Polypeptide translocation by the AAA+ ClpXP protease machine

Abstract

In the AAA+ ClpXP protease, ClpX uses repeated cycles of ATP hydrolysis to pull native proteins apart and to translocate the denatured polypeptide into ClpP for degradation. Here, we probe polypeptide features important for translocation. ClpXP degrades diverse synthetic peptide substrates despite major differences in side-chain chirality, size, and polarity. Moreover, translocation occurs without a peptide -NH and with 10 methylenes between successive peptide bonds. Pulling on homopolymeric tracts of glycine, proline, and lysine also allows efficient ClpXP degradation of a stably folded protein. Thus, minimal chemical features of a polypeptide chain are sufficient for translocation and protein unfolding by the ClpX machine. These results suggest that the translocation pore of ClpX is highly elastic, allowing interactions with a wide range of chemical groups, a feature likely to be shared by many AAA+ unfoldases.

Figure 1

Top — peptide substrates contained an N-terminal sequence (FAPHMALVP) that is cleaved efficiently by a ClpP, a central guest region of variable composition, and a C-terminal ssrA tag (AANDENYALAA). The cleavage cassette had an amino-benzoic acid fluorophore (ABZ) on the N-terminal side and a nitro-tyrosine quencher (Y^NO2) on the C-terminal side to allow detection of ClpP proteolysis. Bottom — ClpP cleavage between the ABZ and Y^NO2 groups of peptide substrates requires prior translocation of the guest region through the axial pore of ClpX.

Figure 2

(A) Efficient cleavage of the [G₁₀] peptide by ClpP was observed in the presence of wild-type ClpX but not in the absence of ClpX or with ClpX^E185Q, which cannot hydrolyze ATP. All reactions contained 10 µM of the [G₁₀] substrate and 300 nM ClpP₁₄. When present, the concentration of ClpX₆ or the ATPase-defective mutant was 800 nM. (B) Degradation of different concentrations of the [VG]₅ peptide by 800 nM ClpX₆ and 300 nM ClpP₁₄. (C) Steady-state rates of [VG]₅ peptide degradation by ClpXP were calculated from the data in panel C and fit to the Michaelis-Menten equation (K_M = 3.1 µM; V_max = 12.7 min⁻¹ ClpP⁻¹).

Figure 3

Maximum rates of ClpXP degradation of peptide substrates with different guest regions were determined from multiple experiments like those shown in Fig. 2B and Fig. 2C. See Table 1 for sequences of individual peptides and definition of error bars.

Figure 4

(A) Michaelis-Menten analysis of ClpXP degradation of GFP-[K₁₅]-ssrA. The solid line is a non-linear least-squares fit (K_M = 3.3 ± 0.3 µM; V_max = 1.44 ± 0.05 min⁻¹ ClpP⁻¹ ). (B) Maximum rates of ClpXP degradation of native GFP substrates with homopolymeric sequences of lysine, proline, or glycine between the folded body of GFP and the ssrA degradation tag. Error bars represent the uncertainty of a non-linear least-squares fit of experimental data to the Michaelis-Menten equation. K_M’s for the fits not shown in panel A were GFP-ssrA (3.3 ± 0.4 µM); GFP-[P₁₅]-ssrA (7 ± 2 µM); GFP-[GV]₅-[G₁₀]-ssrA (2.4 ± 0.4 µM); GFP-[GV]₃-[G₁₀]-ssrA (2.0 ± 0.2 µM); GFP-[G₁₅]-ssrA (2.1 ± 1 µM); GFP-[G₁₀]-ssrA (4.1 ± 0.5 µM).