Showing papers by "Robert T. Sauer published in 2014"

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery.

Abstract: Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein–substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.

Mechanochemical basis of protein degradation by a double-ring AAA+ machine.

Abstract: A single-molecule optical-trapping approach is used to examine protein unfolding and translocation by double-ring AAA+ machine ClpA. Although ClpA can unfold some substrates faster than ClpX can, it translocates the unfolded polypeptide more slowly.

Restriction of the Conformational Dynamics of the Cyclic Acyldepsipeptide Antibiotics Improves Their Antibacterial Activity

Abstract: The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP–ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentratio...

Mechanochemical basis of protein degradation by a double-ring AAA+ machine

Abstract: A single-molecule optical-trapping approach is used to examine protein unfolding and translocation by double-ring AAA+ machine ClpA. Although ClpA can unfold some substrates faster than ClpX can, it translocates the unfolded polypeptide more slowly.

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

Abstract: 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.

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Abstract: ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48⋅20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.

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-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.

Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity

Abstract: Summary Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross-linking and scanning for mutations that prevent sul20-peptide binding These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation

A simple fragment of cyclic acyldepsipeptides is necessary and sufficient for ClpP activation and antibacterial activity.

Abstract: The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.

Distinct regulatory mechanisms balance DegP proteolysis to maintain cellular fitness during heat stress

Abstract: Protein degradation is an essential cellular function. In the absence of degradation, toxic misfolded proteins or aggregates can accumulate after heat shock or other environmental stresses, resulting in reduced viability or cell death. Protein quality control (PQC) proteases play critical biological roles by degrading diverse misfolded proteins but also require careful regulation because proteolysis of functional proteins can be cytotoxic. Intracellular proteases whose activities are allosterically regulated have evolved to meet these conflicting needs. DegP is a member of the highly conserved HtrA family of proteases, is overexpressed at high temperatures as the major PQC protease in the periplasmic compartment of Escherichia coli and many other Gram-negative bacteria, and is implicated in the virulence of several pathogens (Lipinska et al. 1989; Strauch et al. 1989; Pallen and Wren 1997; Clausen et al. 2011). Each subunit of DegP contains a trypsin-like protease domain and two PDZ domains (Krojer et al. 2002, 2008b; Kim et al. 2011). The protease domains of three subunits pack together to form a stable trimer, which can switch between a proteolytically active conformation with high substrate affinity and a proteolytically inactive conformation with low substrate affinity (Fig. 1A; Kim et al. 2011). DegP trimers display positively cooperative substrate binding to the active enzyme, a hallmark of Monod-Wyman-Changeux (MWC) allostery, which allows proteolysis to be controlled. Substrate binding also triggers the assembly of DegP trimers and hexamers into large polyhedral cages that restrict the access of native proteins to the active sites within these cages (Fig. 1A; Jiang et al. 2008; Krojer et al. 2008b; Kim et al. 2011). Figure 1. The R207P mutation stabilizes the high-affinity active conformation of DegP. (A) Substrate-binding triggers proteolytic activation and cage assembly. The fundamental unit of DegP structure is a trimer, which reversibly transforms between an inactive conformation ... Distinct modes of substrate binding control DegP activation and assembly. In unfolded or misfolded substrates, two degron sequences in a single polypeptide interact with DegP to promote allosteric activation (Kim et al. 2011). Substrate residues flanking scissile peptide bonds bind to the active site cleft and oxyanion hole of the active protease domain, whereas a hydrophobic residue at the substrate C terminus binds to a site in the PDZ1 domain. These linked interactions also result in the assembly of trimers into hollow polyhedral cages with 12, 18, or 24 subunits by stabilizing interactions between the PDZ1 domains of one trimer and the PDZ2′ domains of neighboring trimers (Kim and Sauer 2012). Substrate cleavage eventually generates peptide products that bind too weakly to maintain DegP in the active conformation, resulting in cage disassembly and a return to the inactive conformation. Thus, bivalent substrate binding helps ensure that DegP is activated and assembled only in the presence of appropriate substrates. Several observations demonstrate the coupling of DegP activation and cage assembly. Crystal structures are known for a DegP hexamer in which trimers assume an inactive conformation with a malformed Ser–His–Asp catalytic triad and oxyanion hole and for substrate-bound cages with 12 or 24 subunits in which trimers assume an active conformation (Krojer et al. 2002, 2008b; Kim et al. 2011). In solution, proteolytic activation and cage assembly of DegP also occur synchronously (Krojer et al. 2008b; Kim et al. 2011), which led to the proposal that cage assembly was the molecular switch for activation (Krojer et al. 2008b). We found, however, that cage assembly was not required for either proteolytic activity in vitro or suppression of proteotoxic heat stress in vivo (Kim and Sauer 2012). At present, it is unclear how allosteric activation and cage assembly contribute to DegP function in vivo and cellular fitness. Here, we show that mutations that increase or decrease the equilibrium population of active DegP trimers result in small decreases in high-temperature fitness. Combining a mutation that allosterically activates DegP with one that prevents cage formation converts the enzyme into a lethal rogue protease. This lethality appears to be caused by excessive proteolysis of proteins associated with cell envelope maintenance and can be relieved by an extragenic suppressor that alters the cell envelope and directly inhibits the rogue protease or by intragenic mutations that stabilize inactive DegP trimers. We propose that substrate-dependent allosteric control of the conformation and activity of DegP trimers represents the primary form of proteolytic regulation, whereas cage assembly protects cells from promiscuous proteolysis that would otherwise be triggered under heat stress and other conditions that result in protein misfolding and allosteric activation of DegP.

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

Abstract: 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.

Overexpression of CupB5 activates alginate overproduction in Pseudomonas aeruginosa by a novel AlgW-dependent mechanism.

Abstract: Summary In Pseudomonas aeruginosa, alginate overproduction, also known as mucoidy, is negatively regulated by the transmembrane protein MucA, which sequesters the alternative sigma factor AlgU. MucA is degraded via a proteolysis pathway that frees AlgU from sequestration, activating alginate biosynthesis. Initiation of this pathway normally requires two signals: peptide sequences in unassembled outer-membrane proteins (OMPs) activate the AlgW protease, and unassembled lipopolysaccharides bind periplasmic MucB, releasing MucA and facilitating its proteolysis by activated AlgW. To search for novel alginate regulators, we screened a transposon library in the non-mucoid reference strain PAO1, and identified a mutant that confers mucoidy through overexpression of a protein encoded by the chaperone-usher pathway gene cupB5. CupB5-dependent mucoidy occurs through the AlgU pathway and can be reversed by overexpression of MucA or MucB. In the presence of activating OMP peptides, peptides corresponding to a region of CupB5 needed for mucoidy further stimulated AlgW cleavage of MucA in vitro. Moreover, the CupB5 peptide allowed OMP-activated AlgW cleavage of MucA in the presence of the MucB inhibitor. These results support a novel mechanism for conversion to mucoidy in which the proteolytic activity of AlgW and its ability to compete with MucB for MucA is mediated by independent peptide signals.

Overexpression of CupB5 activates alginate overproduction in Pseudomonas aeruginosa by a novel AlgW-dependent mechanism

Abstract: United States. National Aeronautics and Space Administration. West Virginia Space Grant Consortium

A Simple Fragment of Cyclic Acyldepsipeptides Is Necessary and Sufficient for ClpP Activation and Antibacterial Activity

Abstract: The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N‐acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N‐acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N‐acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non‐peptide activators of ClpP that can be used as antibacterial agents.