The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.014739
ISSN1083-351X
AutoresVanessa Carvalho, Irfan Prabudiansyah, Ľubomír Kováčik, Mohamed Chami, Roland Kieffer, Ramon van der Valk, Nick de Lange, Andreas Engel, Marie‐Eve Aubin‐Tam,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoAAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH. AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH. Cells are complex systems that rely on numerous tightly regulated vital processes. For instance, protein quality control is crucial for maintaining the cell's proteome. To avoid the lethal accumulation of misfolded or nonfunctional proteins, eukaryotes as well as prokaryotes use proteolysis (1Barrett A. Rawlings N. Woessner J. Handbook of Proteolytic Enzymes. Academic Press, London, UK2012Google Scholar). In this process, peptide bonds are cleaved by proteases and the resulting amino acids (aa) are recycled to build new and functional proteins. This cycle allows cells to maintain their homeostasis. It is then understandable that malfunctions in proteolysis lead to diverse forms of disease (2López-Otín C. Bond J.S. Proteases: multifunctional enzymes in life and disease.J. Biol. Chem. 2008; 283: 30433-30437Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 3Richard I. The genetic and molecular bases of monogenic disorders affecting proteolytic systems.J. Med. Genet. 2005; 42: 529-539Crossref PubMed Scopus (16) Google Scholar). AAA+ proteases belong to the family of ATPases associated with various cellular activities and are molecular machines capable of unfolding and degrading proteins (4Olivares A.O. Baker T.A. Sauer R.T. Mechanistic insights into bacterial AAA+ proteases and protein- remodelling machines.Nat. Rev. Microbiol. 2016; 14: 33-44Crossref PubMed Scopus (148) Google Scholar). AAA+ proteases share several structural and functional characteristics. They assemble into a barrel-shaped chamber with a central pore formed by the ATP-binding domains. The pore entrance exhibits translocating loops with highly conserved residues, which bind to target substrates. ATP-driven conformational changes of the ATP-binding domain unfold the bound substrate and translocate it through the central pore into the proteolytic chamber for degradation. In general, bacterial AAA+ protease malfunctions can lead to a complete discoordination of the cell homeostasis. From the five AAA+ proteases in Escherichia coli, FtsH is the only one that is anchored to the membrane and that is essential (5Bittner L.M. Arends J. Narberhaus F. When, how and why? Regulated proteolysis by the essential FtsH protease in Escherichia coli.Biol. Chem. 2017; 398: 625-635Crossref PubMed Scopus (35) Google Scholar). FtsH plays a crucial role in membrane protein quality control (6Hari S.B. Sauer R.T. The AAA+ FtsH protease degrades an ssrA-tagged model protein in the inner membrane of Escherichia coli.Biochemistry. 2016; 55: 5649-5652Crossref PubMed Scopus (10) Google Scholar) and in aminoglycoside antibiotic resistance, possibly by eliminating misfolded proteins disruptive to the membrane (7Hinz A. Lee S. Jacoby K. Manoil C. Membrane proteases and aminoglycoside antibiotic resistance.J. Bacteriol. 2011; 193: 4790-4797Crossref PubMed Scopus (52) Google Scholar). FtsH also regulates the phospholipid to lipopolysaccharide ratio in the outer membrane by degrading LpxC, the key enzyme of lipopolysaccharide biosynthesis (8Schäkermann M. Langklotz S. Narberhaus F. FtsH-mediated coordination of lipopolysaccharide biosynthesis in Escherichia coli correlates with the growth rate and the alarmone (p) ppGpp.J. Bacteriol. 2013; 195: 1912-1919Crossref PubMed Scopus (28) Google Scholar). In mitochondria, the FtsH ortholog called i-AAA protease translocates polynucleotide phosphorylase into the intermembrane space (9Rainey R.N. Glavin J.D. Chen H. French S.W. Teitell M.A. Koehler C.M. A new function in translocation for the mitochondrial i-AAA protease Yme1: import of polynucleotide phosphorylase into the intermembrane space.Mol. Cell. Biol. 2006; 26: 8488-8497Crossref PubMed Scopus (84) Google Scholar), whereas the hydrophobicity of a specific transmembrane segment dictates its dislocation from the inner membrane by the mitochondrial m-AAA protease, another FtsH ortholog (10Botelho S.C. Tatsuta T. von Heijne G. Kim H. Dislocation by the m-AAA protease increases the threshold hydrophobicity for retention of transmembrane