Showing papers by "Robert T. Sauer published in 2013"
TL;DR: In this article, the authors found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal for the degradation of a negative regulator of the σ(E) transcription factor.
Abstract: In Gram-negative bacteria, outer-membrane integrity is essential for survival and is monitored by the σ(E) stress-response system, which initiates damage-repair pathways. One activating signal is unassembled outer-membrane proteins. Using biochemical and genetic experiments in Escherichia coli, we found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal. These distinct signals, arising from disruptions in the transport and assembly of the major outer-membrane components, jointly determined the rate of proteolytic destruction of a negative regulator of the σ(E) transcription factor, thereby modulating the expression of stress-response genes. This dual-signal system permits a rapid response to dysfunction in outer-membrane biogenesis, while buffering responses to transient fluctuations in individual components, and may represent a broad strategy for bacteria to monitor their interface with the environment.
155 citations
TL;DR: It is demonstrated that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work.
120 citations
TL;DR: It is found that Lon hexamers assemble via a matrix of N-domain interactions to form a dodecamer with altered substrate-degradation properties, which supports a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.
Abstract: Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamer–hexamer interface, with portals of ∼45 A providing access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.
65 citations
TL;DR: In this paper, the authors report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase.
Abstract: There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. β-Lactones with certain substituents on the α- and β-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analogue of a bioactive β-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the grow...
62 citations
TL;DR: It is suggested that eukaryotic Cdc48 orthologs function directly with 20S to maintain intracellular protein quality control and that similar dual determinants mediate PAN–20S interactions and Rpt1–6– 20S interactions in the 26S proteasome.
Abstract: Proteasomes are essential and ubiquitous ATP-dependent proteases that function in eukarya, archaea, and some bacteria. These destructive but critically important proteolytic machines use a 20S core peptidase and a hexameric ATPase associated with a variety of cellular activities (AAA+) unfolding ring that unfolds and spools substrates into the peptidase chamber. In archaea, 20S can function with the AAA+ Cdc48 or proteasome-activating nucleotidase (PAN) unfoldases. Both interactions are stabilized by C-terminal tripeptides in AAA+ subunits that dock into pockets on the 20S periphery. Here, we provide evidence that archaeal Cdc48 also uses a distinct set of near-axial interactions to bind 20S and propose that similar dual determinants mediate PAN–20S interactions and Rpt1–6–20S interactions in the 26S proteasome. Current dogma holds that the Rpt1–6 unfolding ring of the 19S regulatory particle is the only AAA+ partner of eukaryotic 20S. By contrast, we show that mammalian Cdc48, a key player in cell-cycle regulation, membrane fusion, and endoplasmic-reticulum–associated degradation, activates mammalian 20S and find that a mouse Cdc48 variant supports protein degradation in combination with 20S. Our results suggest that eukaryotic Cdc48 orthologs function directly with 20S to maintain intracellular protein quality control.
61 citations
01 Sep 2013
TL;DR: Chemical syntheses and evaluations of structurally diverse β-lactones are reported, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase, and this work defines a mechanism by which bacteria could resist the toxic effects of ClPP inhibitors.
Abstract: There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. β-Lactones with certain substituents on the α- and β-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analogue of a bioactive β-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the grow...
48 citations
TL;DR: In this paper, allosteric conformations and proteolytic activities of each subunit of the E. coli DegS protease share a cooperatively coupled energy landscape that allows regulation via the binding of substrate and OMP peptides.
Abstract: Allosteric conformations and proteolytic activities of each subunit of the trimeric E. coli DegS protease share a cooperatively coupled energy landscape that allows regulation via the binding of substrate and OMP peptides.
44 citations
01 Nov 2013
TL;DR: The sul20 degron from the cell-division inhibitor SulA is shown to bind to the N domain of Escherichia coli protease, and the recognition site is identified by cross-linking and scanning for mutations that prevent sul20-peptide binding as discussed by the authors.
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.
33 citations
TL;DR: It is demonstrated that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a β20 degron appended to the N terminus or a sul20 degrons appendedto the C terminus, which allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays in vivo.
Abstract: AAA+ proteases, such as Escherichia coli Lon, recognize protein substrates by binding to specific peptide degrons and then unfold and translocate the protein into an internal degradation chamber for proteolysis. For some AAA+ proteases, attaching specific degrons to the N- or C-terminus of green fluorescent protein (GFP) generates useful substrates, whose unfolding and degradation can be monitored by loss of fluorescence, but Lon fails to degrade appropriately tagged GFP variants at a significant rate. Here, we demonstrate that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a β20 degron appended to the N terminus or a sul20 degron appended to the C terminus. Lon degradation of non-permuted GFP-sul20 is very slow, in part because the enzyme cannot efficiently extract the degron-proximal C-terminal β-strand to initiate denaturation. The circularly permuted GFP substrates described here allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays of Lon proteolysis in vivo.
24 citations
TL;DR: The results suggest that the Lon sequence segment near residue 240 may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity.
Abstract: Escherichia coli Lon, an ATP-dependent AAA+ protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, LonE240K exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, LonE240K displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity.
9 citations
TL;DR: Cases are reviewed in which mutagenesis, biochemistry, structure determination, protein engineering, and single‐molecule biophysics have illuminated the sequence determinants of folding, binding specificity, and biological function for DNA‐binding proteins and ATP‐fueled machines that forcibly unfold native proteins as a prelude to degradation.
Abstract: Understanding the relationship between the amino-acid sequence of a protein and its ability to fold and to function is one of the major challenges of protein science. Here, cases are reviewed in which mutagenesis, biochemistry, structure determination, protein engineering, and single-molecule biophysics have illuminated the sequence determinants of folding, binding specificity, and biological function for DNA-binding proteins and ATP-fueled machines that forcibly unfold native proteins as a prelude to degradation. In addition to structure-function relationships, these studies provide information about folding intermediates, mutations that accelerate folding, slow unfolding, and stabilize proteins against denaturation, show how new binding specificities and folds can evolve, and reveal strategies that proteolytic machines use to recognize, unfold, and degrade thousands of distinct substrates.