The ClpS adaptor mediates staged delivery of N-end rule substrates to the AAA+ ClpAP protease

The ClpS adaptor delivers N-end rule substrates to ClpAP, an energy-dependent AAA+ protease, for degradation. How ClpS binds specific N-end residues is known in atomic detail and clarified here, but the delivery mechanism is poorly understood. We show that substrate binding is enhanced when ClpS binds hexameric ClpA. Reciprocally, N-end rule substrates increase ClpS affinity for ClpA(6). Enhanced binding requires the N-end residue and a peptide bond of the substrate, as well as multiple aspects of ClpS, including a side chain that contacts the substrate α-amino group and the flexible N-terminal extension (NTE). Finally, enhancement also needs the N domain and AAA+ rings of ClpA, connected by a long linker. The NTE can be engaged by the ClpA translocation pore, but ClpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the adaptor to the protease.     

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA₆). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA₆ with a t_½ of ∼3 min at 37 °C. Maximum activation of degradation of the N-end rule substrate, LR-GFP_Venus, occurs with a single ClpS bound per ClpA₆; one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA₆, ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA₆. Limiting ClpS-mediated substrate delivery to one per ClpA₆ avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.     

The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies

The N-end rule states that the half-life of a protein is determined by the nature of its N-terminal residue. This fundamental principle of regulated proteolysis is conserved from bacteria to mammals. Although prokaryotes and eukaryotes employ distinct proteolytic machineries for degradation of N-end rule substrates, recent findings indicate that they share common principles of substrate recognition. In eukaryotes substrate recognition is mediated by N-recognins, a class of E3 ligases that labels N-end rule substrates via covalent linkage to ubiquitin, allowing the subsequent substrate delivery to the 26S proteasome. In bacteria, the adaptor protein ClpS exhibits homology to the substrate binding site of N-recognin. ClpS binds to the destabilizing N-termini of N-end rule substrates and directly transfers them to the ClpAP protease.     

An intrinsic degradation tag on the ClpA C-terminus regulates the balance of ClpAP complexes with different substrate specificity

ATP-dependent protein degradation in bacteria is carried out by barrel-shaped proteases architecturally related to the proteasome. In Escherichia coli, ClpP interacts with two alternative ATPases, ClpA or ClpX, to form active protease complexes. ClpAP and ClpXP show different but overlapping substrate specificities. ClpXP is considered the primary recipient of ssrA-tagged substrates while ClpAP in complex with ClpS processes N-end rule substrates. Notably, in its free form, but not in complex with ClpS, ClpAP also degrades ssrA-tagged substrates and its own chaperone component, ClpA. To reveal the mechanism of ClpAP-mediated ClpA degradation, termed autodegradation, and its possible role in regulating ClpAP levels, we dissected ClpA to show that the flexible C-terminus of the second AAA module serves as the degradation signal. We demonstrate that ClpA becomes largely resistant to autodegradation in the absence of its C-terminus and, conversely, transfer of the last 11 residues of ClpA to the C-terminus of green fluorescent protein (GFP) renders GFP a substrate of ClpAP. This autodegradation tag bears similarity to the ssrA-tag in its degradation behavior, displaying similar catalytic turnover rates when coupled to GFP but a twofold lower apparent affinity constant compared to ssrA-tagged GFP. We show that, in analogy to the prevention of ssrA-mediated recognition, the adaptor ClpS inhibits autodegradation by a specificity switch as opposed to direct masking of the degradation signal. Our results demonstrate that in the presence of ssrA-tagged substrates, ClpA autodegradation will be competitively reduced. This simple mechanism allows for dynamic reallocation of free ClpAP versus ClpAPS in response to the presence of ssrA-tagged substrates.     

Tuning the strength of a bacterial N-end rule degradation signal

The N-end rule is a degradation pathway conserved from bacteria to mammals that links a protein's stability in vivo to the identity of its N-terminal residue. In Escherichia coli, the components of this pathway directly responsible for protein degradation are the ClpAP protease and its adaptor ClpS. We recently demonstrated that ClpAP is able to recognize N-end motifs in the absence of ClpS although with significantly reduced substrate affinity. In this study, a systematic sequence analysis reveals new features of N-end rule degradation signals. To achieve specificity, recognition of an N-end motif by the protease-adaptor complex uses both the identity of the N-terminal residue and a free alpha-amino group. Acidic residues near the first residue decrease substrate affinity, demonstrating that the identity of adjacent residues can affect recognition although significant flexibility is tolerated. However, shortening the distance between the N-end residue and the stably folded portion of a protein prevents degradation entirely, indicating that an N-end signal alone is not always sufficient for degradation. Together, these data define in vitro the sequence and structural requirements for the function of bacterial N-end signals.     

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.     

Bioinformatic analysis of ClpS,a protein module involved in prokaryotic and eukaryotic protein degradation

ClpS is a small protein, usually encoded immediately upstream of ClpA in the genomes of proteobacteria. Recent results show that it is a molecular adaptor for substrate recognition by ClpA in Escherichia coli. We analyzed ClpS by bioinformatic methods and found that ClpS homologs are also found in organisms that lack ClpA, such as actinobacteria, cyanobacteria, and plant chloroplasts. Furthermore, ClpS is homologous to a domain in the eukaryotic E3 ubiquitin ligase, N-recognin. This domain has previously been described as responsible for the recognition of type 2 N-end rule substrates. Despite very low levels of sequence similarity to proteins of known structure, there appears to be substantial structural similarity between ClpS and the C-terminal domain of ribosomal protein L7/12 (1CTF).     

