Multiple sequence signals direct recognition and degradation of protein substrates by the AAA+ protease HslUV

Abstract

Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V(max). By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease.

Figure 1

C-terminal λcI^N sequences alter HslUV degradation. ( A) Degradation of λcI^N variants (10 μM) bearing RSEYE or ISVTL C-terminal sequences by 300 nM HslU₆, 800 nM HslV₁₂ was analyzed by SDS-PAGE. (B, C) Rates of steady-state degradation of different concentrations of ^35S-λcI^N variants by 100 nM HslU₆, 300 nM HslV₁₂ were determined by assaying acid-soluble radioactivity. Lines represent non-linear-least-squares fits to the Michaelis-Menten equation: rate = V_max· [S]/([S]+K_M). Kinetic parameters are listed in Table 1.

Figure 2

C- and N-terminal sequences of Arc substrates affect HslUV degradation.Steady-state rates of HslUV degradation of ³⁵S-Arc variants were determined as described in the Fig. 1 legend. Lines are fits to the Michaelis-Menten equation; kinetic parameters are listed in Table 1.

Figure 3

Occluding the C-terminus of λcI^N and Arc substrates inhibits HslUV degradation. (A) Degradation of ³⁵S-labeled λcI^N substrates (50 μM) by 0.5 μM HslU₆, 1.5 μM HslV₁₂ was measured at different concentrations of the DegS PDZ domain, which binds strongly to the C-terminal tripeptide YYF but weakly to EYE. Rates are expressed as a percentage of the rate with no PDZ domain. (B) Degradation of ³⁵S-labeled Arc variants (4 μM) by 0.5 μM HslU₆, 1.5 μM HslV₁₂ was measured at different concentrations of SspB, which binds strongly to the wild-type ssrA tag but weakly to the ssrA^N3A mutant. Rates are expressed as a percentage of the rate with no SspB.

Figure 4

ClpS binding to the N-terminus of Arc or λcI^N substrates slows HslUV degradation. (A) Proteolysis of ³⁵S-labeled Arc variants (4 μM) by 0.5 μM HslU₆, 1.5 μM HslV₁₂ was measured at different concentrations of ClpS, which binds strongly to the N-terminal leucine of ^M1LArc-ssrA but weakly to the N-terminal methionine of Arc-ssrA. (B) ClpS inhibits HslUV (0.5 μM HslU₆, 1.5 μM HslV₁₂) degradation of λcI^N substrates (50 μM) with N-terminal leucines (^S1L λcI^N-RSYYF – open circles; ^S1L λcI^N-RSEYE – closed circles) more efficiently than variants with an N-terminal serine (λcI^N-RSYYF – open squares) or glycine (ext1-λcI^N-RSEYE – closed squares).

Figure 5

Combinatorial and single-turnover inhibition.( A) (top) A combination of SspB and ClpS inhibited degradation of ³⁵S-^M1LArc-ssrA (15 μM) by 0.5 μM HslU₆, 1.5 μM HslV₁₂ to a greater extent than either individual protein. (bottom) A combination of the DegS PDZ domain and ClpS also inhibited degradation of ³⁵S-^S1L λcI^N-RSYYF (50 μM) by 0.5 μM HslU₆, 1.5 μM HslV₁₂ more than either single protein alone. (B) Degradation of different ³⁵S-labeled substrates (2 μM) by 5 μM HslU₆, 6 μM HslV₁₂ was assayed in the presence the indicated concentrations of SspB, ClpS, or the DegS PDZ domain.

Figure 6

Model of HslUV recognition of a substrate (S) with degrons at each terminus. One degron (shown as a triangle) binds to a tethering site on the HslUV enzyme (E). The other degron (shown as a circle) binds to a site in the translocation pore of HslU. Because binding to the pore is a prerequisite for degradation, the enzyme·substrate complexes marked P and TP are proteolytically active but the T complex is inactive. K₁ ([E][S]/[T]), K₂ ([E][S]/[P]), K₃ ([T]/[TP]), and K₄ ([P]/[TP]) are equilibrium dissociation constants. Note that K₁·K₃ = K₂·K₄. If the rate of substrate dissociation is fast compared to k_deg, then the apparent K_M ([S] at half maximal velocity) is 1/(1/K₁+1/K₂+1/(K₁·K₃)). At substrate saturation, the fraction of active enzymes (f_act) = ([P]+[TP])/([T]+[P]+[TP]) = (1/K₂+1/(K₁·K₃)/(1/K₁+1/K₂+1/(K₁·K₃)). V_max/[E_total] equals f_act·k_deg. The K₃-k_deg pathway of degradation corresponds to single-degron recognition.