The crystal structure of apo -FtsH reveals domain movements necessary for substrate unfolding and translocation

Abstract

The hexameric membrane-spanning ATP-dependent metalloprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA؉ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 Å resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo-and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA؉ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH.AAAϩ protease ͉ conformational rearrangement ͉ pore residue ͉ FtsH ͉ metalloprotease T he ATPases associated with various cellular activities (AAAϩ) family comprises a large group of proteins which share a common ATPase module, carrying the Walker A and B and other conserved sequence motifs (1, 2). They function as oligomers and usually form hexa-or heptameric rings, whereas in some cases oligomerization is ATP-dependent. AAAϩ proteins are present in all kingdoms of life and participate in a wide range of different actions such as protein disaggregation, complex remodeling, and protein degradation (reviewed in refs. 3-5). In these processes, the energy of ATP is converted into a mechanical force by large conformational rearrangements. Until now, information on these immense structural changes is sparse; x-ray studies on HslU (6) and p97 (7) revealed only relatively small changes between the conformations of apo, ADP, and ATP state. In contrast, electron microscopic studies of p97 (8) and of the chaperone ClpB (9) displayed very large conformational rearrangements of the AAA domains.The membrane-bound metalloprotease FtsH (10, 11) is a homohexameric complex that carries, contrary to most other AAAϩ proteases, the AAA and proteolytic domain on the same polypeptide chain. The AAA domain bears the Walker A and B motifs necessary for nucleotide binding and hydrolysis; the second region of homology (SRH), which carries conserved arginine residues (''arginine fingers'') for oligomerization and nucleotide hydrolysis; and the conserved FGV pore motif required for substrate recognition and translocation. The protease domain exhibits the zincbinding HEXXH finger print. The N-terminal AAA domain is preceded by 2 transmembrane helices flanking a s...

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

“…The resolution of the structure, determined at 12 Å, was sufficient to confirm the presence of six FtsH protomers within each complex, obtained by fitting the atomic structures of cytosolic fragments of FtsH into the cryo-EM model (Bieniossek et al, 2006(Bieniossek et al, , 2009, but was insufficient to determine the structural organization of the two types of subunit within the ring.…”

“…To aid interpretation of the three-dimensional reconstruction, the crystallographically derived hexameric structure of a soluble fragment of FtsH, containing residues 146 to 603 of the 610-amino acid sequence of Thermotoga maritima FtsH (Bieniossek et al, 2009), encompassing the conserved AAA domain and PD, was modeled by visual inspection into the molecular envelope, contoured at a threshold of 2.5 s ( Figure 7A). The crystal structure fitted remarkably well into the central core, providing clear evidence to support the presence of six FtsH subunits in the GST-tagged FtsH complex.…”

“…Coordinate data sets were obtained from the Research Collaboratory Structural Bioinformatics Data Bank (www.rcsb.org) for 3KDS.pdb (structure of the cytosolic region of the Thermotoga maritima FtsH protease at 2.60 Å; Bieniossek et al, 2009) and 1GTA.pdb (structure of GST at 2.4 Å; McTigue et al, 1995). Structures were modeled into the final calculated three-dimensional map using PyMol (DeLano, 2008).…”

Electron Cryomicroscopy Structure of a Membrane-anchored Mitochondrial AAA Protease

“…Next, to improve the envelope fit, we replaced the AAA domain of apo-FtsH Cyt (7) with that of the nucleotide-bound structure (6) by superimposing the small helical domain from each structure. Because the pore loop-2 (residues 393-404 in Yta10 and 453-463 in Yta12) is disordered in the T. maritima FtsH Cyt crystal structure, the loop was modeled according to the conformation seen in the x-ray structure of Thermus thermophilus FtsH Cyt (PDB code 2DHR (5)), followed by manually adjusting the position of the AAA domains.…”

“…X-ray crystal structures of the soluble cytosolic domains of the bacterial AAA protease FtsH (FtsH Cyt ) bound to ADP and in the absence of nucleotide (apo) have been reported (5)(6)(7). Although the AAA ring of the apo-FtsH Cyt hexamer was 6-fold symmetric (7), the ADP-bound structures revealed a 2-(6) and 3-fold symmetrical hexamer (5).…”