Large conformational changes in FtsH create an opening for substrate entry

AAA+ proteases are degradation machines, which exploit ATP hydrolysis to unfold protein substrates and translocate them through a central pore towards a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. Although cytoplasmic structures are described, the full-length structure of bacterial FtsH is unknown, and the route by which substrates reach the central pore remains unclear. We use electron microscopy to determine the 3D map of the full-length Aquifex aeolicus FtsH hexamer. Moreover, detergent solubilisation induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. FtsH structures reveal that the cytosolic domain can tilt with respect to the membrane. A flexible linker of ~20 residues between the second transmembrane helix and the cytosolic domain permits the observed large tilting movements, thereby facilitating the entry of substrate proteins towards the central pore for translocation.


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Cells are complex systems that rely on numerous tightly controlled vital processes. For instance, protein 22 quality control is crucial to the maintenance of the cell's proteome. To avoid the lethal accumulation of 23 misfolded or non-functional proteins, eukaryotes as well as prokaryotes use proteolysis (Barrett et al., 24 2012). In this process, peptide bonds are cleaved by proteases, and resulting amino acids (aa) are reused 25 2 to build new and functional proteins. This cycle allows cells to maintain their homeostasis and to keep them 26 healthy. It is then understandable that malfunctions in proteolysis lead to diverse forms of diseases (López-27 Otín and Bond, 2008;Richard, 2005).

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AAA+ proteases belong to the family of ATPases associated with various cellular activities, and are 29 molecular machines capable of unfolding and degrading proteins (Olivares et al., 2016). AAA+ proteases 30 share several structural and functional characteristics. They assemble into a barrel-shaped chamber with 31 a central pore formed by the ATP-binding domains. The pore entrance exhibits translocating loops with 32 highly conserved residues, which bind to target substrates. ATP-driven conformational changes of the ATP-33 binding domain unfold the bound substrate and translocate it through the central pore into the proteolytic 34 chamber for degradation. In general, bacterial AAA+ protease malfunctions can lead to a complete 35 discoordination of the cell homeostasis.

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From the five AAA+ proteases in E. Coli, FtsH is the only one that is anchored to the membrane and that 37 is essential (Bittner et al., 2017). FtsH plays a crucial role in membrane protein quality control (Hari and

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In mitochondria, the i-AAA protease, a FtsH ortholog, translocates polynucleotide phosphorylase into the 43 intermembrane space (Rainey et al., 2006), while the hydrophobicity of a specific transmembrane segment 44 dictates its dislocation from the inner membrane by the mitochondrial m-AAA protease, another FtsH 45 ortholog (Botelho et al., 2013). In humans, mutations in the gene coding for paraplegin, a subunit of m-46 AAA, are related to the severe disease spastic paraplegia (Nolden et al., 2005). Therefore, and because 47 the FtsH mechanics and structure are less well understood than those of cytoplasmic AAA+ proteases, 48 increasing our knowledge on the workings of FtsH are of both medical and fundamental interest.

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The FtsH protein comprises a N-terminal transmembrane helix, a ~75 aa periplasmic domain, a second 50 transmembrane helix (Scharfenberg et al., 2015), and the larger cytoplasmic AAA+ and protease domains 51 (Bieniossek et al., 2009). FtsH proteins assemble into hexamers, with 12 transmembrane helices inserting 52 3 into the lipid bilayer. The ATPase domain has conserved arginine residues that compose the second region 53 of homology (SRH), which is believed to be crucial for FtsH oligomerization. This domain also houses the 54 highly conserved Walker A and Walker B domains, which bind and hydrolyse nucleotides (Bieniossek et 55 al., 2009;Vostrukhina et al., 2015).

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Accordingly, a 13 Å gap between the membrane and the cytosolic domain observed by cryo-electron 72 microscopy would provide access to substrate, which implies that only (partly) unfolded proteins can reach 73 the translocating loops and be moved through the pore for degradation (Lee et al., 2011).

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Here we report the first full-length structure of Aquifex aeolicus FtsH (AaFtsH), which we determined by MgCl 2 and 25 µM ZnCl 2 just before the SEC purification. The SEC profile shows a first peak that is centred 93 at 12.1 mL ± 0.2 mL (SD, N=10) and a second peak at 13.4 mL ± 0.2 mL (SD, N=10) ( Figure 1A). Peak 94 positions are determined by simultaneously fitting two Gaussian functions. Native gel electrophoresis 95 suggests that the second peak likely contains hexameric AaFtsH as it runs at ~700 kDa, while the first peak 96 indicates a higher oligomeric state ( Figure 1B). The molecular weight of the eluted complexes was 97 estimated using the partition coefficient (K av ) values extracted from a calibration curve of the Superose 6 98 column, and the positions of fitted Gauss functions. The second peak is centred at a molecular weight of 99 730 kDa, which is larger than the size expected for AaFtsH hexamers (~432 kDa). Extrapolating the 100 calibration curve to smaller elution volumes, the first peak corresponds to a molecular weight of 940 kDa.

