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[Preprint]. 2023 Dec 15:2023.12.14.571662.
doi: 10.1101/2023.12.14.571662.

A proteolytic AAA+ machine poised to unfold a protein substrate

Alireza Ghanbarpour  1 , Robert T Sauer  1 , Joseph H Davis  1   2
Affiliations

A proteolytic AAA+ machine poised to unfold a protein substrate

Alireza Ghanbarpour et al. bioRxiv. .

Update in

Abstract

AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1:
Figure 1:. ClpXP bound to degron-tagged DHFR.
(a) Cartoon of steps in ClpXP degradation of a protein substrate. (b) Overlay of cryo-EM map and model of ClpXP bound to the degron-tagged DHFR substrate (left). Density for the degron tail could be modeled extending from the folded domain of DHFR to the ClpP entry portal (center). The insets (right) display density for methotrexate and the DHFR Trp30 and Phe31 side chains (top), and the last two residues of native DHFR, Arg158, and Arg159, contacted by the pore-1 loop of ClpX subunit A, which includes Tyr153.
Figure 2:
Figure 2:. ClpX•DHFR interactions and the consequences of binding.
(a) Front and back views of contacts between pore-1 loops of ClpX (residues 150-155; sphere representation), pore-2 loops of ClpX (residues 198-205; cartoon representation), and DHFR and its degron tail (red surface representation). The six ClpX subunits are annotated A-F and colored purple, salmon, green, wheat, orange, and grey, respectively. The ClpX subunit F pore-1 loop is disengaged from the degron tail but adjacent to the folded domain of DHFR. (b) Top view showing packing of the ClpX RKH loops (residues 220-238; sphere representation colored by subunit as above) around native DHFR (red cartoon representation). (c) Side views of ClpX complexes with DHFR (left) and an ssrA degron (right; pdb 6WRF) illustrating major upward movement of subunit F. (d) Bars show mean rates of ATP hydrolysis for ClpX, ClpXP, or ClpXP in the presence of degron tagged DHFR•MTX ± 1 SD, with symbols denoting replicate assays.
See this image and copyright information in PMC

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