1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Interactions between a subset of substrate side chains and AAA+ motor pore loops determine grip during protein unfolding

  1. Tristan A Bell  Is a corresponding author
  2. Tania A Baker
  3. Robert T Sauer  Is a corresponding author
  1. Massachusetts Institute of Technology, United States
  2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
Research Article
Cite this article as: eLife 2019;8:e46808 doi: 10.7554/eLife.46808
5 figures, 3 tables and 1 additional file

Figures

Effects of cassette sequence on GFP unfolding and degradation.

(A) Starting at the N terminus, substrates contained residues 1–229 of A.victoria GFP (PDB 1GFL, Yang et al., 1996), a cassette with 12 variable residues, and a partial ssrA degron. (B) Method for measuring intracellular degradation of substrates by ClpXΔN/ClpP. (C) Cellular fluorescence depends upon ClpXΔN/ClpP expression and cassette sequence (listed in Table 1). (D) Fraction intracellular degradation for substrates bearing different cassettes. (E) Fits of the substrate dependence of degradation in vitro to a hyperbolic Michaelis-Menten equation. (F) Vmax values for different substrates. In panels, C–F, values represent averages (± S.D.) of three biological replicates.

https://doi.org/10.7554/eLife.46808.003
Figure 2 with 2 supplements
A small subset of tail residues mediate grip during GFP unfolding.

(A) Fraction intracellular degradation for substrates with tails containing LYV tripeptides in otherwise all-glycine cassettes. Gly12 and GA substrates were included as internal controls. (B) Fraction intracellular degradation for substrates with tails containing one tyrosine (Y) in otherwise all-glycine cassettes. Gly12 and GA substrates were included as internal controls. (C) Vmax values from Michaelis-Menten analysis of degradation of purified substrates with single-tyrosine cassettes. (D) Rates of ATP hydrolysis by ClpXΔN (0.1 μM hexamer) in the presence of ClpP (0.3 μM 14-mer) in the absence (–) or presence of different substrates (15 μM monomer). (E) ATP cost of degrading substrates with single-tyrosine cassettes. Note that the Y-axis is logarithmic. In all panels, values represent averages (± S.D.) of three biological replicates.

https://doi.org/10.7554/eLife.46808.006
Figure 2—source data 1

Stimulation of ClpXP ATP hydrolysis by purified substrates.

Values are averages of three biological replicates ± S.D.

https://doi.org/10.7554/eLife.46808.009
Figure 2—figure supplement 1
Comparison of KM values for substrates tested in vitro; comparison of fitted values for KM for substrate degradation.

Values are the average of three biological replicates ± S.D. None of the substrates exhibited a substantial increase in KM, indicating that differences in degradation rates result from differences in grip rather than in initial substrate recognition.

https://doi.org/10.7554/eLife.46808.007
Figure 2—figure supplement 2
Stimulation of ClpXP ATP hydrolysis by purified substrates.

(A) Rates of ATP hydrolysis by ClpXΔN (0.1 μM hexamer) in the presence of ClpP (0.3 μM 14-mer) in the absence (–) or presence of different substrates (15 μM monomer). (B) ATP cost of degrading substrates. In both panels, values represent averages (± S.D.) of three biological replicates.

https://doi.org/10.7554/eLife.46808.008
Side-chain grip effects at tail-position 4.

(A) In substrates with otherwise all-glycine cassettes, fraction intracellular degradation depends on side-chain identity at tail-position 4. (B) Comparison of degradation in vivo for substrates with Thr or Val at tail-position four or Glu or Gln at tail-position 4 (Student’s two-tailed t-test significance; Val/Thr: t = 6.37, df = 4; Glu/Gln: t = 5.47, df = 4). (C) Vmax values from Michaelis-Menten analysis of degradation of purified substrates. (D) Effects of position-4 residues, color-coded by side-chain properties, on Vmax. (E) Comparison of degradation in vitro between substrates with Ala, Ser, Cys, Thr, or Val at tail-position four or Glu or Gln at tail-position 4 (Student’s two-tailed t-test significance; Val/Thr: t = 13.3, df = 4; Glu/Gln: t = 5.49, df = 4). (F) ATP cost of degrading substrates with Ala, Cys, Thr, Val, Glu, or Gln at tail-position 4. With the exception of panel A, where Gly12 and GA values represent averages (± S.D.) of nine biological replicates, all values represent three biological replicates.

https://doi.org/10.7554/eLife.46808.010
Figure 4 with 1 supplement
Multiple substrate residues contribute synergistically to grip.

