The N-degron pathways are a set of proteolytic systems that relate the half-life of a protein to its N-terminal (Nt) residue. In Escherichia coli the principal N-degron pathway is known as the Leu/N-degron pathway. Proteins degraded by this pathway contain an Nt degradation signal (N-degron) composed of an Nt primary destabilizing (Nd1) residue (Leu, Phe, Trp or Tyr). All Leu/N-degron substrates are recognized by the adaptor protein, ClpS and delivered to the ClpAP protease for degradation. Although many components of the pathway are well defined, the physiological role of this pathway remains poorly understood. To address this gap in knowledge we developed a biospecific affinity chromatography technique to isolate physiological substrates of the Leu/N-degron pathway. In this chapter we describe the use of peptide arrays to determine the binding specificity of ClpS. We demonstrate how the information obtained from the peptide array, when coupled with ClpS affinity chromatography, can be used to specifically elute physiological Leu/N-degron ligands from a bacterial lysate. These techniques are illustrated using E. coli ClpS (EcClpS), but both are broadly suitable for application to related N-recognins and systems, not only for the determination of N-recognin specificity, but also for the identification of natural Leu/N-degron ligands from various bacterial and plant species that contain ClpS homologs.

EDITORIAL article Front. Mol. Biosci., 19 December 2023Sec. Protein Biochemistry for Basic and Applied Sciences Volume 10 - 2023 | https://doi.org/10.3389/fmolb.2023.1350666

ABSTRACTThe N-degron pathways are a set of proteolytic systems that relate the half-life of a protein to its N-terminal (Nt) residue. In Escherchia coli the principal N-degron pathway is known as the Leu/N-degron pathway of which an Nt Leu is a key feature of the degron. Although the physiological role of the Leu/N-degron pathway is currently unclear, many of the components of the pathway are well defined. Proteins degraded by this pathway contain an Nt degradation signal (N-degron) composed of an Nt primary destabilizing (Nd1) residue (Leu, Phe, Trp or Tyr) and an unstructured region which generally contains a hydrophobic element. Most N-degrons are generated from a pro-N-degron, either by endoproteolytic cleavage, or by enzymatic attachment of a Nd1 residue (Leu or Phe) to the N-terminus of a protein (or protein fragment) by the enzyme Leu/Phe tRNA protein transferase (LFTR) in a non-ribosomal manner. Regardless of the mode of generation, all Leu/N-degrons are recognized by ClpS and delivered to the ClpAP protease for degradation. To date, only two physiological Leu/N-degron bearing substrates have been verified, one of which (PATase) is modified by LFTR. In this study, we have examined the substrate proteome of LFTR during stationary phase. From this analysis, we have identified several additional physiological Leu/N-degron ligands, including AldB, which is modified by a previously undescribed activity of LFTR. Importantly, the novel specificity of LFTR was confirmed in vitro, using a range of model proteins. Our data shows that processing of the Nt-Met of AldB generates a novel substrate for LFTR. Importantly, the LFTR-dependent modification of T2-AldB is essential for its turnover by ClpAPS, in vitro. To further examine the acceptor specificity of LFTR, we performed a systematic analysis using a series of peptide arrays. These data reveal that the identity of the second residue modulates substrate conjugation with positively charged residues being favored and negatively charged and aromatic residues being disfavored. Collectively, these findings extend our understanding of LFTR specificity and the Leu/N-degron pathway in E. coli.

In Escherichia coli, SigmaS (σS) is the master regulator of the general stress response. The cellular levels of σS are controlled by transcription, translation and protein stability. The turnover of σS, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)—which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σS recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssBN and RssBC) and the SAXS analysis of full-length RssB (both free and in complex with σS). Together with our biochemical analysis we propose a model for the recognition and delivery of σS by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (βα)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of σS, much of the direct binding to σS occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a “closed” state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σS interaction, as σS binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σS, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σS at a distal site.

