Cryo-EM structure of the entire FtsH-HflKC AAA protease complex

Zhu, Qiao; Yokoyama, Takako; Yan, Xin-Fu; Beh, Ing Tsyr; Shi, Jing; Basak, Sandip; Akiyama, Yoshinori; Gao, Yong‐Gui

doi:10.1016/j.celrep.2022.110890

Cited by 29 publications

(47 citation statements)

References 55 publications

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“…Our sample preparation also preserved membrane-bound complexes. As an example, the AAA protease complex, formed by four hexamers of the AAA protease (ftsH) and 12 copies of each single-pass membrane proteins (HflK and HflC)[23], was recovered at high molecular weight in a broad peak as shown in Fig.2D. The large molecular weight range and sensitivity covered by our separation approach was also demonstrated in the recovery of more transient complexes such as the DNA polymerase III (dnaA, dnaE, and dnaQ) loaded with the γ complex (holA and dnaX) which plays a key role at the replication fork[24] (Fig.2E).…”

Section: Resultsmentioning

confidence: 99%

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Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

Fossati

Mozumdar

Kokontis

et al. 2023

Preprint

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Host-pathogen interactions (HPIs) are pivotal in regulating establishment, progression, and outcome of an infection. Affinity-purification mass spectrometry has become instrumental for the characterization of HPIs, however the targeted nature of exogenously expressing individual viral proteins has limited its utility to the analysis of relatively small pathogens. Here we present the use of co-fractionation mass spectrometry (SEC-MS) for the high-throughput analysis of HPIs from native viral infections of two jumbophages (phiKZ and phiPA3) in Pseudomonas aeruginosa. This enabled the detection >6000 unique host-pathogen and >200 pathogen-pathogen interactions for each phage, encompassing more then 50% of the phage proteome. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. Prediction of novel ORFs revealed a phiPA3 complex showing strong structural and sequence similarity to phiKZ nvRNAp, suggesting phiPA3 also possesses two RNA polymerases acting at different stages of the infection cycle. We further expanded our understanding on the molecular organization of the injected phage proteome by providing 23 novel virion components and 5 novel injected proteins, as well as providing the first evidence for phage manipulation of the host ribosome. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction \url{http://phagemap.ucsf.edu/}. We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of host-pathogen interactomes and protein complex dynamics upon infection.

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Section: Resultsmentioning

confidence: 99%

Section: A Cross-phage Study Of Viral Infection Cyclementioning

confidence: 99%

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

Fossati

Mozumdar

Kokontis

et al. 2023

Preprint

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“…Various important questions remain, such as the biophysical mechanism by which flotillins fluidize the membrane and how flotillins interact with other membrane proteins and lipids. An important clue may be found in the recently described structures of the HflK/C‐FtsH protease complex (Daumke & Lewin, 2022; Ma et al, 2022; Qiao et al, 2022). HflK and HflC are both proteins with a single transmembrane segment and an SPFH domain, and 12 subunits of each protein together were found to form a large membrane embedded ring that forms a cage like structure on top of the membrane outer leaflet which encloses four FtsH hexamers (Ma et al, 2022; Qiao et al, 2022).…”

Section: Current Questions Surrounding Flotillin Function and Fmmsmentioning

confidence: 99%

“…An important clue may be found in the recently described structures of the HflK/C‐FtsH protease complex (Daumke & Lewin, 2022; Ma et al, 2022; Qiao et al, 2022). HflK and HflC are both proteins with a single transmembrane segment and an SPFH domain, and 12 subunits of each protein together were found to form a large membrane embedded ring that forms a cage like structure on top of the membrane outer leaflet which encloses four FtsH hexamers (Ma et al, 2022; Qiao et al, 2022). The SPFH domains interact at the base of this cage, close to the membrane, with long α‐helices forming the barrel of the cage, which is closed on top by a β‐barrel formed of β‐strands from the individual subunits.…”

Section: Current Questions Surrounding Flotillin Function and Fmmsmentioning

confidence: 99%

Bacterial membrane dynamics: Compartmentalization and repair

Bramkamp

Scheffers

2023

Molecular Microbiology

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In every bacterial cell, the plasma membrane plays a key role in viability as it forms a selective barrier between the inside of the cell and its environment. This barrier function depends on the physical state of the lipid bilayer and the proteins embedded or associated with the bilayer. Over the past decade or so, it has become apparent that many membrane‐organizing proteins and principles, which were described in eukaryote systems, are ubiquitous and play important roles in bacterial cells. In this minireview, we focus on the enigmatic roles of bacterial flotillins in membrane compartmentalization and bacterial dynamins and ESCRT‐like systems in membrane repair and remodeling.

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“…Complexes of HflK and HflC, which are SPFH domain proteins, and FtsH, which is a membrane‐anchored AAA + protease, were purified from the E. coli membrane fraction after solubilization with a detergent and subjected to cryo‐electron microscopy (cryo‐EM), which revealed their high‐resolution supra‐assembly [ 6 , 7 ]. The HflK‐HflC and FtsH (KCF) complex has a 2.7 MDa structure, displaying a gigantic architecture with 12 copies of the HflK‐HflC dimer to provide a circular assembly embedded in detergent molecules.…”

mentioning

confidence: 99%

Higher‐order structure formation using refined monomer structures of lipid raft markers, Stomatin, Prohibitin, Flotillin, and HflK/C‐related proteins

Yokoyama

Matsui

2023

FEBS Open Bio

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Currently, information on the higher‐order structure of Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)‐domain proteins is limited. Briefly, the coordinate information (Refined PH1511.pdb) of the stomatin ortholog, PH1511 monomer, was obtained using the artificial intelligence, ColabFold: AlphaFold2. Thereafter, the 24mer homo‐oligomer structure of PH1511 was constructed using the superposing method, with HflK/C and FtsH (KCF complex) as templates. The 9mer‐12mer homo‐oligomer structures of PH1511 were also constructed using the ab initio docking method, with the GalaxyHomomer server for artificiality elimination. The features and functional validity of the higher‐order structures were discussed. The coordinate information (Refined PH1510.pdb) of the membrane protease PH1510 monomer, which specifically cleaves the C‐terminal hydrophobic region of PH1511, was obtained. Thereafter, the PH1510 12mer structure was constructed by superposing 12 molecules of the Refined PH1510.pdb monomer onto a 1510‐C prism‐like 12mer structure formed along the crystallographic threefold helical axis. The 12mer PH1510 (prism) structure revealed the spatial arrangement of membrane‐spanning regions between the 1510‐N and 1510‐C domains within the membrane tube complex. Based on these refined 3D homo‐oligomeric structures, the substrate recognition mechanism of the membrane protease was investigated. These refined 3D homo‐oligomer structures are provided via PDB files as Supplementary data and can be used for further reference.

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Cryo-EM structure of the entire FtsH-HflKC AAA protease complex

Cited by 29 publications

References 55 publications

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

Bacterial membrane dynamics: Compartmentalization and repair

Higher‐order structure formation using refined monomer structures of lipid raft markers, Stomatin, Prohibitin, Flotillin, and HflK/C‐related proteins

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