ClpS, a substrate modulator of the ClpAP machine

In the bacterial cytosol, ATP-dependent protein degradation is performed by several different chaperone-protease pairs, including ClpAP. The mechanism by which these machines specifically recognize substrates remains unclear. Here, we report the identification of a ClpA cofactor from Escherichia coli, ClpS, which directly influences the ClpAP machine by binding to the N-terminal domain of the chaperone ClpA. The degradation of ClpAP substrates, both SsrA-tagged proteins and ClpA itself, is specifically inhibited by ClpS. In contrast, ClpS enhanced ClpA recognition of two heat-aggregated proteins in vitro and, consequently, the ClpAP-mediated disaggregation and degradation of these substrates. We conclude that ClpS modifies ClpA substrate specificity, potentially redirecting degradation by ClpAP toward aggregated proteins.     

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 A resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.     

Crystal structure of the heterodimeric complex of the adaptor,ClpS, with the N-domain of the AAA+ chaperone,ClpA

Substrate selectivity and proteolytic activity for the E. coli ATP-dependent protease, ClpAP, is modulated by an adaptor protein, ClpS. ClpS binds to ClpA, the regulatory component of the ClpAP complex. We report the crystal structure of ClpS in complex with the isolated N-terminal domain of ClpA in two different crystal forms at 2.3- and 3.3-A resolution. The ClpS structure forms an alpha/beta-sandwich and is topologically analogous to the C-terminal domain of the ribosomal protein L7/L12. ClpS contacts two surfaces on the N-terminal domain in both crystal forms; the more extensive interface was shown to be favored in solution by protease protection experiments. The N-terminal 20 residues of ClpS are not visible in the crystal structures; the removal of the first 17 residues produces ClpSDeltaN, which binds to the ClpA N-domain but no longer inhibits ClpA activity. A zinc binding site involving two His and one Glu residue was identified crystallographically in the N-terminal domain of ClpA. In a model of ClpS bound to hexameric ClpA, ClpS is oriented with its N terminus directed toward the distal surface of ClpA, suggesting that the N-terminal region of ClpS may affect productive substrate interactions at the apical surface or substrate entry into the ClpA translocation channel.     

The N-end rule pathway controls the import of peptides through degradation of a transcriptional repressor.

Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress. One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues. Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein. Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import. We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p. Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter. In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal. The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway. An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae. Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis.     

The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades.     

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.     

ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis

The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 A, reveals specific recognition of the peptide alpha-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.     

A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons

A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1(-/-) UBR2(-/-) mice die as early embryos, the rescued UBR1(-/-) UBR2(-/-) fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a approximately 70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1(-/-) UBR2(-/-) fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.     

Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease

ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA’s substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to “tug on” and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines.     

The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule pathway, stabilizes Tex19.1 during spermatogenesis

Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation or altered biochemical properties. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule proteolytic pathway, recognizes proteins with N-terminal destabilizing residues and plays an important role in spermatogenesis. Tex19.1 (also known as Tex19) has been previously identified as a germ cell-specific protein in mouse testis. Here we report that Tex19.1 forms a stable protein complex with Ubr2 in mouse testes. The binding of Tex19.1 to Ubr2 is independent of the second position cysteine of Tex19.1, a putative target for arginylation by the N-end rule pathway R-transferase. The Tex19.1-null mouse mutant phenocopies the Ubr2-deficient mutant in three aspects: heterogeneity of spermatogenic defects, meiotic chromosomal asynapsis, and embryonic lethality preferentially affecting females. In Ubr2-deficient germ cells, Tex19.1 is transcribed, but Tex19.1 protein is absent. Our results suggest that the binding of Ubr2 to Tex19.1 metabolically stabilizes Tex19.1 during spermatogenesis, revealing a new function for Ubr2 outside the conventional N-end rule pathway.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

Characterization of the N-terminal repeat domain of Escherichia coli ClpA-A class I Clp/HSP100 ATPase

The ClpA, ClpB, and ClpC subfamilies of the Clp/HSP100 ATPases contain a conserved N-terminal region of approximately 150 residues that consists of two approximate sequence repeats. This sequence from the Escherichia coli ClpA enzyme is shown to encode an independent structural domain (the R domain) that is monomeric and approximately 40% alpha-helical. A ClpA fragment lacking the R domain showed ATP-dependent oligomerization, protein-stimulated ATPase activity, and the ability to complex with the ClpP peptidase and mediate degradation of peptide and protein substrates, including casein and ssrA-tagged proteins. Compared with the activities of the wild-type ClpA, however, those of the ClpA fragment missing the R domain were reduced. These results indicate that the R domain is not required for the basic recognition, unfolding, and translocation functions that allow ClpA-ClpP to degrade some protein substrates, but they suggest that it may play a role in modulating these activities.