(A) GA and Ala-4 cassette sequences. A heatmap of Vmax values from Figure 2C is overlaid to show contribution of single tyrosine residues as each tail position. (B) Fraction intracellular degradation of substrates with one alanine at tail-position 4 and a second alanine at a variable position in otherwise all-glycine cassettes. (C) Comparison of intracellular degradation for a subset of substrates, including Ala-1. (D) Vmax values from Michaelis-Menten analysis of degradation of purified substrates. (E and F) Michaelis-Menten Vmax values for purified substrates with one tyrosine (E) or valine (F) at tail-position four and a second tyrosine (E) or valine (F) at each tail position in otherwise all-glycine cassettes. Overlaid dashed lines indicate degradation rate for the parental Tyr-4 (E) or Val-4 (F) substrates. In all panels, values represent averages (± S.D.) of three biological replicates.

https://doi.org/10.7554/eLife.46808.011
Figure 4—figure supplement 1
Degradation of Dual-Tyr substrates centered at tail position 3.

Vmax values for degradation from Michaelis-Menten analysis of purified substrates with one tyrosine at tail-position three and a second tyrosine at a variable position in otherwise all-glycine cassettes.

Relative degradation for substrate tails with a single Tyr residue at position 3 or 4 indicated by dashed lines. Values represent averages (± S.D.) of three biological replicates.

https://doi.org/10.7554/eLife.46808.012
Only a subset of pore-1 loops in ClpX appear to mediate substrate grip.

(A) Model of an extended poly-alanine substrate in the axial pore of ClpX and its interactions with different pore-1 loops based on cryo-EM structures of ClpXP (X.Fei, T.A. Bell, B.M. Stinson, S. Jenni, T.A. Baker, S.C. Harrison, and R.T. Sauer, in preparation). Similar loop-substrate interactions are observed in the yeast AAA+ protease Yme1 (Puchades et al., 2017). On the right, a heatmap of Vmax values from Figure 2C is shown. The substrate tail residues are numbered relative to where a folded domain would be expected to sit at the apical surface of the AAA+ ring during unfolding. Tail residues 2–6, which promote strong grip in ClpX, are positioned to interact with the three pore-1 loops at the top of the axial pore. (B) Two models for asymmetric contribution of pore-1 loops to substrate grip.

https://doi.org/10.7554/eLife.46808.013

Tables

Table 1
Degradation of variable-tail substrates in the bacterial cytoplasm.

Sequences of all substrate tails tested and the extent of degradation by ClpXΔNP in E. coli after 35 min. For substrates tested in multiple panels, the value presented is from the panel in which they first appear. Values are the average of three biological replicates ± S.D.