Summary Polymerase δ interacting protein of 38 kDa (PDIP38) was originally identified in a yeast two hybrid screen as an interacting protein of DNA polymerase delta, more than a decade ago. Since this time several subcellular locations have been reported and hence its function remains controversial. Our current understanding of PDIP38 function has also been hampered by a lack of detailed biochemical or structural analysis of this protein. Here we show, that human PDIP38 is directed to the mitochondrion, where it resides in the matrix compartment, together with its partner protein CLPX. PDIP38 is a bifunctional protein, composed of two conserved domains separated by an α-helical hinge region (or middle domain). The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain (ZBD) of CLPX. In contrast, the C-terminal (DUF525) domain forms an Immunoglobin-like β-sandwich fold, which contains a highly conserved hydrophobic groove. Based on the physicochemical properties of this groove, we propose that PDIP38 is required for the recognition (and delivery to CLPXP) of proteins bearing specific hydrophobic degrons, potentially located at the termini of the target protein. Significantly, interaction with PDIP38 stabilizes the steady state levels of CLPX in vivo . Consistent with these data, PDIP38 inhibits the LONM-mediated turnover of CLPX in vitro. Collectively, our findings shed new light on the mechanistic and functional significance of PDIP38, indicating that in contrast to its initial identification as a nuclear protein, PIDP38 is a bona fide mitochondrial adaptor protein for the CLPXP protease.

Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/β linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like β-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.

The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis ( Msm ) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of Msm ClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.

The maintenance of mitochondrial protein homeostasis (proteostasis) is crucial for correct cellular function. Recently, several mutations in the mitochondrial protease CLPP have been identified in patients with Perrault syndrome 3 (PRLTS3). These mutations can be arranged into two groups, those that cluster near the docking site (hydrophobic pocket, Hp) for the cognate unfoldase CLPX (i.e. T145P and C147S) and those that are adjacent to the active site of the peptidase (i.e. Y229D). Here we report the biochemical consequence of mutations in both regions. The Y229D mutant not only inhibited CLPP-peptidase activity, but unexpectedly also prevented CLPX-docking, thereby blocking the turnover of both peptide and protein substrates. In contrast, Hp mutations cause a range of biochemical defects in CLPP, from no observable change to CLPP activity for the C147S mutant, to dramatic disruption of most activities for the "gain-of-function" mutant T145P - including loss of oligomeric assembly and enhanced peptidase activity.

Significance Assembly factors play key roles in the biogenesis of many multisubunit protein complexes regulating their stability, activity, or incorporation of essential cofactors. The bacterial assembly factor SdhE (also known as Sdh5 or SDHAF2 in mitochondria) promotes covalent attachment of flavin adenine dinucleotide (FAD) to SdhA and hence the assembly of functional succinate:quinone oxidoreductase (also known as complex II). Here, we present the crystal structure of Escherichia coli SdhE bound to its client protein SdhA. This structure provides unique insight into SdhA assembly, whereby SdhE constrains unassembled SdhA in an “open” conformation, promoting covalent attachment of FAD, but renders the holoprotein incapable of substrate catalysis. These data also provide a structural explanation for the loss-of-function mutation, Gly78Arg, in SDHAF2, which causes hereditary paraganglioma 2.

The pupylation of cellular proteins plays a crucial role in the degradation cascade via the Pup-Proteasome system (PPS). It is essential for the survival of Mycobacterium smegmatis under nutrient starvation and, as such, the activity of many components of the pathway is tightly regulated. Here, we show that Pup, like ubiquitin, can form polyPup chains primarily through K61 and that this form of Pup inhibits the ATPase-mediated turnover of pupylated substrates by the 20S proteasome. Similarly, the autopupylation of PafA (the sole Pup ligase found in mycobacteria) inhibits its own enzyme activity; hence, pupylation of PafA may act as a negative feedback mechanism to prevent substrate pupylation under specific cellular conditions.

The N-end rule is a conserved protein degradation pathway that relates the metabolic stability of a protein to the identity of its N-terminal residue. Proteins bearing a destabilising N-terminal residue (N-degron) are recognised by specialised components of the pathway (N-recognins) and degraded by cellular proteases. In bacteria, the N-recognin ClpS is responsible for the specific recognition of proteins bearing an N-terminal destabilising residue such as leucine, phenylalanine, tyrosine or tryptophan. In this study, we show that the putative apicoplast N-recognin from Plasmodium falciparum (PfClpS), in contrast to its bacterial homologues, exhibits an expanded substrate specificity that includes recognition of the branched chain amino acid isoleucine.

Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPR mt ). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPR mt . Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPR mt , this pathway may preferentially act to promote chaperone mediated refolding of proteins.

