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Results: 6

1.
Figure 4

Figure 4. From: Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine.

Closed hexamers with disulfide bonds across all rigid-body subunit interfaces. (a) Non-reducing SDS-PAGE showing end-point disulfide-bond formation of T66C P388C ClpXΔN after oxidation with copper phenanthroline. (b) Michaelis-Menten plots of GFP-ssrA degradation by ClpXΔN, oxidized T66C P388C ClpXΔN, or reduced T66C P388C ClpXΔN (0.3 μM pseudo hexamer) and ClpP14 (0.9 μM). Symbols represent averages of three independent measurements. Table 1 lists values of KM and Vmax obtained by curve fitting. (c) Non-reducing SDS-PAGE showing end-point disulfide-bond formation of T66C P388C E109C N331C ClpXΔN after oxidation with copper phenanthroline. Symbols represent averages of three independent measurements. (d) Michaelis-Menten plots of GFP-ssrA or cp7-GFP-ssrA degradation by ClpXΔN or oxidized T66C P388C E109C N331C ClpXΔN (0.3 μM pseudo hexamer) and ClpP14 (0.9 μM). Table 1 lists KM and Vmax values.

Steven E. Glynn, et al. Nat Struct Mol Biol. ;19(6):616-622.
2.
Figure 5

Figure 5. From: Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine.

Unfolding and translocation by ClpXΔN circular hexamers. (a) Irradiation of Kaede-ssrA with UV light results in photo-cleavage of the polypeptide chain at a single site and a change in fluorescence24. Following photo-cleavage, ClpXΔN unfolding of Kaede-ssrA is effectively irreversible and eliminates native fluorescence. (b) Rates of Kaede-ssrA (10 μM) unfolding were determined for ClpXΔN, oxidized T66C P388 ClpXΔN, and oxidized T66C P388C E109C N331C ClpXΔN (0.3 μM pseudo hexamer) with or without ClpP14 (0.9 μM). Values are averages of 3 independent experiments ± 1 standard deviation. (c) ClpXP degradation of disulfide-bonded substrates requires concurrent translocation of multiple polypeptide chains. (d) Non-reducing SDS-PAGE showing that degradation of disulfide-bonded N11CArc-ssrA (10 μM) by ClpXΔN or by oxidized T66C P388C E109C N331C ClpXΔN (0.3 μM hexamer) and ClpP14 (0.9 μM) occurs with comparable kinetics.

Steven E. Glynn, et al. Nat Struct Mol Biol. ;19(6):616-622.
3.
Figure 6

Figure 6. From: Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine.

ClpX hinge mutations uncouple ATP hydrolysis and substrate unfolding. (a) The upper panel shows that the nucleotide-binding site in a loadable ClpX subunit (pdb code 3HWS, chain A)5 lies in a cleft formed by the large AAA+ domain, the hinge, and the small AAA+ domain. In E. coli ClpX, the hinge sequence is Asn315-Glu316-Leu317-Ser318. The lower panel is a histogram of sequence conservation from an alignment of >200 ClpX orthologs and was prepared using WebLogo39. The length of the hinge is highly conserved, but only Leu317, which contacts the nucleotide base, shows strong sequence conservation. Numbers represent residue positions in E. coli ClpX. (b) Rates of ATP hydrolysis by 0.3 μM ClpXΔN hexamer, Δ315N ClpXΔN hexamer, or +315N ClpXΔN hexamer were assayed in the absence or presence of carboxymethylated-titinI27-ssrA (20 μM). Values are averages of 3 independent experiments ± 1 standard deviation. (c) Michaelis-Menten plots of the rate of degradation of different concentrations of GFP-ssrA in the presence of 0.9 μM ClpP14 and 0.3 μM ClpXΔN hexamer (KM = 0.8 μM; Vmax = 0.92−1 min ClpX6−1), Δ315N ClpXΔN hexamer (KM = 0.6 μM; Vmax = 0.08 min−1 ClpX6−1) or +315N ClpXΔN hexamer (KM = 8 μM; Vmax = 0.04 min−1 ClpX6−1). (d) Rates of degradation of 10 μM cp7-GFP-ssrA in the presence of 0.9 μM ClpP14 and 0.3 μM ClpXΔN hexamer, Δ315N ClpXΔN hexamer, or +315N ClpXΔN hexamer. Values are averages of 3 independent experiments ± 1 standard deviation.
(e) SDS-PAGE assay of the degradation of an unfolded substrate, carboxymethylated-titinI27-ssrA (20 μM), by 0.9 μM ClpP14 and either 0.3 μM ClpXΔN hexamer or +315N ClpXΔN hexamer.

Steven E. Glynn, et al. Nat Struct Mol Biol. ;19(6):616-622.
4.
Figure 1

Figure 1. From: Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine.