https://doi.org/10.7554/eLife.46808.004
SubstrateVariable tail sequenceFraction degraded in vivo
Gly12GGGG GGGG GGGG0.20 ± 0.05
GAAGAG GGAG AGGA0.88 ± 0.07
TitinHLGL IEVE KPLY0.78 ± 0.01
BasicGKGR GKGR GKGR0.83 ± 0.05
AcidicGEGD GEGD GEGD0.96 ± 0.01
LYV2-4GLYV GGGG GGGG0.82 ± 0.03
LYV4-6GGGL YVGG GGGG0.83 ± 0.01
LYV6-8GGGG GLYV GGGG0.65 ± 0.08
LYV8-10GGGG GGGL YVGG0.23 ± 0.01
LYV10-12GGGG GGGG GLYV0.2 ± 0.1
Tyr1YGGG GGGG GGGG0.3 ± 0.1
Tyr2GYGG GGGG GGGG0.49 ± 0.08
Tyr3GGYG GGGG GGGG0.80 ± 0.01
Tyr4GGGY GGGG GGGG0.80 ± 0.02
Tyr5GGGG YGGG GGGG0.8 ± 0.1
Tyr6GGGG GYGG GGGG0.4 ± 0.1
Tyr7GGGG GGYG GGGG0.32 ± 0.05
Tyr8GGGG GGGY GGGG0.20 ± 0.03
Ala4GGGA GGGG GGGG0.28 ± 0.03
Arg4GGGR GGGG GGGG0.39 ± 0.03
Asn4GGGN GGGG GGGG0.23 ± 0.01
Asp4GGGD GGGG GGGG0.20 ± 0.02
Cys4GGGC GGGG GGGG0.35 ± 0.02
Glu4GGGE GGGG GGGG0.27 ± 0.02
Gln4GGGQ GGGG GGGG0.41 ± 0.04
His4GGGH GGGG GGGG0.26 ± 0.01
Ile4GGGI GGGG GGGG0.78 ± 0.05
Leu4GGGL GGGG GGGG0.7 ± 0.1
Lys4GGGK GGGG GGGG0.36 ± 0.02
Met4GGGM GGGG GGGG0.6 ± 0.2
Phe4GGGF GGGG GGGG0.7 ± 0.1
Pro4GGGP GGGG GGGG0.19 ± 0.03
Ser4GGGS GGGG GGGG0.24 ± 0.04
Thr4GGGT GGGG GGGG0.24 ± 0.01
Trp4GGGW GGGG GGGG0.5 ± 0.1
Val4GGGV GGGG GGGG0.7 ± 0.1
Ala1AGGG GGGG GGGG0.41 ± 0.03
Ala1 + 4AGGA GGGG GGGG0.84 ± 0.03
Ala2 + 4GAGA GGGG GGGG0.88 ± 0.01
Ala3 + 4GGAA GGGG GGGG0.88 ± 0.01
Ala4 + 5GGGA AGGG GGGG0.87 ± 0.02
Ala4 + 6GGGA GAGG GGGG0.7 ± 0.1
Ala4 + 7GGGA GGAG GGGG0.51 ± 0.09
Ala4 + 8GGGA GGGA GGGG0.31 ± 0.04
Ala4 + 9GGGA GGGG AGGG0.24 ± 0.07
Ala4 + 10GGGA GGGG GAGG0.29 ± 0.07
Ala4 + 11GGGA GGGG GGAG0.33 ± 0.06
Ala4 + 12GGGA GGGG GGGA0.31 ± 0.04
Table 2
Degradation of purified variable-tail substrates in vitro.

Fitted parameters from Michaelis-Menten analysis of substrate degradation by ClpXΔNP. No fit – substrate degradation too slow to be accurately fit. Values are the average of three biological replicates ± S.D.

