Structural basis of N‐end rule substrate recognition in Escherichia coli by the ClpAP adaptor protein ClpS

In Escherichia coli, the ClpAP protease, together with the adaptor protein ClpS, is responsible for the degradation of proteins bearing an amino‐terminal destabilizing amino acid (N‐degron). Here, we determined the three‐dimensional structures of ClpS in complex with three peptides, each having a different destabilizing residue—Leu, Phe or Trp—at its N terminus. All peptides, regardless of the identity of their N‐terminal residue, are bound in a surface pocket on ClpS in a stereo‐specific manner. Several highly conserved residues in this binding pocket interact directly with the backbone of the N‐degron peptide and hence are crucial for the binding of all N‐degrons. By contrast, two hydrophobic residues define the volume of the binding pocket and influence the specificity of ClpS. Taken together, our data suggest that ClpS has been optimized for the binding and delivery of N‐degrons containing an N‐terminal Phe or Leu.     

The N-end rule pathway and regulation by proteolysis

The N‐end rule relates the regulation of the in vivo half‐life of a protein to the identity of its N‐terminal residue. Degradation signals (degrons) that are targeted by the N‐end rule pathway include a set called N‐degrons. The main determinant of an N‐degron is a destabilizing N‐terminal residue of a protein. In eukaryotes, the N‐end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N‐end rule and the Arg/N‐end rule pathways. The Ac/N‐end rule pathway targets proteins containing N^α‐terminally acetylated (Nt‐acetylated) residues. The Arg/N‐end rule pathway recognizes unacetylated N‐terminal residues and involves N‐terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt‐acetylated. Thus, most proteins harbor a specific degradation signal, termed ^AcN‐degron, from the moment of their birth. Specific N‐end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N‐degrons include methionine‐aminopeptidases, caspases, calpains, Nt‐acetylases, Nt‐amidases, arginyl‐transferases, and leucyl‐transferases. Regulated degradation of specific proteins by the N‐end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights.     

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

Clp proteases are found in prokaryotes, mitochondria, and plastids where they play crucial roles in maintaining protein homeostasis (proteostasis). The plant plastid Clp machinery comprises a hetero-oligomeric ClpPRT proteolytic core, ATP-dependent chaperones ClpC and ClpD, and an adaptor protein, ClpS1. ClpS1 selects substrates to the ClpPR protease-ClpC chaperone complex for degradation, but the underlying substrate recognition and delivery mechanisms are currently unclear. Here, we characterize a ClpS1-interacting protein in Arabidopsis thaliana, ClpF, which can interact with the Clp substrate glutamyl-tRNA reductase. ClpF and ClpS1 mutually stimulate their association with ClpC. ClpF, which is only found in photosynthetic eukaryotes, contains bacterial uvrB/C and YccV protein domains and a unique N-terminal domain. We propose a testable model in which ClpS1 and ClpF form a binary adaptor for selective substrate recognition and delivery to ClpC, reflecting an evolutionary adaptation of the Clp system to the plastid proteome.     

Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design

Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains—a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs.     

Modification of PATase by L/F‐transferase generates a ClpS‐dependent N‐end rule substrate in Escherichia coli

The N‐end rule pathway is conserved from bacteria to man and determines the half‐life of a protein based on its N‐terminal amino acid. In Escherichia coli, model substrates bearing an N‐degron are recognised by ClpS and degraded by ClpAP in an ATP‐dependent manner. Here, we report the isolation of 23 ClpS‐interacting proteins from E. coli. Our data show that at least one of these interacting proteins—putrescine aminotransferase (PATase)—is post‐translationally modified to generate a primary N‐degron. Remarkably, the N‐terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl‐tRNA‐protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N‐terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N‐end rule pathway, through the ClpAPS‐mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N‐degron, which is required for substrate delivery to ClpA.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

Does N‐Terminal Protein Acetylation Lead to Protein Degradation?

The N‐end rule denotes the relationship between the identity of the amino‐terminal residue of a protein and its in vivo half‐life. Since its discovery in 1986, the N‐end rule has generally been described by a defined set of rules for determining whether an amino‐terminal residue is stabilizing or not. However, recent studies are revealing that this N‐end rule (or N‐degron concept) is less straightforward than previously appreciated. For instance, it is unveiled that N‐terminal acetylation of N‐terminal residues may create a degradation signal (Ac‐degron) that promotes the degradation of target proteins. A recent high‐throughput dissection of degrons in yeast proteins amino termini intriguingly suggested that the hydrophobicity of amino‐terminal residues—but not the N‐terminal acetylation status—may be the indispensable feature of amino‐terminal degrons. Herein, these recent advances in N‐terminal acetylation and the complexity of N‐terminal degradation signals in the context of the N‐degron pathway are analyzed.     

GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

Background information. The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo‐erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. Results. In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo‐erythrocytic schizogony in vitro, leading to impaired parasite maturation. Conclusions. Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red‐blood‐cell‐infective merozoites.     