helices in the inner membrane of yeast mitochondria.J. Biol. Chem. 2013; 288: 4792-4798Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). In humans, mutations in the gene coding for paraplegin, a subunit of m-AAA, are related to the severe disease spastic paraplegia (11Nolden M. Ehses S. Koppen M. Bernacchia A. Rugarli E.I. Langer T. The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria.Cell. 2005; 123: 277-289Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Therefore, and because the FtsH mechanics and structure are less well understood than those of cytoplasmic AAA+ proteases, increasing our knowledge on the mechanisms of FtsH is of both medical and fundamental interest. The FtsH protein comprises an N-terminal transmembrane helix, an ∼75-aa periplasmic domain, a second transmembrane helix (12Scharfenberg F. Serek-heuberger J. Coles M. Hartmann M.D. Habeck M. Martin J. Lupas A.N. Alva V. Structure and evolution of N-domains in AAA metalloproteases.J. Mol. Biol. 2015; 427: 910-923Crossref PubMed Scopus (13) Google Scholar), and the larger cytoplasmic AAA+ and protease domains (13Bieniossek C. Niederhauser B. Baumann U.M. The crystal structure of apo-FtsH reveals domain movements necessary for substrate unfolding and translocation.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 21579-21584Crossref PubMed Scopus (64) Google Scholar). FtsH proteins assemble into hexamers, with 12 transmembrane helices inserting into the lipid bilayer. The ATPase domain has conserved arginine residues that compose the second region of homology, which is believed to be crucial for FtsH oligomerization. This domain also houses the highly conserved Walker A and Walker B domains, which bind and hydrolyze nucleotides (13Bieniossek C. Niederhauser B. Baumann U.M. The crystal structure of apo-FtsH reveals domain movements necessary for substrate unfolding and translocation.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 21579-21584Crossref PubMed Scopus (64) Google Scholar, 14Vostrukhina M. Popov A. Brunstein E. Lanz M.A. Baumgartner R. Bieniossek C. Schacherl M. Baumann U. The structure of Aquifex aeolicus FtsH in the ADP-bound state reveals a C2-symmetric hexamer.Acta Crystallogr. D Biol. Crystallogr. 2015; D71: 1307-1318Crossref Scopus (10) Google Scholar). Structural studies have used truncated FtsH forms with only the soluble C-terminal (cytosolic) part (13Bieniossek C. Niederhauser B. Baumann U.M. The crystal structure of apo-FtsH reveals domain movements necessary for substrate unfolding and translocation.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 21579-21584Crossref PubMed Scopus (64) Google Scholar, 14Vostrukhina M. Popov A. Brunstein E. Lanz M.A. Baumgartner R. Bieniossek C. Schacherl M. Baumann U. The structure of Aquifex aeolicus FtsH in the ADP-bound state reveals a C2-symmetric hexamer.Acta Crystallogr. D Biol. Crystallogr. 2015; D71: 1307-1318Crossref Scopus (10) Google Scholar, 15Bieniossek C. Schalch T. Bumann M. Meister M. Meier R. Baumann U. The molecular architecture of the metalloprotease FtsH.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3066-3071Crossref PubMed Scopus (121) Google Scholar, 16Kim S.H. Kang G.B. Song H.E. Park S.J. Bea M.H. Eom S.H. Structural studies on Helicobacter pylori ATP-dependent protease, FtsH.J. Synchrotron Radiat. 2008; 15: 208-210Crossref PubMed Scopus (5) Google Scholar, 17Niwa H. Tsuchiya D. Makyio H. Yoshida M. Morikawa K. Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8.Structure. 2002; 10: 1415-1423Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Suno R. Niwa H. Tsuchiya D. Zhang X. Yoshida M. Morikawa K. Structure of the whole cytosolic region of ATP-dependent protease FtsH.Mol. Cell. 2006; 22: 575-585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 19Suno R. Shimoyama M. Abe A. Shimamura T. Shimodate N. Watanabe Y. Akiyama Y. Yoshida M. Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH.FEBS Lett. 2012; 586: 3117-3121Crossref PubMed Scopus (3) Google Scholar, 20Puchades C. Rampello A.J. Shin M. Giuliano C.J. Wiseman R.L. Glynn S.E. Lander G.C. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.Science. 