The ClpX6 ring is stabilized by rigid-body packing between subunits. (a) In the ATP-dependent ClpXP protease, a ClpX hexamer recognizes and unfolds protein substrates, and then translocates them into the degradation chamber of ClpP14 for proteolysis. The unfolding and translocation reactions require ATP hydrolysis. (b) Crystal structure of a ring hexamer of ClpXΔN (pdb code 3HWS)5. Four subunits are shown in cartoon representation. The large AAA+ domain of one subunit (yellow; surface representation) packs against the small AAA+ domain of the counterclockwise neighboring subunit (green; surface representation). Similar rigid-body packing interactions occur between all subunits in the ring and comprise the major subunit-subunit interfaces of the hexamer. (c) In this diagram, each ClpXΔN subunit is a different color. Four loadable (L) subunits contain a binding site for ATP or ADP (red oval) in a cleft between the large and small AAA+ domains. In two unloadable (U) subunits, a rotation around the intradomain hinge destroys the nucleotide binding site. (d) Each rigid-body unit is a different color in this representation, which shows that the ClpXΔN ring consists of six rigid-body units connected by six hinges. (e) Comparison of individual rigid-body units from nucleotide-free and nucleotide-bound crystal structures of ClpXΔN hexamers (pdb codes 3HTE and 3HWS, respectively)5. For each rigid-body unit, the large AAA+ domains were structurally aligned (the large domain of subunit A from 3HWS is shown in gray surface representation) and the position of the small AAA+ domain of the rigid-body unit was shown in ribbon representation without alignment (each individual small domain is a different color).

Steven E. Glynn, et al. Nat Struct Mol Biol. ;19(6):616-622.
5.
Figure 2

Figure 2. From: Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine.

Fusing rigid-body units with tethers of different lengths. (a) A single-chain ClpXΔN trimer contains three subunits and two polypeptide linkers that tether adjacent subunits. Each trimer contains two rigid-body units, which are fused by the tethers. (b) The position of one single-chain trimer in a pseudo hexamer is marked by the red outline. Shortening the tethers constrains potential movements of the large and small AAA+ domains of the rigid-body unit. (c) Single-chain ClpXΔN trimers with tethers of 20 residues (T20), 4 residues (T4), or zero residues (T0) chromatographed at similar positions on a Superose-6 gel-filtration column (void volume ~8 mL; GE Healthcare). A previous study15 showed that the T20 variant chromatographed identically to non tethered ClpXΔN at the position expected for a pseudo hexamer. (d) Pseudo hexamers formed from T0, T4, or T20 single-chain trimers catalyzed similar basal levels of ATP hydrolysis. This activity was repressed slightly by addition of ClpP and was stimulated markedly by addition of an unfolded protein substrate (carboxymethylated-titin-ssrA)20. (e) Pseudo hexamers (0.3 μM) formed from T0, T4, or T20 single-chain ClpXΔN trimers supported very similar levels of ClpP (0.9 μM) degradation of GFP-ssrA over a wide range of substrate concentrations. Symbols represent the average of three independent measurements. Lines are non-linear least-squares fits to the Michaelis-Menten equation. Values of KM and Vmax are listed in Table 1.

Steven E. Glynn, et al. Nat Struct Mol Biol. ;19(6):616-622.
6.
Figure 3

Figure 3. From: Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine.

Stabilizing rigid-body packing between single-chain dimers by disulfide bonds. (a) Diagram of a single-chain ClpXΔN dimer with cysteines introduced into the large AAA+ domain of the first subunit and the small AAA+ domain of the second subunit. These cysteines were designed to allow disulfide-bond formation within the rigid-body units that form the subunit interfaces between different single-chain ClpXΔN dimers in pseudo hexamers. (b) Cartoon showing how disulfide bonds across rigid-body units and tethers would covalently connect each subunit in the ClpXΔN ring to both neighboring subunits, resulting in topological closure of the ring. (c) Non-reducing SDS-PAGE showing the progressive formation of disulfide-bonded species of the (T66C P388)–(T66 P388C) single-chain dimer following initiation of oxidation by addition of copper phenanthroline. The kinetics of formation and disappearance of different species show that the highest band corresponds to the circular hexamer with three disulfide bonds. Bands were visualized by staining with Coomassie Blue. The leftmost lane shows the positions of molecular weight standards. (d) The oxidized (T66C P388)–(T66 P388C) single-chain dimer was incubated with 10 mM DTT at 37 °C and the progressive disassembly of disulfide-bonded species was assayed by non-reducing SDS-PAGE. The kinetics of reduction support the pathway: circular hexamer → linear hexamer → linear tetramer → linear dimer. (e) Non-reducing SDS-PAGE showing oxidation of (E109C N331)–(E109 N331C) to form the circular hexamer. (f) Mixtures containing >90% disulfide-bond circular hexamers of (T66C P388)–(T66 P388C) and (E109C N331)–(E109 N331C) (0.3 μM) supported similar levels of ClpP (0.9 μM) degradation of GFP-ssrA over a range of substrate concentrations. Symbols are averages of three independent measurements. Lines are fits to the Michaelis-Menten equation, with values of KM and Vmax listed in Table 1.

Steven E. Glynn, et al. Nat Struct Mol Biol. ;19(6):616-622.

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