1. Nucleotide-Dependent Substrate Handoff from the SspB Adaptor to the AAA+ ClpXP Protease 
Daniel N. Bolon, Robert A. Grant, Tania A. Baker, Robert T. Sauer 
Molecular Cell 
Volume 16, Issue 3, Pages (November 2004)
DOI: /j.molcel

2. Figure 1 Structure of the Covalent Adaptor-Substrate Complex
(A) Aligned structures of the disulfide-linked SspB-SBDS-SssrA complex (pink/red) with the noncovalent wild-type complex (cyan/blue; PDB code 1OU8) (Levchenko et al., 2003). SspB is displayed in ribbon representation, and the main chain atoms of the ssrA peptide are in stick representation. The disulfide bond (yellow) is displayed in CPK representation.
(B) Simulated-annealing omit map of electron density for a portion of the ssrA peptide and the disulfide crosslink to SspB. The ssrA peptide and Cys44 of SspB were omitted during map calculations. The 1.9 Å 2F0 − FC map is contoured at 0.7σ.
(C) Sequence of the ssrA tag showing sites of contact with SspB or ClpX and the mutant cysteine used for crosslinking to SspB.
(D) Surface/stick representation of one subunit of the covalent SspB-SBDS-SssrA complex. Tag residues 9–11 (shown in purple) were not visible in the crystal structure and were modeled in an energetically favorable conformation for this figure.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Thermodynamic Stability of the Covalent Adaptor-Substrate Complex
(A) Temperature denaturation monitored by differential scanning calorimetry. Tm was 62.2°C for SspBSH and 85.9°C for the disulfide-bonded SspBS-SssrA complex.
(B) GuHCl denaturation monitored by tryptophan fluorescence (the sole tryptophan in SspB is buried in the core far from the ssrA peptide binding groove). For the fits shown, ΔG was 20.0 kcal/mol dimer for SspBSH, 21.2 kcal/mol dimer for the nonspecific (NS) peptide control (SspBSH disulfide-bonded to the peptide CWEEGLPLVGRVAA), and 27.6 kcal/mol dimer for SspBS-SssrA; m values were between 5.2 and 5.8 kcal/mol·M for all proteins.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
ClpX Binding Assayed by Fluorescence Anisotropy of Fluorescein-Labeled SspB Variants
Experiments were performed using 25 nM concentrations of the fluorescein-labeled proteins at 30°C in PD200 buffer with 5 mM ATPγS (upper panel) or 5 mM ADP (lower panel). Competition experiments contained ClpX6 (125 nM in ATPγS experiments; 4 μM in ADP experiments) and FLSspBS-SssrA (25 nM), in the presence or absence of competitors (500 μM XB peptide; 500 μM ssrA peptide).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 ClpXP Degradation
(A) Degradation of 35S-SspBS-SGFP-ssrA (0.6 μM) by ClpXP (0.3 μM ClpX6; 0.8 μM ClpP14) assayed by changes in GFP fluorescence (♦) or acid-soluble 35S-peptides (■). 35S-SspBSH (▴) was degraded very slowly, and changing the C-terminal residues of the ssrA tag from AA to DD (•) prevented degradation of the covalent adaptor-substrate complex.
(B) ClpXP degradation rates as a function of the concentration of the covalent adaptor-substrate complexes. Degradation of 35S-SspBS-SssrA (Vmax = 0.25 ± 0.01 min−1ClpX6−1) was monitored by acid-soluble radioactivity; degradation of SspBS-SGFP-ssrA (Vmax = 0.05 ± 0.01 min−1ClpX6−1) was monitored by fluorescence. KM was less than 50 nM for both substrates, but precise values could not be obtained because the concentrations of total substrate and enzyme bound substrate were too close.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Cartoon Representation of the Assembly and Processing of Delivery Complexes Containing ClpX and the SspB Adaptor with the Disulfide-Linked ssrA Peptide
Note that tethering interactions between SspB and ClpX are present in both the ADP and ATP bound enzyme states, whereas interactions between the end of the ssrA tag and ClpX occur only in the ATP state. Following ATP hydrolysis, ClpX applies a pulling force to the ssrA tag and the attached protein. This highly strained state is very short-lived, leading to denaturation only infrequently and to release of the tag most of the time. Following slippage or release of the tag, it can reengage ClpX in a unimolecular reaction following nucleotide exchange or the adaptor can dissociate. The ratio of these rates determines the number of denaturation attempts per binding event.
Molecular Cell  , DOI: ( /j.molcel )