Cryo-EM structure of transmembrane AAA+ protease FtsH in the ADP state

Abstract

AAA+ proteases regulate numerous physiological and cellular processes through tightly regulated proteolytic cleavage of protein substrates driven by ATP hydrolysis. FtsH is the only known family of membrane-anchored AAA+ proteases essential for membrane protein quality control. Although a spiral staircase rotation mechanism for substrate translocation across the FtsH pore has been proposed, the detailed conformational changes among various states have not been clear due to absence of FtsH structures in these states. We report here the cryo-EM structure for Thermotoga maritima FtsH (TmFtsH) in a fully ADP-bound symmetric state. Comparisons of the ADP-state structure with its apo-state and a substrate-engaged yeast YME1 structure show conformational changes in the ATPase domains, rather than the protease domains. A reconstruction of the full-length TmFtsH provides structural insights for the dynamic transmembrane and the periplasmic domains. Our structural analyses expand the understanding of conformational switches between different nucleotide states in ATP hydrolysis by FtsH.

Fig. 1. Production and purification, and characterization of TmFtsH.

a Schematic diagram of TmFtsH domains. PD periplasmic domain; TM transmembrane. b Size-exclusion chromatography and c SDS-PAGE analysis for the purified TmFtsH in MSP1D1 nanodiscs. d Negative-staining micrograph of TmFtsH reconstituted in MSP1D1 nanodiscs.

Fig. 2. Determination of TmFtsH structure in ADP state.

a A typical motion-corrected cryo-EM micrograph from a total of 11,169 micrographs. b 2D class averages. c Three views for the reconstructed map colored with local resolutions. d Fourier Shell Correlation (FSC) curve for the 3D reconstruction to determine the structure resolution. e Orientation distribution for particles used in the final 3D reconstruction.

Fig. 3. TmFtsH structure in a symmetric ADP state.

a, b Two views of the overall structure of symmetric protease and ATPases domains. One subunit was colored orange for protease and marine for ATPase, and the rest subunits were colored in gray. ADP molecules are shown as spheres. c–e Three views of the superimposition of the ADP-state structure (colored in the same way as in a and b) with its apo-state crystal structure (PDB code 3KDS, colored in green). Both structures have a six-fold symmetry. The ADP-state structure was colored as in a, and the apo-state structure was colored in green. ADP molecules are shown as sticks. Red arrows indicate the Cα atom positions for measuring the distance between two V235 residues. Blue arrows indicate the loops connecting the protease and ATPase domains. f Superimposition of ADP-state structure and the apo structure for the binding sites as well as their next clockwise neighboring region. Arginine finger residue R318′ is shown as magenta (ADP-state) and green (apo-state). Red arrows indicated the Cα atom positions for measuring distance. Cryo-EM densities for ADP and the R318′ regions were colored as gray and orange isomeshes, respectively.

Fig. 4. Structural comparison of ADP-state structure with a substrate loaded yeast homolog structure YME1.

a Top view to show the alignment for the protease domain. YME1 structure (PDB code 6AZ0) is colored in green; ADP-state TmFtsH structure is colored in orange and gray. b Side view to show both protease and ATPase domains. Red arrows indicate the loops connecting the protease and ATPase domains. c Bottom view to show the alignment for the ATPase domains. ADP and ATP molecules are shown as sticks (magenta for TmFtsH and green for YME1). Substrate is colored in black. Colors of the rest structures are the same as those of Fig. 3c–e. A magenta hexagon indicates the center of the symmetric ADP-state hexamer.

Fig. 5. Structural comparison of ADP-state cryo-EM and crystal structures.

The crystal structure used for comparison is TmFtsH intracellular domains (ATPase and protease) crystallized in a mixed C2/C6 symmetry (PDB code 2CEA). Two views to show the alignments for the protease (a) and ATPase (b) domains. ADP molecules are shown as sticks in cryo-EM structure and spheres in the crystal structure. Colors of the two structures are the same as in Fig. 3c–e. c Bottom view of the ADP-bound crystal structure for the ATPase domains to show the relative separation of ADP (orange spheres for carbon) and arginine finger residue R318 (green spheres for carbon). Circles and rectangular boxes indicate nearby and far away ADP-R318 pairs, respectively. d–f Side-view comparisons of one ADP-state subunit with three non-symmetric equivalents in the crystal structure. ADP molecules are shown as sticks: magenta for cryo-EM structure and green for crystal structure. Red arrows indicate the loops (disordered in the crystal structure) between the protease and ATPase domains.

Fig. 6. Full-length TmFtsH reconstruction at a low resolution.

a Selected views of 2D class averages showing full-length TmFtsH). b Four views of the reconstructed 3D map colored with local resolutions. c Gold-standard Fourier Shell Correlation. d A full-length TmFtsH model with the transmembrane and periplasmic domains built by AlphaFold. e Fitting of the full-length TmFtsH model into the density map.