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. 2014 Sep 16;111(37):E3853-9.
doi: 10.1073/pnas.1414933111. Epub 2014 Sep 3.

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

Izarys Rivera-Rivera  1 , Giselle Román-Hernández  1 , Robert T Sauer  1 , Tania A Baker  2
Affiliations

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

Izarys Rivera-Rivera et al. Proc Natl Acad Sci U S A. .

Abstract

The ClpS adaptor collaborates with the AAA+ ClpAP protease to recognize and degrade N-degron substrates. ClpS binds the substrate N-degron and assembles into a high-affinity ClpS-substrate-ClpA complex, but how the N-degron is transferred from ClpS to the axial pore of the AAA+ ClpA unfoldase to initiate degradation is not known. Here we demonstrate that the unstructured N-terminal extension (NTE) of ClpS enters the ClpA processing pore in the active ternary complex. We establish that ClpS promotes delivery only in cis, as demonstrated by mixing ClpS variants with distinct substrate specificity and either active or inactive NTE truncations. Importantly, we find that ClpA engagement of the ClpS NTE is crucial for ClpS-mediated substrate delivery by using ClpS variants carrying "blocking" elements that prevent the NTE from entering the pore. These results support models in which enzymatic activity of ClpA actively remodels ClpS to promote substrate transfer, and highlight how ATPase/motor activities of AAA+ proteases can be critical for substrate selection as well as protein degradation.

