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[Preprint]. 2024 Aug 10:2024.08.09.604662.
doi: 10.1101/2024.08.09.604662.

An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

Alireza Ghanbarpour  1   2 , Bertina Telusma  1 , Barrett M Powell  1 , Jia Jia Zhang  1 , Isabella Bolstad  1 , Carolyn Vargas  3   4   5 , Sandro Keller  3   4   5 , Tania Baker  1 , Robert T Sauer  1 , Joseph H Davis  1   6
Affiliations

An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

Alireza Ghanbarpour et al. bioRxiv. .

Abstract

FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli. How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. The membrane curvature in our FtsH•HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsH's function in the degradation of membrane-embedded proteins.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.. Nautilus-like structure of a FtsH•HflK/C complex.
(A) Density map (unsharpened) and cartoon models of HflK (blue) and HflC (cyan) viewed from top. Nautilus opening into the HflK/C chamber noted. Map was resolved to a GS-FSC resolution of ~4.5Å (B) A sliced top view of the complex highlighting two FtsH hexamers (pink) interacting with SPFH domains of HflK within the nautilus chamber. (C) A side view of the nautilus super-complex, as shown in (A). Approximate location of the membrane depicted with periplasmic and cytoplasmic faces of the inner membrane noted. (D) A hat-like portion of the structure locally refined to ~3.5Å GS-FSC resolution, depicted as in (A). This map allowed for clear identification of HflK and HflC. The inset displays is a sharpened map and the model built using this map, highlighting bulky residues in helical regions of HflK and HflC.
Figure 2.
Figure 2.. HflK/C structures with different numbers of FtsH hexamers.
Density maps were low-pass filtered to 10 Å and colored using the underlying atomic model (HflK – blue; HflC – cyan, FtsH – pink; unassigned membrane or detergent – grey). (A) Two FtsH hexamers per super-complex as refined in Supplementary Figure 10. (B) One FtsH hexamer per super-complex as refined in Supplementary Figure 9. (C) FtsH-free super-complex affinity-purified from cells overexpressing HflC-FLAG and HflK, and refined as depicted in Supplementary Figures 11-12. (D) C4-symmetric structure with four FtsH hexamers (Qiao et al., 2022).
Figure 3.
Figure 3.. Nautilus-like FtsH•HflK/C structure isolated with native lipids via detergent-free extraction.
(A Density map highlighting the nautilus-like FtsH•HflK/C structure determined after affnity purification following detergent-free extractoin using Carboxy-DIBMA (see Methods). Map is color-coded (HflK, blue; HflC cyan; FtsH, pink) using a docked atomic model and is displayed from the top (left) and as a sliced side view (right). Key structural elements and scale bar noted. (B) 2D-class average of the Carboxy-DIBMA-extracted sample, highlighting membrane reshaping, as evidenced by the belt-like membrane structure that exhibits dual curvature as it passes through and extends beyond the particle.
Figure 4.
Figure 4.. FtsH-catalyzed lipid scramblase activity.
(A) Schematic representation of the scramblase activity assay, with expected fluorescence level in the absence (left) or presence (right) of enzymatic lipid scrambling activity. Scramblase activity assay of the FtsH•HflK/C complex (B) or isolated FtsH6 (C) using NBD-PE as a substrate. Note that the reported protein concentrations do not consider potential differences in proteoliposome reconstitution efficiency between samples, and that not all liposomes necessarily contain protein. Control reactions to asssess the FtsH-dependence of the measured scramblase activity include: 1) absense of scramblase activity using detergent-solubilized GlpG membrane protein (Ghanbarpour et al., 2021); and 2) NBD-glucose and BSA-back-extraction assays demonstrating that the measured scramblase activity was not a result of partially disrupted, leaky liposomes (Supplementary Figure 15).
Figure 5.
Figure 5.. Steady-state protein abundance measurements in wild-type and ΔhflK/C cells.
(A) Semi-quantitative proteomic measurements of protein abundance in wild-type or ΔhflK/C strains of E. coli. Proteins (n=274) exhibiting statistically significant changes (p<0.01) across 6 replicates are plotted following the measured rank order abundance change between cell types. A subset of proteins are labeled by gene name and colored according to their annotated localization. Among the highlighted ΔhflK/C-enriched proteins are the putative FtsH substrates lpxC, dadA, secY, and ylaC. (B) Signal intensity (quantified using precursor ion intensities with Spectronaut®) compared between wild-type and ΔhflK/C cells. Inner-quartile ranges are depicted as box plots, with dots indicating the abundance measured in each of 6 replicates. Changes in protein levels were statistically significant, with multiple hypothesis testing corrected q-values of 7.9*10−5 (secY), 3.6*10−11 (dadA), and 8.2*10−07 (ylaC).
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