2017; 358eaao0464Crossref PubMed Scopus (98) Google Scholar) or with only the periplasmic domain (12Scharfenberg F. Serek-heuberger J. Coles M. Hartmann M.D. Habeck M. Martin J. Lupas A.N. Alva V. Structure and evolution of N-domains in AAA metalloproteases.J. Mol. Biol. 2015; 427: 910-923Crossref PubMed Scopus (13) Google Scholar). The single full-length structure known concerns m-AAA, the yeast mitochondrial ortholog of bacterial FtsH, which has been resolved at 12 Å resolution by cryo-electron microscopy (cryo-EM) (21Lee S. Augustin S. Tatsuta T. Gerdes F. Langer T. Tsai F.T.F. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.J. Biol. Chem. 2011; 286: 4404-4411Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, no information on the conformational rearrangement of full-length FtsH in relation to the membrane when bound to nucleotides or to a substrate is available. Crystal structures of the cytosolic domain of FtsH exhibit a six- (13Bieniossek C. Niederhauser B. Baumann U.M. The crystal structure of apo-FtsH reveals domain movements necessary for substrate unfolding and translocation.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 21579-21584Crossref PubMed Scopus (64) Google Scholar), two- (14Vostrukhina M. Popov A. Brunstein E. Lanz M.A. Baumgartner R. Bieniossek C. Schacherl M. Baumann U. The structure of Aquifex aeolicus FtsH in the ADP-bound state reveals a C2-symmetric hexamer.Acta Crystallogr. D Biol. Crystallogr. 2015; D71: 1307-1318Crossref Scopus (10) Google Scholar, 15Bieniossek C. Schalch T. Bumann M. Meister M. Meier R. Baumann U. The molecular architecture of the metalloprotease FtsH.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3066-3071Crossref PubMed Scopus (121) Google Scholar), or threefold (18Suno R. Niwa H. Tsuchiya D. Zhang X. Yoshida M. Morikawa K. Structure of the whole cytosolic region of ATP-dependent protease FtsH.Mol. Cell. 2006; 22: 575-585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) symmetric conformation of the ATPase domain. These different conformations suggest that the ATPase domain could move polypeptides in steps as long as 45 Å into the central cavity during ATP hydrolysis cycles (13Bieniossek C. Niederhauser B. Baumann U.M. The crystal structure of apo-FtsH reveals domain movements necessary for substrate unfolding and translocation.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 21579-21584Crossref PubMed Scopus (64) Google Scholar). In contrast, the C-terminal protease domain always shows a sixfold symmetry for all crystal structures, i.e., the cytosolic domain of Thermus thermophiles FtsH (19Suno R. Shimoyama M. Abe A. Shimamura T. Shimodate N. Watanabe Y. Akiyama Y. Yoshida M. Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH.FEBS Lett. 2012; 586: 3117-3121Crossref PubMed Scopus (3) Google Scholar), Thermotoga maritima FtsH (13Bieniossek C. Niederhauser B. Baumann U.M. The crystal structure of apo-FtsH reveals domain movements necessary for substrate unfolding and translocation.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 21579-21584Crossref PubMed Scopus (64) Google Scholar, 15Bieniossek C. Schalch T. Bumann M. Meister M. Meier R. Baumann U. The molecular architecture of the metalloprotease FtsH.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3066-3071Crossref PubMed Scopus (121) Google Scholar), and Aquifex aeolicus FtsH (14Vostrukhina M. Popov A. Brunstein E. Lanz M.A. Baumgartner R. Bieniossek C. Schacherl M. Baumann U. The structure of Aquifex aeolicus FtsH in the ADP-bound state reveals a C2-symmetric hexamer.Acta Crystallogr. D Biol. Crystallogr. 2015; D71: 1307-1318Crossref Scopus (10) Google Scholar, 18Suno R. Niwa H. Tsuchiya D. Zhang X. Yoshida M. Morikawa K. Structure of the whole cytosolic region of ATP-dependent protease FtsH.Mol. Cell. 2006; 22: 575-585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The proposed mechanism for substrate entry in m-AAA is based on substrate recognition by solvent-exposed lateral regions of the FtsH cytosolic domain. Accordingly, a 13-Å gap between the membrane and the cytosolic domain observed by cryo-electron microscopy would provide access to substrate, which implies that only (partly) unfolded proteins can reach the translocating loops and be moved through the pore for degradation (21Lee S. Augustin S. Tatsuta T. Gerdes F. Langer T. Tsai F.T.F. