of the cryo-EM map (left) or top view of the model (right) of the closed PHB1/2 complex with the elements that form four structural layers at the narrow end of the cage colored as indicated. g Hydrogen bonding interactions involving the CC2 domains of PHB1 (mauve) and PHB2 (orange or robin blue), shown on the model of the closed PHB1/2 complex. Arrow indicates counter-clockwise (CCW) direction, when viewed from the top of the complex. h Hydrophobic packing interactions involving the CC2 and CT domains of PHB1 (mauve) and PHB2 (colored according to the indicated 4-layer feature or robin blue). Select residues of PHB1 (mauve), the PHB2 subunit in the clockwise direction (orange), or the PHB2 subunit in the counter-clockwise direction (robin blue) are indicated.

Fig. 7. Comparison of SPFH protein complexes.

a Side and b top views of the indicated membrane-associated SPFH protein complexes. c Side (left) and top (right) view of the major vault complex. Unique subunits are colored purple and pink (for SPFH proteins) or gray (for other associated proteins). All other copies of SPFH proteins in the complex are colored according to domain as indicated. The total number of SPFH protein subunits (in parentheses) and the relative stoichiometry of proteins (colored) in each complex are listed. d Plot of the length of the CC1 helix (based on the number of amino acids, aa) versus the number of subunits observed in experimentally determined structures of SPFH complexes, fitted with a linear regression (y = 0.1782x + 10.22). Source data are provided as a Source Data file.