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Particles from the first peak appear to house two AaFtsH oligomers from the second peak, although their 114 average length is less than twice the average length of the smaller particles.

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The class average corresponding to the infrequent, approximately axial projections is compatible with the 125 hexameric nature of the small AaFtsH complex ( Figure 3A), while Figure 3B-D display representative side 7 or tilted views of AaFtsH hexamers. From such class averages EMAN2.12 calculated starting models and 127 refined one of them against all particle projections, imposing 6-fold symmetry. Figure

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Dimensions of FtsH subunits and their conformational changes. Using ten class averages from both 153 AaFtsH hexamers and dodecamers we measured that the detergent micelle, highlighted in green ( Figure   154 3E,L), has a thickness of 40 ± 4 Å for the hexamer and 43 ± 2 Å for the dodecamer, which is close to the 155 lipid bilayer thickness. The measured micelle/helical bundle width is 100 ± 18 Å for the hexamer and 126 ± 156 7 Å for the dodecamer. The estimated length of the 3D map for the full hexamer is 167 ± 5 Å (SD, N = 10) 157 and its width is 131 ± 7 Å (SD, N = 10). When compared with the dimensions of A. aeolicus FtsH crystal 158 structure, a similar width is reported (120 Å) (Suno et al., 2006). In hexamers, the cytoplasmic domain has 159 a height of 83 ± 7 Å, and the periplasmic domain has a height of 31 ± 3 Å and a width of 63 ± 6 Å. The

F F I G I W I F L L R Q M S G G G N V N R A F N F G K S R A K V Y I E E K P K SCRATCH: H H H H H H H H H H H H H H O O O O O O O E E E O O O O O O E E E E E E O O O O H:helical ; E:exten PREDICTPROTEIN: H H H H H H H H H H H H H H H L L L L L L L E E E E L L L L L E E E E L L L L L H:helical ; E:exten PRE-FOLD 3: H H H H H H H H H H H H C C C C C C C C C C E C C C C C H E E E C C C C C C C C H:helical (red) ; C:
CONSURF: conservation scale: dodecamers (Figures 2 and 3). Importantly, both fractions show highly similar specific ATPase activities:  29˚. TM2 corresponds to TM6 of AQP1 and is thus probably tilted by 36˚. Modelling helices as 10 Å wide 296 tilted cylinders, the cross section of the helical bundle is 6 x π x 5 2 (1/cos (29˚) + 1/ cos(36˚) ) Å 2 , and the 297 bilayer cross section inside the ring then amounts to 1700 Å 2 . Taking 50 Å 2 per lipid molecule into account,

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the ring houses about 34 lipid pairs, which make ~50 kDa. From the dimension of the membrane domain 299 with its LMNG belt measured by EM, the LMNG micelle and the lipid molecules are estimated to represent 300 19 ~290kDa, close to the molecular weight estimate from SEC. Such a molecular weight estimate is less 301 accurate for the AaFtsH dodecamer, because it elutes outside the range of calibration proteins used. In 302 addition, the hexamer has possibly different migration properties compared to the compact elongated 303 dodecamer composed of two intertwined hexamers (Figure 3).

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Further we report here the first full length bacterial FtsH 3D map at a resolution of 20 Å, which is related to 305 the negative staining used for visualizing the protein complexes, as well as their intrinsic flexibility. As 306 documented in Figure 3G this map accommodates X-ray structures of cytosolic and periplasmic fragments.

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The density of the X-ray structure of Aquifex aeolicus cytosolic domain (PDB 4WW0) rendered at 20 Å 308 resolution fits well with the cytosolic domain of the 3D map presented here. There is also a similar match

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The unexpected AaFtsH dodecamers consistently observed (Figures 1 and 2) exhibit highly variable 314 conformations ( Figure 3H and I), with periplasmic domains strongly tilted with respect to cytosolic domains.

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The resulting cell debris was purified at 20000 g for 15 min and membranes were isolated at 125000 g for

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The incubated sample was concentrated to 500 µL and loaded into a SEC Superose 6 Increase 10/300 GL 357 column (GE Healthcare) pre-equilibrated with 10 mM Tris-HCl pH8.0; 150 mM NaCl; 0.01%(w/v) LMNG; 358 5% Glycerol. AaFtsH monomer concentration was measured with a Nanodrop. A representative SEC run 359 is plotted in Figure 1A. The centre position of the two largest peaks was determined by fitting Gaussian

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Transmission Electron Microscopy analysis. 3 µL of AaFtsH dodecamer or hexamer fractions was 365 loaded on a carbon-coated 400 square mesh copper grid (Aurion) previously glow-discharged for 1 min.

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The liquid drop was absorbed with filter paper after 1 min and quickly washed with a drop of water that was 367 again blotted with filter paper. This procedure was repeated 3 times to rinse all the detergent present in the 368 samples. Finally, a 3 µL drop of 3 % uranyl-acetate was added to the grid, incubated for 1 min and absorbed 369 with a filter paper.