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.J. Biol. Chem. 2011; 286: 4404-4411Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Here we report the first full-length structure of A. aeolicus FtsH (AaFtsH), which we determined with negative stain electron microscopy to a resolution of 20 Å and with cryo-electron microscopy to resolutions of 6.6 Å with sixfold symmetry and 16 Å without the symmetry imposed. Unexpectedly, upon detergent solubilization we found not only AaFtsH hexamers but also fully stable and active AaFtsH dodecamers. The dodecamer structure from negatively stained specimen was solved to a resolution of 25 Å, showing two AaFtsH hexamers sharing a single lauryl maltose neopentyl glycol (LMNG) micelle. This arrangement induces a tilt of the periplasmic domain with respect to the cytosolic domain. In this conformation, the periplasmic domain of one hexamer touches the cytosolic domain of the other hexamer. Cryo-EM analysis revealed a variety of S-shaped or V-shaped dodecameric structures with differing N-terminal interactions at resolutions from 12.3 to 20.5 Å. Since both AaFtsH hexamers and dodecamers have similar ATPase and proteolytic activities, we propose that the cytosolic domain tilts with respect to the membrane plane so that substrates can reach the translocating pore loops, as required for substrate unfolding and degradation. Such a large conformational change relates to the unique properties of the 20-aa linker between the end of the second transmembrane helix and the ATPase domain. Eliminating this linker leads to inactive AaFtsH hexamers that are not able to form dodecamers. AaFtsH with a C-terminal His-tag was overexpressed in E. coli cells and extracted from purified membranes with the use of a mild detergent, LMNG (22Chae P.S. Rasmussen S.G.F. Rana R.R. Gotfryd K. Chandra R. Goren M.A. Kruse A.C. Nurva S. Loland C.J. Pierre Y. Drew D. Popot J. Picot D. Fox B.G. Guan L. et al.Maltose–neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.Nat. Methods. 2010; 7: 1003-1008Crossref PubMed Scopus (286) Google Scholar). Ni-NTA chromatography followed by size-exclusion chromatography (SEC) using Superose 6 10/300 GL yielded pure AaFtsH complexes (Fig. 1). Best results were obtained by incubating the AaFtsH-containing fractions, collected from Ni-NTA chromatography, overnight at 60 °C in the presence of 20 mM ATP, 10 mM MgCl2, and 25 μM ZnCl2 before the SEC purification. The SEC profile shows a first peak that is centered at 12.1 ± 0.2 ml (SD, N = 10) and a second peak at 13.4 ± 0.2 ml (SD, N = 10) (Fig. 1A). The peak positions were determined by simultaneously fitting two Gaussian functions. Native gel electrophoresis shows that the second peak has an approximate molecular weight of ∼700 kDa, whereas the first peak indicates a larger complex (Fig. 1B). The molecular weight of the eluted complexes was estimated using the partition coefficient (Kav) values extracted from a calibration curve of the Superose 6 column and the positions of fitted Gaussian functions. The second peak is centered at a molecular weight of 730 kDa, which is larger than the size expected for AaFtsH hexamers (∼430 kDa), as expected owing to the weight contribution of the bound detergent micelle. On extrapolation of the calibration curve to smaller elution volumes, the first peak corresponds to a molecular weight of 940 kDa. To determine more precisely the molecular weight of AaFtsH oligomers and LMNG micelles, size-exclusion chromatography combined with static light scattering (SEC-MALS) was used. SEC-MALS accounts for the amount of detergent bound to a membrane protein and allows for a determination of the molecular weight of a protein in a protein-detergent mixed micelle (23Slotboom D.J. Duurkens R.H. Olieman K. Erkens G.B. Static light scattering to characterize membrane proteins in detergent solution.Methods. 2008; 46: 73-82Crossref PubMed Scopus (120) Google Scholar). SEC-MALS experiments of AaFtsH resulted in a molecular weight of 427 kDa for the protein in the second peak, which corresponds well to the molecular weight expected for an AaFtsH hexamer (430 kDa). The first peak has a molecular weight of 810 kDa, indicating a higher oligomeric state, close to the molecular weight expected for two hexamers. The molecular weight of LMNG micelle in the first and second peaks determined by SEC-MALS was 286 and 218 kDa, respectively (Fig. S1 and Table S1). These values are close to the molecular weight expected for LMNG micelles calculated with MALDI-TOF MS (24Chaptal V. Delolme F. Kilburg A. Magnard S. Montigny C. Picard M. Prier C. Monticelli L. Bornert O. Agez M. Ravaud S. Orelle C. Wagner R. Jawhari A. Broutin I. et al.Quantification of detergents complexed with membrane proteins.Sci. Rep. 2017; 7: 41751Crossref PubMed Scopus (32) Google Scholar). Transmission electron microscopy (TEM) was used to visualize samples from each SEC peak. Negative stain EM analysis of particles from the second peak, with molecular weight compatible with AaFtsH hexamers, exhibits structures with an average length of 154 ± 13 Å (SD, N = 280) (Fig. 1C). 