Cryo-EM analysis of the TClpB hexamer.
(A) Representative area of a digital micrograph of the Trap-ATP hexamer in vitreous ice.
(B) The left panels show selected projection views of the Trap-ATP reconstruction. The corresponding class averages are shown in the right panels.

Comparison of the cryo-EM reconstruction of TClpB in different nucleotide states. Top down and side views of the isosurface representation of different nucleotide states, which were superimposed pairwise via the AAA-2 ring. The TClpB-AMPPNP hexamer is shown in grey, the Trap-ATP hexamer in magenta, the TClpB-ADP hexamer in cyan, and the TClpB-apo hexamer in gold. The arrows point to the six protruding mass densities on the top surface of the AAA-1 ring in the Trap-ATP hexamer, which likely account for the beginning of the flexible linker that connects the N-terminal domain to the AAA-1 domain.

Substrate binding to the TClpB hexamer is ATP- and temperature-dependent.
(A) Structural basis for high-affinity substrate binding. The figure shows an enlarged top down and cut-away side view of the pore region of the fitted Trap-ATP and TClpB-AMPPNP hexamer. The cryo-EM reconstruction is shown as a semitransparent surface, the hexamer model as a ribbon diagram, Tyr243 as a ball-and-stick model, and the bound nucleotides as CPK models. Residues 238-250 in the pore region, which lie outside the mass density in the TClpB-AMPPNP reconstruction, are colored purple. The figure shows that ATP-activation stabilizes the D1 loop at the central pore. The positions of the Tyr243 side-chains are shown for clarity.
(B) Binding isotherm of FITC-casein and TClpB. To measure fluorescence polarization, the Trap and TClpB hexamers were pre-assembled at 55 °C in the presence or absence of nucleotide, and mixed with FITC-casein. All measurements were carried out at 55 °C. The binding curve for Trap-ATP is shown in red, TClpB-ATPγS in purple, TClpB-AMPPNP in yellow, TClpB-ATP in cyan, TClpB-ADP in green, and TClpB-apo in blue. The curves represent least-square nonlinear regression fits of the change in fluorescence polarization obtained from three independent measurements. Standard deviations, if larger than the size of the symbols, are shown. The apparent K_D was calculated where possible and is 0.147 ± 0.010 μM for Trap-ATP and 0.378 ± 0.009 μM for TClpB-ATPγS, corresponding to a calculated K_D of 0.025 μM and 0.063 μM for the respective hexamers.
(C) Binding isotherm of FAM-TrfA and TClpB. Trap-ATP and TClpB-AMPPNP hexamers were generated as described in (B). The Trap_25°C-ATP sample was prepared by pre-incubation of Trap mutant at 25 °C in the presence of 1 mM ATP. To measure fluorescence polarization, the pre-assembled Trap-ATP and TClpB-AMPPNP samples were mixed with FAM-TrfA, incubated for 5 min at either 37 °C or 25 °C as shown, and measured at the corresponding temperature. The binding curve for Trap-ATP at 37 °C is shown in red, Trap-ATP at 25 °C in purple, Trap_25°C-ATP at 37 °C in green, and TClpB-AMPPNP at 37 °C in yellow. The curves represent leastsquare nonlinear regression fits of the change in fluorescence polarization from three independent measurements. Standard deviations, if larger than the size of the symbols, are shown. The apparent K_D for the binding of FAM-TrfA to Trap-ATP was calculated to be 0.267 ± 0.022 μMat 37 °C and 0.308 ± 0.014 μM at 25 °C, corresponding to 0.045 μM and 0.051 μM for the respective hexamers.

Cryo-EM reconstruction of the TClpB hexamer in different nucleotide states. The figure shows top down, side, and cut-away side views of the TClpB-AMPPNP, Trap-ATP, TClpB-ADP, and TClpB-apo hexamer obtained by cryo-EM and single particle reconstruction techniques. The TClpB hexamer has a height of 90 Å and a diameter of 140 Å (bottom ring). The AAA-1 and AAA-2 rings are stacked on top of each other with the top ring rotated ∼23° counterclockwise with respect to the bottom ring. While the central pore of the bottom ring is closed, the pore is open in the top ring and leads to a large internal cavity. The size of the central pore in the top ring varies in diameter and is the widest in the AMPPNP-bound state (28 Å) and the narrowest in the ATP-activated state (13 Å). It is noteworthy that, in the ADP-bound state, the narrowest part of the pore is located ∼13 Å below the narrowest part in the Trap-ATP hexamer.

Atomic models of the fitted TClpB-AMPPNP and Trap-ATP hexamer. The cryo-EM reconstruction is shown as a semitransparent surface (top down, side, and cut-away side view), with the structure of an N-terminal domain-truncated TClpB hexamer docked in, demonstrating the goodness of fit. The hexamer model is depicted as a ribbon diagram. One subunit of the hexamer is highlighted in red, illustrating the staggered conformation of the AAA-1 and AAA-2 domains. The bound nucleotides are shown as CPK models and are colored yellow (AAA-1) and blue (AAA-2), respectively.

The M-domain undergoes a large motion.
(A) Top down view of the AAA-1 ring of an N-terminal domain-truncated Trap-ATP hexamer fitted into the corresponding cryo-EM reconstruction shown as a semitransparent surface. The figure illustrates the position of the ClpB M-domain in the ATP-activated state. The Trap-ATP hexamer model is depicted as a ribbon diagram and is colored cyan, with the AAA-1 domain of one subunit highlighted red and its corresponding M-domain blue. The bound ATP molecules are shown as CPK models and are colored yellow.
(B) Enlarged view of the M-domain position in the Trap-ATP hexamer model as seen in (A). The figure shows that motif 2 of the M-domain is positioned between the D1-large domains of neighboring subunits. The position of motif 2 is supported by an independent sulfhydryl crosslinking study using E. coli ClpB (Haslberger et al., 2007). The position of G167 (G175 in E. coli ClpB) and A490 (S499 in E. coli ClpB), which were mutated to form a disulfide cross-link are depicted as green spheres.
(C) Enlarged view of the M-domain position as seen in the crystal structure of a TClpBAMPPNP monomer (Lee et al., 2003),. For clarity, only the AAA-1 domain of the TClpB-AMPPNP monomer is shown, which was superimposed via the D1-large domain onto the atomic model of the Trap-ATP hexamer. The AAA-1 domain is colored orange with the M-domain highlighted blue. Motif 1 and motif 2 of the M-domain are labeled accordingly. The figure shows the position of G167 and R475 (green spheres), which were mutated to form a disulfide crosslink (Lee et al., 2003).