In Escherichia coli, σ(S) is the master regulator of the general stress response. The level of σ(S) changes in response to multiple stress conditions and it is regulated at many levels including protein turnover. In the absence of stress, σ(S) is rapidly degraded by the AAA+ protease, ClpXP in a regulated manner that depends on the adaptor protein RssB. This two-component response regulator mediates the recognition of σ(S) and its delivery to ClpXP. The turnover of σ(S) however, can be inhibited in a stress specific manner, by one of three anti-adaptor proteins. Each anti-adaptor binds to RssB and inhibits its activity, but how this is achieved is not fully understood at a molecular level. Here, we describe details of the interaction between each anti-adaptor and RssB that leads to the stabilization of σ(S). By defining the domains of RssB using partial proteolysis we demonstrate that each anti-adaptor uses a distinct mode of binding to inhibit RssB activity. IraD docks specifically to the N-terminal domain of RssB, IraP interacts primarily with the C-terminal domain, while IraM interacts with both domains. Despite these differences in binding, we propose that docking of each anti-adaptor induces a conformational change in RssB, which resembles the inactive dimer of RssB. This dimer-like state of RssB not only prevents substrate binding but also triggers substrate release from a pre-bound complex.

Mutations in succinate dehydrogenase (SDH) subunits and assembly factors cause a range of clinical conditions. One such condition, hereditary paraganglioma 2 (PGL2), is caused by a G78R mutation in the assembly factor SDH5. Although SDH5(G78R) is deficient in its ability to promote SDHA flavinylation, it has remained unclear whether impairment to its import, structure, or stability contributes to its loss of function. Using import-chase analysis in human mitochondria isolated from HeLa cells, we found that the import and maturation of human SDH5(G78R) was normal, while its stability was reduced significantly, with ~25% of the protein remaining after 180 min compared to ~85% for the wild-type protein. Notably, the metabolic stability of SDH5(G78R) was restored to wild-type levels by depleting mitochondrial LON (LONM). Degradation of SDH5(G78R) by LONM was confirmed in vitro; however, in contrast to the in organello analysis, wild-type SDH5 was also rapidly degraded by LONM. SDH5 instability was confirmed in SDHA-depleted mitochondria. Blue native PAGE showed that imported SDH5(G78R) formed a transient complex with SDHA; however, this complex was stabilized in LONM depleted mitochondria. These data demonstrate that SDH5 is protected from LONM-mediated degradation in mitochondria by its stable interaction with SDHA, a state that is dysregulated in PGL2.

Mitochondria are specialised organelles that are structurally and functionally integrated into cells in the vast majority of eukaryotes. They are the site of numerous enzymatic reactions, some of which are essential for life. The double lipid membrane of the mitochondrion, that spatially defines the organelle and is necessary for some functions, also creates a physical but semi-permeable barrier to the rest of the cell. Thus to ensure the biogenesis, regulation and maintenance of a functional population of proteins, an autonomous protein handling network within mitochondria is required. This includes resident mitochondrial protein translocation machinery, processing peptidases, molecular chaperones and proteases. This review highlights the contribution of proteases of the AAA+ superfamily to protein quality and activity control within the mitochondrion. Here they are responsible for the degradation of unfolded, unassembled and oxidatively damaged proteins as well as the activity control of some enzymes. Since most knowledge about these proteases has been gained from studies in the eukaryotic microorganism Saccharomyces cerevisiae, much of the discussion here centres on their role in this organism. However, reference is made to mitochondrial AAA+ proteases in other organisms, particularly in cases where they play a unique role such as the mitochondrial unfolded protein response. As these proteases influence mitochondrial function in both health and disease in humans, an understanding of their regulation and diverse activities is necessary.

Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest.

The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, including CLPXP, some of which contribute to protein maturation and quality control. Currently however, the substrates and the physiological roles of mitochondrial CLPXP in humans, has remained elusive. Similarly, the mechanism by which these ATP-dependent proteases recognize their substrates currently remains unclear. Here we report the characterization of a Walker B mutation in human CLPX, in which the highly conserved glutamate was replaced with alanine. This mutant protein exhibits improved interaction with the model unfolded substrate casein and several putative physiological substrates in vitro. Although this mutant lacks ATPase activity, it retains the ability to mediate casein degradation by hCLPP, in a fashion similar to the small molecule ClpP-activator, ADEP. Our functional dissection of hCLPX structure, also identified that most model substrates are recognized by the N-terminal domain, although some substrates bypass this step and dock, directly to the pore-1 motif. Collectively these data reveal, that despite the difference between bacterial and human CLPXP complexes, human CLPXP exhibits a similar mode of substrate recognition and is deregulated by ADEPs.

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40-substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40-substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.