2D Class averages of the negative stain preparation were calculated from 15,000 images of AaFtsH hexamer particles using the image processing software packages Scipion1.1 (25Rosa-trevín J. M. De Quintana A. del Cano L. Zaldívar A. Foche I. Gutiérrez J. Gómez-blanco J. Burguet-castell J. Cuenca-alba J. Abrishami V. Vargas J. Otón J. Sharov G. Vilas J.L. Navas J. et al.Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy.J. Struct. Biol. 2016; 195: 93-99Crossref PubMed Scopus (213) Google Scholar) and EMAN2.12 (26Ludtke S.J. Single-particle refinement and variability analysis in EMAN2.1.Methods Enzymol. 2016; 579: 159-189Crossref PubMed Scopus (48) Google Scholar). Figure 2, A–D displays representative side or tilted views of AaFtsH hexamers. Using 10 class averages from AaFtsH hexamers, we measured that the estimated length for the full hexamer is 167 ± 5 Å (SD, N = 10) and its width is 131 ± 7 Å (SD, N = 10). When compared with the dimensions of A. aeolicus FtsH cytosolic crystal structure, a similar width is reported (120 Å) (18Suno R. Niwa H. Tsuchiya D. Zhang X. Yoshida M. Morikawa K. Structure of the whole cytosolic region of ATP-dependent protease FtsH.Mol. Cell. 2006; 22: 575-585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The cytosolic domain has a height of 83 ± 7 Å, and the periplasmic domain has a height of 31 ± 3 Å and a width of 63 ± 6 Å (Table S5). Finally, the detergent micelle, highlighted in green (Fig. 2E), has a thickness of 40 ± 4 Å, which is close to the lipid bilayer thickness, and width of 100 ± 18 Å. From the 2D class averages, EMAN2.12 calculated starting models and refined one of them against all particles, imposing a sixfold symmetry. Figure 2F displays the 20-Å resolution 3D map of the AaFtsH hexamer, which accommodates the crystal structure of the AaFtsH cytosolic domain (PDB 4WW0) and the E. coli FtsH periplasmic domain (PDB 4V0B; Fig. 2G). Albeit the resolution achieved is only 20 Å, the orientation of the cytosolic domain with respect to the detergent micelle is clearly visible. Some of the hexamer classes show that the cytosolic domain is tilted in relation to the detergent micelle, creating a gap for substrate entry (Fig. 2, A–D). Cryo-EM was then performed to visualize samples from the second SEC peak. 2D Class averages calculated from the initial dataset of 35,048 images of AaFtsH hexamer particles (Fig. 1E) using Relion 3.0.8 showed a large structural variability. In full-hexameric classes, the well-formed cytosolic domain displays high-resolution features (e.g., C-terminal helices), whereas the N termini are fuzzy (Fig. 2H). Owing to a small number of particles remaining in the dataset after 3D classification (5649), 3D refinement was initially performed in C6 symmetry. The acquired 3D map revealed a protein structure similar to the negatively stained one, with clearly discernible cytosolic and N-terminal (periplasmic + transmembrane) domains (Fig. 2I, Table S5). The diameter of the cytosolic domain is 140 Å, length along the C6 axis is 134 Å, and the nominal FSC0.143 resolution determined by Relion is 6.6 Å. The cytosolic chamber could be fitted with six copies of the 4WW0 X-ray model comprising residues 141 to 608 in a similar way to the negatively stained 3D structure. The fitted 4WW0 subunits suggest the presence of a central orifice in the proteolytic domain, but the cryo-EM structure lacks it, likely owing to insufficient resolution and the introduction of the sixfold symmetry. On the other hand, the fitted ATPase domains form an ∼20-Å ring entrance into the proteolytic chamber, in agreement to structures of other proteases (20Puchades C. Rampello A.J. Shin M. Giuliano C.J. Wiseman R.L. Glynn S.E. Lander G.C. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.Science. 2017; 358eaao0464Crossref PubMed Scopus (98) Google Scholar, 27Botos I. Lountos G.T. Wu W. Cherry S. Ghirlando R. Kudzhaev A.M. Rotanova T.V. de Val N. Tropea J.E. Gustchina A. Wlodawer A. Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery.Curr. Res. Struct. Biol. 2019; 1: 13-20Crossref PubMed Scopus (5) Google Scholar, 28Steele T.E. Glynn S.E. Mitochondrial AAA proteases: a stairway to degradation.Mitochondrion. 