Structure of the Whole Cytosolic Region of ATP-Dependent Protease FtsH Ryoji Suno, Hajime Niwa, Daisuke Tsuchiya, Xiaodong Zhang, Masasuke Yoshida, Kosuke Morikawa  Molecular Cell  Volume 22, Issue 5, Pages 575-585 (June 2006) DOI: 10.1016/j.molcel.2006.04.020 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 FtsH Protease Domain (A) Schematic drawing of the constructions for FtsH. Loop regions between rigid domains are colored red, and the large and small subdomains of the AAA+ domain are shown in cyan and green, respectively. (B) Multiple sequence alignment of the protease domain. T. thermophilus FtsH (accession code AB032368) aligned with A. aeolicus FtsH (AE000714-12), E. coli FtsH (U01376), and yeast Yme1 (L14616). Cylinders and arrows indicate the secondary structure elements (α helices and β strands, respectively) of T. thermophilus FtsH. All secondary structures in A. aeolicus pFtsH are the same as those in T. thermophilus FtsH, except the disordered regions. The conserved glycine (G399) in the loop between the AAA+ and protease domains is highlighted in white. Limited proteolysis of A. aeolicus FtsH revealed that trypsin preferably digested the loop region (red arrowhead). The lid helix (helix 14) appears to be kinked at the conserved glycine residue in green. The zinc binding motif (HEXXH) and the third coordinating aspartic acid residue are shown in red. (C) Hexameric structure of A. aeolicus pFtsH from the top (left) and side (right) views. In the side view, the protease structure is superposed onto T. thermophilus sFtsH (transparent molecular surface). (D) A stereopair diagram of the pFtsH monomer. The catalytic residues are shown in stick models. The Zn binding site (gray sphere) was actually occupied by an Hg atom in the crystal. The dotted lines indicate the disordered regions. (E) Stereo view of the catalytic residues in the pFtsH (orange) superposed onto the corresponding residues of thermolysin (cyan) and tricorn interacting factor F3 (green). Molecular Cell 2006 22, 575-585DOI: (10.1016/j.molcel.2006.04.020) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Characterization of sFtsH (A) The wild-type (thin line) and G399L mutant (thick line) analyzed by gel filtration chromatography (protein concentration, 7.5 mg/ml). The molecular weight marker was purchased from Amersham Biosciences. (B) Protease activities of sFtsH (black arrow) in the presence (lanes 1–6) or absence (lanes 7–12) of ATP, and the full-length FtsH (open arrow) in the presence of ATP (lanes 13–18). Substrate digestion (α-casein; asterisk) for each reaction was monitored at the incubation times of 0, 2, 4, 8, 16, and 32 min, respectively. (C) ATP-dependent protease activities of the wild-type (lanes 1–5) and the G399L mutant (lanes 6–10) in the presence of ATP. These enzymes (black arrow) were incubated with α-casein (asterisk) for 0, 5, 10, 15, and 30 min, respectively. For reference, α-casein (lane 11) and the G399L mutant (lane 12) were loaded separately. Molecular Cell 2006 22, 575-585DOI: (10.1016/j.molcel.2006.04.020) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Structure of the Hexameric sFtsH (A) Entire structure of the ADP bound sFtsH hexamer. Cylinders and arrows represent α helices and β strands, respectively. An open (green) and a closed (orange) subunit are surrounded by broken lines. These two structures are used for the following illustrations with the same coloring. Bound ADP molecules and the arginine finger (R313) are depicted in blue and red space-filling models, respectively. (B) Structural difference between the open and closed subunits, shown by the superposition at their protease domains (cyan). The mobile regions in the protease domain are highlighted by the secondary-structure elements with the corresponding subunit color. (C) Stereo view of the AAA+ domains in the open and closed subunits, superposed onto the structures observed in the nucleotide-free crystals (Niwa et al., 2002) with P65 (red) and C2221 (gray) symmetries. The AAA+-domain structure complexed with AMPPNP is basically the same as that for the nucleotide-free state in P65 symmetry, due to the same crystal packing (Niwa et al., 2002), and thus is not drawn for clarity. The blue stick model represents the bound ADP molecule at the closed subunit. Molecular Cell 2006 22, 575-585DOI: (10.1016/j.molcel.2006.04.020) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Catalytic Environments in sFtsH (A) Stereo view of the nucleotide binding regions in the open (top) and closed (bottom) subunits. Each ADP molecule fits into the Fo−Fc omit map (transparent surface, contoured at the 1.8 σ level). (B) Stereopair diagrams of the interdomain regions in the open (top) and closed (bottom) subunits. The transparent surface represents the Fo−Fc omit map of N302 contoured at the 1.8 σ level. The lid helix modulating the AAA+ domain and the protease active site is highlighted with bright coloring. The Zn ion (gray sphere) is modeled at the predicted position in Figure 1D. Molecular Cell 2006 22, 575-585DOI: (10.1016/j.molcel.2006.04.020) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Disulfide Crosslinking Experiment (A) Close-up view of A310 and L527 in the open (left) and closed (right) subunits, from the same direction as in Figure 3B, where these residues are similarly illustrated with spheres. The broken lines indicate the Cα distances between the two residues. (B) Intrasubunit disulfide crosslinking of the C250A/A310C/L527C triple mutant. The protein was prepared by gel filtration chromatography under a denaturing condition in the absence of DTT (see Experimental Procedures). The major peak was eluted at the corresponding position to monomeric FtsH and analyzed by SDS-PAGE under reducing (lane 1) and nonreducing (lane 2) conditions. In the latter, a new band (black arrow) appeared in the higher molecular weight range than the predicted position for monomeric FtsH (open arrow). (C) ATP-dependent protease activity of the triple mutant in the presence and the absence of DTT, as monitored by the fluorescence increase ascribed to release of small proteolytic fragments of BODIPY-labeled casein. ATP was added at 100 s (open arrow) to start the reaction. Molecular Cell 2006 22, 575-585DOI: (10.1016/j.molcel.2006.04.020) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 The ATPase Cycle and Putative Polypeptide Translocation Pathway (A) Schematic diagram of the ATPase cycle in the hexameric FtsH. The cycle for the blue subunit is described in the text, where step one corresponds to the upper left. T, D, and R represent ATP, ADP, and the arginine finger, respectively. The catalytic cycle for the red subunit is supplemented, because the binding of ATP might help transition from the lower right to the lower left. (B) A diagram representing a cross-section of three-fold-symmetric FtsH, including the three-fold axis (black line) and two protease active sites (blue arrowheads). The axial area of the AAA+ domains is closed by three D273 residues of the closed subunits (green). The β hairpin structures composed of β9 and β10 (cyan) interfere with the direct access to the open protease active site from the central chamber. F229 (red) of the closed subunit is located at the entrance of the putative polypeptide translocation tunnel (bold arrow in dark magenta), whereas the residue in the open subunit is fully exposed on the top of the AAA+-domain hexamer. The pathway connecting the two F229 residues (light magenta) appears to reflect the unit length of polypeptide substrate, which one open-to-close transition can send into the protease active site. Molecular Cell 2006 22, 575-585DOI: (10.1016/j.molcel.2006.04.020) Copyright © 2006 Elsevier Inc. Terms and Conditions