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

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-substrateClpA 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.AAA+ ATPase adaptor | N-degron substrate selection | adaptor remodeling | AAA+ unfoldase/translocase A AA+ molecular machines power cellular processes as diverse as protein degradation, microtubule severing, membrane fusion, and initiation of DNA replication, with the common theme that macromolecules are actively remodeled (1-3). Furthermore, protein quality control in all organisms involves deployment of ATPdependent proteases, consisting of hexameric AAA+ rings that unfold and translocate specific substrates into an associated peptidase barrel (3, 4). Adaptor proteins are known to aid recognition and degradation of certain substrates (5-8), but how enzyme-adaptor pairs ensure proper substrate selection is poorly understood.In prokaryotes and eukaryotes, the N-end rule pathway governs degradation of proteins with specific N-terminal amino acids (9, 10). In Escherichia coli, the primary destabilizing N-degron amino acids are Phe, Tyr, Trp, and Leu (11,12). ClpS, a widespread bacterial adaptor, recognizes and delivers N-degron substrates to the ClpAP or ClpCP AAA+ proteases (6,11,13). These enzymes consist of the AAA+ ClpA or ClpC unfoldases coaxially stacked with the ClpP peptidase (14-16). In eukaryotes, a family of E3 ligases shares homology with the substrate-binding region of ClpS (17, 18). These ligases recognize N-degron substrates and promote ubiquitination, which then targets the modified protein to the 26S proteasome (17,18).Multiple crystal structures reveal the regions of ClpS that bind to the N-degron, as well as a patch that binds the N-terminal domain of . This bivalent binding to the substrate and the enzyme tethers N-degron substrates to ClpAP. Tethering alone is insufficient for ClpS to promote substrate delivery however, given that deletion of 12 amino acids of the ClpS unstructured N-terminal extension (NTE; residues 1-25 in E. coli ClpS) (Fig. 1A) prevents N-degron substrate degradation, but does not block format...

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

“…ClpC/D can recognize substrates directly (Rosano et al, 2011;Bruch et al, 2012;Huang et al, 2016), although recognition of a subset of proteins is mediated by a dedicated binary adaptor consisting of ClpS1 and ClpF (Nishimura et al, 2013. The basic ClpS structure has an N-terminal extension for substrate delivery to the chaperone and a C-terminal core domain for substrate recognition and chaperone binding Zeth et al, 2002;Erbse et al, 2006;Rivera-Rivera et al, 2014). ClpF is a plastidspecific ClpS1-interacting protein with a tripartite structure harboring a unique N-terminal domain for adaptor-substrate-chaperone binding, a uvrB/C motif for chaperone interaction, and a YccV-like domain of unknown function, the last two of which likely are derived from two distinct proteins of bacterial origin .…”

Discovery of a Unique Clp Component, ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery

“…Escherichia coli ClpS recognizes substrates containing N-degrons and delivers them to the chaperone's N-terminal domain (N-domain) for degradation (Erbse et al, 2006;Rivera-Rivera et al, 2014). The ClpS core domain is responsible for substrate recognition and N-domain docking (Zeth et al, 2002;Erbse et al, 2006).…”

“…The ClpS core domain is responsible for substrate recognition and N-domain docking (Zeth et al, 2002;Erbse et al, 2006). The substrate delivery into the Clp protease core complex is triggered by ClpA pulling on an unstructured N-terminal extension (NTE) of ClpS (Rivera-Rivera et al, 2014). Notably, ClpS is necessary and sufficient for recognition and delivery of N-end substrates in the bacterial Clp system, without any known additional factors.…”

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

“…It functions as a site of protein polyubiquitylation, is often engaged stochastically (in competition with other "eligible" lysines), and tends to be located in a conformationally disordered region (2,9,19,20). Bacteria also contain the N-end rule pathway, but Ub-independent versions of it (21)(22)(23)(24)(25)(26).…”