Keywords: AAA+ ATPase adaptor; AAA+ unfoldase/translocase; N-degron substrate selection; adaptor remodeling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Model for the active delivery mechanism used by ClpS. (A) The adaptor protein ClpS has a long, flexible N-terminal extension (NTE; residues 1–25) and a folded core domain (ClpScore; residues 26–106). ClpScore binds N-degrons. A substrate Tyr in the binding pocket is shown in red (Protein Data Bank ID code 3O1F). Successful substrate delivery requires that the ClpS NTE be at least 14-aa long (shown in green). (B) Formation of an HADC between ClpS, substrate, and ClpA (19) involves formation of additional contacts among ClpA, ClpS, and the N-degron substrate. Assembly of this complex increases the affinity of the substrate for ClpAS by ∼100-fold (19). (C) The current model for ClpA-driven disassembly of the HADC and N-degron substrate delivery. Translocation-mediated ClpA “pulling” on the NTE remodels the ClpScore structure, weakens the interactions of ClpS with the N-degron, and facilitates its transfer to a site in the ClpA pore. Finally, because ClpS cannot be unfolded by ClpA (19), the adaptor escapes the enzyme, and the substrate is unfolded by ClpA and subsequently degraded by ClpP.
Fig. 2.
Fig. 2.
The ClpS NTE delivers N-degron substrates in cis. (A) Cartoon showing the protein variants for the mixing experiments performed to test cis vs. trans activation by the ClpS-NTE. Although present, the N-degron peptide (Phe-Val) is not depicted. (B) Degradation of the *N-degron substrate (vlfvqela-GFP) by ClpAP. Only when the full-length functional NTE and *N-degron–binding pocket were present on the same ClpS molecule was this substrate efficiently degraded (cis delivery experiment, blue trace). The mixing experiments contained each of the ClpS variants shown in A (1.2 µM each), 1 µM of an N-degron peptide, and 1 µM of *N-degron substrate. (C) Binding of fluorescein-labeled ClpSΔ13/M40A (1.2 µM) to ClpA6 in the presence of ATPγS (2 mM), ClpS (1.2 µM), N-degron peptide (1 µM), and N*-degron peptide (1 µM), as assayed by fluorescence anisotropy (KD = 112 ± 13 nM).
Fig. 3.
Fig. 3.
The ClpS NTE localizes inside the ClpA pore. (A) Cartoon of the protein variants used in the FRET experiments. Three single-cysteine variants of ClpS were labeled with fluorescein (acceptor fluorophore, yellow star). The labeled positions were C5 and C17, both sites in the ClpS NTE (ClpS5-Fl and ClpS17-Fl), and C96, which is in the ClpS core domain (ClpS96-Fl). Unlabeled ClpA was used with a ClpP variant in which residue 17 of each subunit was changed to cysteine and labeled with EDANS (donor fluorophore, green star; ClpPED). This ClpP variant also contains the S97A active-site mutation (45). (B) Emission spectra of the donor fluorophore in ClpPED on excitation at 336 nm in the presence of ClpA6 and ATPγS (black trace); emission spectra of the acceptor fluorophore in ClpS5-Fl on excitation at 336 nm in the presence of ATPγS (green trace); addition spectra of the two independent traces obtained from the emission of the donor and acceptor proteins (gray line); and observed emission spectra characteristic of FRET obtained in reactions containing ATPγS, ClpS5-Fl, ClpAPED, and the N-degron substrate ylfvq-titin I27 (red trace). The red arrow pointing up at ∼525 nm denotes an increase in fluorescence of the acceptor fluorophore, and the red arrow pointing down at ∼475 nm denotes the decreased signal of the donor fluorophore. (C) FRET was also observed when the experiment shown in B was repeated with ClpS17-Fl as the acceptor molecule (Left, red). In contrast, no FRET was detected when the acceptor molecule was ClpS96-Fl (Right, red). (D) ClpS5-Fl fluorescence was insensitive to the fluorescence quencher 4-amino-TEMPO when bound in a complex with ClpAPED and N-degron substrate.
Fig. 4.
Fig. 4.
ClpAP cleaves an extended ClpS NTE. (A) Cartoon of the NTE2-ClpS variant. (B) Delivery and degradation of the N-degron substrate, ylfvqela-GFP, to ClpAP in the absence of ClpS, in the presence of NTE2-ClpS, or in the presence of WT ClpS. Degradation was monitored by the decrease in substrate fluorescence. (C) Truncation of NTE2-ClpS was observed during delivery of N-degron substrates to ClpAP in the presence of ATP (Top), but was not observed without ATP (Middle) or with ATPγS (Bottom). N-terminal sequencing of the lowest molecular weight product revealed an NTE “tail” of 34 amino acids. This “trimmed” NTE2-ClpS truncation product is depicted as a cartoon below the Top panel.
Fig. 5.
Fig. 5.
Engagement of the ClpS NTE is necessary for delivery of N-degron substrates. (A) Cartoon of the H6-DHFR-ClpS fusion protein. (B) Cartoon of results obtained upon addition of ClpAP and ATP to H6-DHFR-ClpS in the absence or presence of methotrexate. Protein processing was monitored by Western blot analysis of the H6-DHFR-ClpS protein with anti-ClpS antisera. ClpAP-dependent cleavage of the fusion protein and release of a truncated ClpS adaptor (with an available NTE) was observed in the absence of methotrexate (Left), whereas no processing of the fusion protein was detected when methotrexate was present (Right). (C) Delivery and degradation of the N-degron substrate ylfvqela-GFP by ClpAP promoted by either H6-DHFR-ClpS or ClpS in the presence and absence of methotrexate. (D) Formation of an HADC by ClpS (Kapp= 35 ± 1 nM), H6-DHFR-ClpS (Kapp= 107 ± 17 nM), and H6-DHFR-ClpS in the presence of methotrexate (Kapp= 119 ± 21 nM) assayed by anisotropy using a fluorescent N-degron peptide.
Fig. 6.
Fig. 6.
Model for ClpA-dependent N-degron substrate transfer. (A) HADC. (B) ClpA-dependent translocation of the ClpS NTE begins to deform the ClpScore by pulling on the middle β1 strand of the three-stranded β-sheet. (C) Extraction of the β1 strand of ClpS facilitates substrate transfer by inverting the adaptor, thereby positioning the N-degron–binding pocket close to the ClpA pore, and by weakening interactions between the substrate and the ClpS-binding pocket. (D) Subsequently, ΔβClpS resists further unfolding and thus is released from the ternary complex, allowing for refolding of the adaptor and translocation and degradation of the N-degron substrate to commence.
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