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.     

ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway

The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its N-terminal residue. In Escherichia coli the adaptor protein ClpS directly interacts with destabilizing N-terminal residues and transfers them to the ClpA/ClpP proteolytic complex for degradation. The crucial role of ClpS in N-end rule degradation is currently under debate, since ClpA/ClpP was shown to process selected N-terminal degrons harbouring destabilizing residues in the absence of ClpS. Here, we investigated the contribution of ClpS to N-end rule degradation by two approaches. First, we performed a systematic mutagenesis of selected N-degron model substrates, demonstrating that ClpS but not ClpA specifically senses the nature of N-terminal residues. Second, we identified two natural N-end rule substrates of E. coli : Dps and PATase (YgjG). The in vivo degradation of both proteins strictly relied on ClpS, thereby establishing the function of ClpS as the essential discriminator of the E. coli N-end rule pathway.     

Structural basis for the N‐degron specificity of ClpS1 from Arabidopsis thaliana

The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein.     

Plasmodium falciparum OTU‐like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with noncanonical role of Atg8

The metabolic pathways associated with the mitochondrion and the apicoplast in Plasmodium, 2 parasite organelles of prokaryotic origin, are considered as suitable drug targets. In the present study, we have identified functional role of a novel ovarian tumour unit (OTU) domain‐containing cysteine protease of Plasmodium falciparum (PfOTU). A C‐terminal regulatable fluorescent affinity tag on native protein was utilised for its localization and functional characterization. Detailed studies showed vesicular localization of PfOTU and its association with the apicoplast. Degradation‐tag mediated knockdown of PfOTU resulted in abnormal apicoplast development and blocked development of parasites beyond early‐schizont stages in subsequent cell cycle; downregulation of PfOTU hindered apicoplast protein import. Further, the isoprenoid precursor‐mediated parasite growth‐rescue experiments confirmed that PfOTU knockdown specifically effect development of functional apicoplast. We also provide evidence for a possible biological function of PfOTU in membrane deconjugation of Atg8, which may be linked with the apicoplast protein import. Overall, our results show that the PfOTU is involved in apicoplast homeostasis and associates with the noncanonical function of Atg8 in maintenance of parasite apicoplast.     

ClpS1 Is a Conserved Substrate Selector for the Chloroplast Clp Protease System in Arabidopsis

Whereas the plastid caseinolytic peptidase (Clp) P protease system is essential for plant development, substrates and substrate selection mechanisms are unknown. Bacterial ClpS is involved in N-degron substrate selection and delivery to the ClpAP protease. Through phylogenetic analysis, we show that all angiosperms contain ClpS1 and some species also contain ClpS1-like protein(s). In silico analysis suggests that ClpS1 is the functional homolog of bacterial ClpS. We show that Arabidopsis thaliana ClpS1 interacts with plastid ClpC1,2 chaperones. The Arabidopsis ClpS1 null mutant (clps1) lacks a visible phenotype, and no genetic interactions with ClpC/D chaperone or ClpPR core mutants were observed. However, clps1, but not clpc1-1, has increased sensitivity to the translational elongation inhibitor chloramphenicol suggesting a link between translational capacity and ClpS1. Moreover, ClpS1 was upregulated in clpc1-1, and quantitative proteomics of clps1, clpc1, and clps1 clpc1 showed specific molecular phenotypes attributed to loss of ClpC1 or ClpS1. In particular, clps1 showed alteration of the tetrapyrrole pathway. Affinity purification identified eight candidate ClpS1 substrates, including plastid DNA repair proteins and Glu tRNA reductase, which is a control point for tetrapyrrole synthesis. ClpS1 interaction with five substrates strictly depended on two conserved ClpS1 residues involved in N-degron recognition. ClpS1 function, substrates, and substrate recognition mechanisms are discussed.     

A genetic screen in rodent malaria parasites identifies five new apicoplast putative membrane transporters,one of which is essential in human malaria parasites

The malaria‐causing parasite, Plasmodium, contains a unique non‐photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the malaria parasite apicoplast membranes. We selected 27 candidate apicoplast membrane proteins, 20 of which are annotated as putative membrane transporters, and performed a genetic screen in Plasmodium berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast‐localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4), and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast‐localised. Interestingly, three apicoplast‐localised candidates lack a canonical apicoplast targeting signal but do contain conserved N‐terminal tyrosines with likely roles in targeting. An inducible knockdown of an essential apicoplast putative membrane transporter, PfDMT2, was only viable when supplemented with isopentenyl diphosphate. Knockdown of PfDMT2 resulted in loss of the apicoplast, identifying PfDMT2 as a crucial apicoplast putative membrane transporter and a candidate for therapeutic intervention.     

