Volume 13, Issue 3, Pages 443-449 (February 2004) Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery  Daniel N. Bolon, David A. Wah, Greg L. Hersch, Tania A. Baker, Robert T. Sauer  Molecular Cell  Volume 13, Issue 3, Pages 443-449 (February 2004) DOI: 10.1016/S1097-2765(04)00027-9 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Properties of the Designed Heterodimer (A) Analytical ultracentrifugation (20°C; 16,000 rpm) of the YGFM/SLA protein (25 μM) after 36 hr. The fitted solid line represents a value of 37.6 kDa for the heterodimer molecular weight. Dashed line, mass distribution expected for a monomer. (B) Far-UV circular dichroism spectra of 25 μM YGFM/SLA or the parental HE SspB protein (25°C). (C) Fluorescence spectra of YGFM/SLA under native and denaturing (5 M GuHCl) conditions (25°C). (D) GuHCl denaturation of 3 μM YGFM/SLA (ΔGu=16.7 kcal/mol; m = 5.7 kcal/mol•M) and HE SspB (ΔGu = 22.4 kcal/mol; m = 6.7 kcal/mol•M) at 25°C. The fitted curves and stability parameters are for native dimer to unfolded monomer reactions. (E) YGFM/SLA heterodimer and E. coli SspB bound equally well to a fluorescent ssrA peptide (50 nM) as assayed by changes in anisotropy at 30°C, pH 7.6, 200 mM KCl. The solid line represents a KD of 3 μM. (F) Degradation of GFP-ssrA (0.6 μM) by ClpXP (0.1 μM ClpX6, 0.3 μM ClpP14) in the absence of SspB or in the presence of YGFM/SLA or E. coli SspB (0.6 μM each). The solid lines are linear fits to the kinetic data. Degradation in the presence of HE SspB was identical to that observed with YGFM/SLA (data not shown). Molecular Cell 2004 13, 443-449DOI: (10.1016/S1097-2765(04)00027-9) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Subunit Exchange Assayed by MonoQ Ion-Exchange Chromatography (A) Rechromatography of a dimer containing one full-length and one substrate binding domain of E. coli SspB immediately following the initial purification of this peak on the same ion-exchange column. (B) Rechromatography of the material from (A) following 24 hr of incubation at 30°C. (C) Rechromatography of a YGFM/SLA heterodimer containing one full-length and one substrate binding domain following initial purification. (D) Rechromatography of the material from (C) following 24 hr of incubation at 30°C. In all experiments, ion-exchange chromatography was performed at 4°C and was monitored by absorbance at 280 nm. The identities of the subunits in each peak were determined by SDS-PAGE. Molecular Cell 2004 13, 443-449DOI: (10.1016/S1097-2765(04)00027-9) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Two SspB Tails Are Required for Full Activity and Strong Binding to ClpX (A) Rate of ClpXP degradation of GFP-ssrA as a function of substrate and SspB concentration for YGFM/SLA heterodimers with two, one, or zero tails and XB modules. (B) Rate of ClpXP degradation of GFP-ssrA as a function of substrate and SspB concentration for E. coli SspB heterodimers with two, one, or zero tails and XB modules. (C) Kinetics of subunit exchange assayed by native gel electrophoresis. For E. coli SspB, a heterodimer containing one full-length subunit and one SBD subunit was incubated at 30°C, and samples were taken after different incubation times and placed on ice until electrophoresis on a native gel. The figure shows the approach to the equilibrium 1:2:1 distribution of subunits. When a YGFM/SLA heterodimer containing full-length subunits was mixed with His6-tagged E. coli SspB, no subunit exchange was detected by native gel following 24 hr of incubation at 30°C. (D) Degradation of GFP-ssrA (0.3 μM) by ClpXP (0.1 μM ClpX6, 0.3 μM ClpP14) in the absence of SspB or in the presence of designed YGFM/SLA heterodimers with two, one, or zero tails/XB modules (0.3 μM each). The solid lines are linear least squares fits to the kinetic data. (E) Degradation of GFP-ssrA (0.2 μM) in the presence of E. coli SspB (0.6 μM) by ClpXP (0.1 μM ClpX6, 0.3 μM ClpP14) was inhibited by increasing concentrations of the XB peptide or the A73Q variants of YGFM/SLA heterodimers containing one or two C-terminal tails/XB modules. The A73Q mutation prevents binding of SspB to ssrA-tagged substrates. (F) The A73Q variants of YGFM/SLA at concentrations of 55 μM did not significantly inhibit ClpXP degradation of GFP-ssrA alone. Degradation conditions were identical to those in (E) except E. coli SspB was not added. Molecular Cell 2004 13, 443-449DOI: (10.1016/S1097-2765(04)00027-9) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 Interactions between the N Domain of ClpX and XB Modules of SspB (A) Equilibrium binding of untagged N domain or ClpX to fluorescent-XB peptide assayed by changes in anisotropy at 20°C (pH 7.6, 200 mM KCl, 10 mM Mg2Cl, 10 mM ATPγS, 2 mM DTT, 10% glycerol). ClpX binding data is from Wah et al. (2003). (B) Stoichiometric binding of untagged N domain to XB peptide assayed by fluorescence anisotropy at 4°C (pH 7.6, 100 mM KCl). Theoretical curves for binding stoichiometries of 1 or 2 XB peptides per N domain dimer are shown. (C) Binding assayed by ITC. Aliquots (7.5 μl) of an XB peptide (1.1 mM) were injected into a 1.4 ml solution containing the His-tagged N domain dimer of E. coli ClpX (80 μM dimer) at 25°C (pH 7.6, 50 mM KCl). (D) Single-species fit of the data shown in (C) gave the KD, ΔH, ΔS, and n values listed. (E) Cartoon representation of SspB, one bound ssrA-tagged substrate, and ClpX, showing how interactions between the XB modules and different ClpX N domains could be shuffled. In populations, the doubly tethered molecules are favored by roughly 10:1. (F) Hierarchy of apparent ClpX affinities (estimated from KM or Ki values) of SspB molecules with different numbers of XB modules and with or without bound ssrA-tagged substrates. Two XB tethering interactions as well as a bound ssrA-tagged substrate are required for strong binding. Molecular Cell 2004 13, 443-449DOI: (10.1016/S1097-2765(04)00027-9) Copyright © 2004 Cell Press Terms and Conditions