https://doi.org/10.7554/eLife.46808.005
SubstrateVmax (min−1 hex−1)KM (μM)
Gly12No fit
GA2.4 ± 0.11.7 ± 0.1
Titin2.3 ± 0.21.1 ± 0.1
Basic1.1 ± 0.11.4 ± 0.1
Acidic2.7 ± 0.31.9 ± 0.2
Tyr1No fit
Tyr20.10 ± 0.030.7 ± 0.2
Tyr30.7 ± 0.21.3 ± 0.2
Tyr41.7 ± 0.11.9 ± 0.2
Tyr51.1 ± 0.11.2 ± 0.1
Tyr60.08 ± 0.020.7 ± 0.3
Tyr7No fit
Tyr8No fit
Ala40.13 ± 0.061.6 ± 0.8
Arg40.4 ± 0.11.8 ± 0.4
Asn4No fit
Asp4No fit
Cys40.16 ± 0.040.9 ± 0.4
Glu40.11 ± 0.061.1 ± 0.6
Gln40.40 ± 0.071.8 ± 0.3
Ile41.4 ± 0.32.2 ± 0.3
Leu41.3 ± 0.12.0 ± 0.2
Lys40.3 ± 0.12.0 ± 0.7
Met41.3 ± 0.12.1 ± 0.3
Phe41.4 ± 0.22.5 ± 0.2
Pro4No fit
Ser4No fit
Thr40.10 ± 0.022.0 ± 0.6
Trp40.48 ± 0.031.4 ± 0.1
Val41.7 ± 0.22.2 ± 0.2
Ala10.19 ± 0.041.8 ± 0.8
Ala1 + 42.3 ± 0.22.2 ± 0.1
Ala3 + 42.4 ± 0.12.1 ± 0.1
Ala4 + 51.7 ± 0.21.5 ± 0.1
Ala4 + 70.38 ± 0.090.8 ± 0.2
Ala4 + 90.09 ± 0.040.5 ± 0.3
Tyr1 + 41.5 ± 0.11.2 ± 0.1
Tyr2 + 42.1 ± 0.11.1 ± 0.1
Tyr3 + 41.2 ± 0.12.4 ± 0.1
Tyr4 + 51.4 ± 0.21.2 ± 0.1
Tyr4 + 62.4 ± 0.21.2 ± 0.1
Tyr4 + 71.0 ± 0.10.67 ± 0.05
Tyr4 + 82.0 ± 0.30.92 ± 0.06
Tyr1 + 31.4 ± 0.10.9 ± 0.1
Tyr2 + 30.81 ± 0.031.1 ± 0.1
Tyr3 + 50.7 ± 0.10.43 ± 0.03
Tyr3 + 61.1 ± 0.10.62 ± 0.05
Tyr3 + 70.97 ± 0.080.61 ± 0.03
Tyr3 + 80.49 ± 0.070.39 ± 0.06
Val1 + 42.3 ± 0.11.6 ± 0.1
Val2 + 41.8 ± 0.11.2 ± 0.1
Val3 + 41.6 ± 0.11.1 ± 0.1
Val4 + 52.1 ± 0.11.5 ± 0.1
Val4 + 61.4 ± 0.11.1 ± 0.1
Val4 + 71.3 ± 0.10.93 ± 0.01
Val4 + 81.0 ± 0.10.88 ± 0.06
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Cell line (Escherichia coli)E. coli T7 Express ΔclpA ΔclpP ΔclpXthis paperE. coli strain lacking the ClpA, ClpP, and ClpX genes. progenitor: E. coli T7 Express (New England Biolabs #C2566)
Recombinant DNA reagentpT7 ClpXΔN(plasmid)Martin et al., 2005N-terminally His6-tagged ClpXΔN (residues 62–424) for overexpression
Recombinant DNA reagentpT7 ClpP (plasmid)Kim et al., 2000C-terminally His6-tagged ClpP for overexpression
Recombinant DNA reagentpBAD ClpP/ClpXΔN(plasmid)this paperfor inducible polycistronic expression of ClpP and ClpXΔN(residues 62–424) for cytoplasmic GFP degradation assays. Progenitor: pBAD (Guzman et al., 1995; jb.177.14.4121–4130.199)
Recombinant DNA reagentpBAD null (plasmid)this papercontrol plasmid for cytoplasmic GFP degradation assays. Progenitor: pBAD (Guzman et al., 1995)
Recombinant DNA reagentProD GFP Gly12 ssrA (plasmid)this paperfor constitutive expression of GFP (residues 1–229) substrates with a 12xGly cassette and partial ssrA (GSENYALAA). All other substrates are derivatives of this construct with different variable cassette sequences. Progenitor: ProD Gemini (Davis et al., 2011; nar/gkq81)
Recombinant DNA reagentpT7 GFP Gly12 ssrA (plasmid)this paperfor overexpression of N-terminally His6-tagged GFP (1-229) substrates with a 12xGly cassette and partial ssrA (GSENYALAA). All other substrates are derivatives of this construct with different variable cassette sequences.

Data availability

All data generated during this study are included in the manuscript and supporting files as Tables 1 and 2 and Figure 2 - source data 1.

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