Protein binding and disruption by Clp/Hsp100 chaperones

Clp/Hsp100 chaperones work with other cellular chaperones and proteases to control the quality and amounts of many intracellular proteins. They employ an ATP-dependent protein unfoldase activity to solubilize protein aggregates or to target specific classes of proteins for degradation. The structural complexity of Clp/Hsp100 proteins combined with the complexity of the interactions with their macromolecular substrates presents a considerable challenge to understanding the mechanisms by which they recognize and unfold substrates and deliver them to downstream enzymes. Fortunately, high-resolution structural data is now available for several of the chaperones and their functional partners, which together with mutational data on the chaperones and their substrates has provided a glimmer of light at the end of the Clp/Hsp100 tunnel.     

ClpAP is an auxiliary protease for DnaA degradation in Caulobacter crescentus

The Clp family of proteases is responsible for controlling both stress responses and normal growth. In Caulobacter crescentus, the ClpXP protease is essential and drives cell cycle progression through adaptor‐mediated degradation. By contrast, the physiological role for the ClpAP protease is less well understood with only minor growth defects previously reported for ΔclpA cells. Here, we show that ClpAP plays an important role in controlling chromosome content and cell fitness during extended growth. Cells lacking ClpA accumulate aberrant numbers of chromosomes upon prolonged growth suggesting a defect in replication control. Levels of the replication initiator DnaA are elevated in ΔclpA cells and degradation of DnaA is more rapid in cells lacking the ClpA inhibitor ClpS. Consistent with this observation, ClpAP degrades DnaA in vitro while ClpS inhibits this degradation. In cells lacking Lon, the protease previously shown to degrade DnaA in Caulobacter, ClpA overexpression rescues defects in fitness and restores degradation of DnaA. Finally, we show that cells lacking ClpA are particularly sensitive to inappropriate increases in DnaA activity. Our work demonstrates an unexpected effect of ClpAP in directly regulating replication through degradation of DnaA and expands the functional role of ClpAP in Caulobacter.     

The Clp ATPases define a novel class of molecular chaperones

The Clp ATPases were originally identified as a regulatory component of the bacterial ATP-dependent Clp serine proteases. Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far. Recent data suggest that the Clp ATPases are not only specificity factors which help to 'present' various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP. This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, λ or P1 DNA replication. A mechanism is suggested to explain how the Clp ATPases 'decide' whether to repair or destroy their protein substrates.     

Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase

Mycobacterial Clp‐family proteases function via collaboration of the heteromeric ClpP1P2 peptidase with a AAA+ partner, ClpX or ClpC1. These enzymes are essential for M. tuberculosis viability and are validated antibacterial drug targets, but the requirements for assembly and regulation of functional proteolytic complexes are poorly understood. Here, we report the reconstitution of protein degradation by mycobacterial Clp proteases in vitro and describe novel features of these enzymes that distinguish them from orthologues in other bacteria. Both ClpX and ClpC1 catalyse ATP‐dependent unfolding and degradation of native protein substrates in conjunction with ClpP1P2, but neither mediates protein degradation with just ClpP1 or ClpP2. ClpP1P2 alone has negligible peptidase activity, but is strongly stimulated by translocation of protein substrates into ClpP1P2 by either AAA+ partner. Interestingly, our results support a model in which both binding of a AAA+ partner and protein‐substrate delivery are required to stabilize active ClpP1P2. Our model has implications for therapeutically targeting ClpP1P2 in dormant M. tuberculosis, and our reconstituted systems should facilitate identification of novel Clp protease inhibitors and activators.     

Structure and evolutionary conservation of the plant N‐end rule pathway

The N‐end rule relates the in vivo half‐life of a protein to the identity of its N‐terminal amino acid residue. While some N‐terminal residues result in metabolically stable proteins, other, so‐called destabilizing residues, lead to rapid protein turnover. The N‐end rule pathway, which mediates the recognition and degradation of proteins with N‐terminal destabilizing residues, is present in all organisms examined, including prokaryotes. This protein degradation pathway has a hierarchical organization in which some N‐terminal residues, called primary destabilizing residues, are directly recognized by specific ubiquitin ligases. Other destabilizing residues, termed secondary and tertiary destabilizing residues, require modifications before the corresponding proteins can be targeted for degradation by ubiquitin ligases. In eukaryotes, the N‐end rule pathway is a part of the ubiquitin/proteasome system and is known to play essential roles in a broad range of biological processes in fungi, animals and plants. While the structure of the N‐end rule pathway has been extensively studied in yeast and mammals, knowledge of its organization in plants is limited. Using both tobacco and Arabidopsis, we identified the complete sets destabilizing and stabilizing N‐terminal residues. We also characterized the hierarchical organization of the plant N‐end rule by identifying and determining the specificity of two distinct N‐terminal amidohydrolases (Nt‐amidases) of Arabidopsis that are essential for the destabilizing activity of the tertiary destabilizing residues Asn and Gln. Our results indicate that both the N‐end rule itself and mechanistic aspects of the N‐end rule pathway in angiosperms are very similar to those of mammals.