Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair

nature structural & molecular biology Article https://doi.org/10.1038/s41594-024-01401-8 Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair Received: 12 October 2023 Accepted: 11 September 2024 Published online: xx xx xxxx Check for updates Souvik Naskar Aurelien Roux , Andrea Merino2,5, Javier Espadas , Adai Colom 2,4 & Harry H. Low , Jayanti Singh1, 1,5 3 3 1 The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems. Endosomal sorting complex required for transport-III (ESCRT-III) family members are ancient membrane remodeling devices with an evolutionary lineage that traces back to the last universal common ancestor of cells1. Over time, the family has radiated across the tree of life, acquiring often essential and conserved functions. In eukaryotes and archaea, ESCRT-III systems drive membrane abscission during cell division2, promote viral replication and budding3,4 and mediate extracellular vesicle biogenesis5,6. Other eukaryotic functions include multivesicular body biogenesis7 and membrane repair8. In bacteria, in which PspA and its paralogue vesicle-inducing protein in plastids 1 (Vipp1/IM30) were discovered as ESCRT-III homologs1,9, these proteins function in membrane stress response and repair. PspA activity is triggered by agents that threaten inner membrane integrity, including phage, mislocalized secretins, and antibiotics10–14, whereas Vipp1 is a plastid component in cyanobacteria, algae, and plants, in which it functions in thylakoid membrane biogenesis and repair15–24. ESCRT-III family members have a conserved fold consisting of five helices, α1–α5 (refs. 1,25,26). Whereas helices α1 and α2 form a characteristic hairpin motif, in some systems helices α3–α5 switch between open, intermediate, and closed conformations 27. Some ESCRT-III family members, such as Vipp1, Vps2 (CHMP2), Vps24 (CHMP3), and Snf7 (CHMP4) supplement this fold with a membrane-binding amino-terminal motif or amphipathic helix (helix α0)13,28–30. Carboxy-terminal to helix α5 are less conserved elements1 that mediate protein interactions in most eukaryotic ESCRT-III systems 25. In this region, Vipp1 has a ~40-amino-acid C-terminal domain (CTD) that is flexible and may incorporate helix α6 (ref. 31). The CTD tunes Vipp1 polymerization dynamics both in vivo and in vitro 1,31–33. Using the core fold as a building block, ESCRT-III family members assemble filaments where the hairpin motif of neighboring subunits stack side by side, with helix α5 binding in a domain swop across the hairpin tip1,7,9,34,35. This filament is used to build different supramolecular structures, including spirals34,36–45, helical filaments9,34,44,46, and dome-shaped rings1,35. In bacteria, although Synechocystis Vipp1 (ref. 47) and PspA9 form planar patches, spiral filaments have not been reported, which Department of Infectious Disease, Imperial College, London, UK. 2Biofisika Institute (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain. 3Biochemistry Department, University of Geneva, Geneva, Switzerland. 4Ikerbasque, Basque Foundation for Science, Bilbao, Spain. 5These authors contributed equally: Souvik Naskar, Andrea Merino. e-mail: ; 1 Nature Structural & Molecular Biology Article https://doi.org/10.1038/s41594-024-01401-8 b a Helical-like ribbon Support bead Filament Ring Rings Helical-like ribbon Lipid film Helical-like ribbon Filament Filament Supported lipid bilayer Filament Unfurling 500 nm 50 nm c Helical-like ribbon 500 nm Vipp1 Membrane Droplet 50 nm Membrane Coverslip d Merge 0s 15 s 30 s 45 s 10 mM NaCl 500 mM NaCl 4 µm 5 µm f t = 45 s t = 30 s t = 15 s 80 60 5.4 s 5.9 s 9s Kymograph Membrane 100 4.9 s 40 20 0 0.75 1.00 1.25 1.50 1.75 Vipp1 Fluorescence intensity (AU) e Length (µm) 5 µm 5 µm Membrane Vipp1 Merge Fig. 1 | Vipp1 is a membrane sensor recruited to highly curved and perturbed membranes. a, Left pair, NS EM images showing Vipp1 purified in low-salt (10 mM NaCl) buffer. The area in the red dashed box is enlarged in the second image. Right pair, Vipp1 in a 500 mM NaCl buffer. The area in the red dashed box is enlarged in the second image. The unfurling of the helical-like ribbon is shown. Experiments were repeated independently more than three times. b, Preparation of SLBs. c, Fluorescent microscopy showing Vipp1Alexa488 recruitment to the highly curved membrane edge. Vipp1Alexa488 recruitment to the membrane edge is unaffected by ionic strength. The experiment was repeated independently three times. The area in the dashed box is shown in d. d, Timecourse showcasing dynamic Vipp1Alexa488 recruitment to the membrane edge. e, Fitted curves of the fluorescence plot profile show increasing Vipp1Alexa488 recruitment to the membrane edge. Measurements were collected from the area in the dashed box in d. f, Timecourse showing fusion of neighboring SLBs with Vipp1Alexa488 lost from the membrane merge point. The kymograph (right) is related to the region enclosed by the dashed box. The experiment was repeated independently three times. currently represents a key differentiating factor from their eukaryotic counterparts. The assembly of ESCRT-III filaments is fundamental to their membrane-remodeling mechanism, with the formation of planar spirals on the membrane being a key step. Current models describe spirals as loaded springs with elastic stress accumulating owing to a preferred radius of curvature. Stress is highest at the spiral perimeter and center, where the filament is under- or over-curved, respectively. This stress, which constitutes an energy store, is theoretically sufficient to bend the membrane41,48. Energy minimization, through the buckling of planar spiral filaments to three-dimensional (3D) polymers such as conical spirals or helices, is directly coupled to the mechanical shaping of the bound membrane. An important component of the (...truncated)