2019; 49: 121-127Crossref PubMed Scopus (4) Google Scholar, 29Rotanova T.V. Andrianova A.G. Kudzhaev A.M. Li M. Botos I. Wlodawer A. Gustchina A. New insights into structural and functional relationships between LonA proteases and ClpB chaperones.FEBS Open Bio. 2019; 9: 1536-1551Crossref PubMed Scopus (7) Google Scholar). Adjacent to the first residue of the fitted crystal structures, M141, a narrow density extends from the cytosolic domain toward the inner part of the N-terminal domain and joins an inner ring of ∼40 Å outer diameter and ∼18 Å inner diameter, which is formed by all six N termini (Fig. 2J). From this ring, six symmetric densities extend further upward. Each of them then makes an outward U-turn of 2 × 90° and subsequently joins the cytosolic domain again on the outer surface. The crystallized 4V0B structure of the periplasmic domain could be fitted in the top of the U-turn density in the expected position between the two transmembrane helices (Figs. 2I and S2). However, the fit was unreliable (avg. CC = 0.0095 by rigid-body fitting in UCSF Chimera). In order to investigate the dynamics of the structure, we performed a local resolution analysis in Relion, which indicated that the resolution varied from the highest 6.6 Å in the periplasmic domain to less than 10 Å in the cytosolic domain. This result, together with the presence of the closed channel in the cytosolic chamber (Fig. 2I), suggested that the symmetry constraint should be released. Therefore, we also resolved the structure of the AaFtsH hexamer without imposing symmetry at 15.9 Å resolution, using only 2129 particles (Fig. 2K). It possesses a distorted cytosolic domain, which could still be fitted with six individual 4WW0 subunits. Its N-terminal domain is fully disordered, and only one subunit seems to partially follow the folding of the C6-symmetric structure. All six N termini bundle into an indistinguishable, tilted off-axis mass, which was already observed in the negatively stained class averages (Fig. 2, A–D). The N termini do not form any inner ring; instead, they give rise to a wide opening between the membrane and the cytosolic domain (Fig. 2K) for substrate entry. Finally, we attempted to find out if the cytosolic domain of the asymmetric FtsH cryo-EM map possesses the staircase arrangement observed in other AAA ATPases (20Puchades C. Rampello A.J. Shin M. Giuliano C.J. Wiseman R.L. Glynn S.E. Lander G.C. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.Science. 2017; 358eaao0464Crossref PubMed Scopus (98) Google Scholar, 27Botos I. Lountos G.T. Wu W. Cherry S. Ghirlando R. Kudzhaev A.M. Rotanova T.V. de Val N. Tropea J.E. Gustchina A. Wlodawer A. Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery.Curr. Res. Struct. Biol. 2019; 1: 13-20Crossref PubMed Scopus (5) Google Scholar) with the help of the high-resolution structure of the YME1 protease (20Puchades C. Rampello A.J. Shin M. Giuliano C.J. Wiseman R.L. Glynn S.E. Lander G.C. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.Science. 2017; 358eaao0464Crossref PubMed Scopus (98) Google Scholar). Its model (PDB ID: 6AZ0) describes an active state of YME1 during substrate processing, with four subunits bound to ATP, one to ADP, and one free of nucleotides, which gave rise to an asymmetric cytosolic domain (Figs. 1, 4, and 5 in (20Puchades C. Rampello A.J. Shin M. Giuliano C.J. Wiseman R.L. Glynn S.E. Lander G.C. Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.Science. 2017; 358eaao0464Crossref PubMed Scopus (98) Google Scholar)). We had incubated the FtsH with ATP; therefore, its individual subunits may be binding ATP, ADP, or no nucleotide. Since the amino acid sequences of the ATPase and proteolytic domains of A. aeolicus and the YME1 protease are highly similar, we attempted to fit the 6AZ0 model of the YME1 cytosolic domain into the cytosolic domain of FtsH cryo-EM map. As a result, the FtsH map and the YME1 model shared the asymmetric appearance induced by the nucleotide-dependent conformational changes (Fig. 2L, CC = 0.13 by rigid-body fitting in UCSF Chimera). Negative stain EM was also performed on the first SEC peak revealing elongated particles with an average length of 231 ± 15 Å (SD, N = 280; Fig. 1D). Particles from the first peak appear to house
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