Biomedical subjects

Robert T Sauer

Publications and source records attributed to Robert T Sauer.

At least 19 recordsLinked to original sources

PubMed2007

Altered specificity of a AAA+ protease.

ClpXP, an ATP-dependent protease, degrades hundreds of different intracellular proteins. ClpX chooses substrates by binding peptide tags, typically displayed at the N or C terminus of the protein to be degraded. Here, we identify a ClpX mutant that displays a 300-fold change in substrate specificity, resulting in decreased degradation of ssrA-tagged substrates but improved degradation of proteins with other classes of degradation signals. The altered-specificity mutation occurs within "RKH" loops, which surround the entrance to the central pore of the ClpX hexamer and are highly conserved in the ClpX subfamily of AAA+ ATPases. These results support a major role for the RKH loops in substrate recognition and suggest that ClpX specificity represents an evolutionary compromise that has optimized degradation of multiple types of substrates rather than any single class.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2006

ATP-dependent proteases of bacteria: recognition logic and operating principles.

ATP-powered AAA+ proteases degrade specific proteins in intracellular environments occupied by thousands of different proteins. These proteases operate as powerful molecular machines that unfold stable native proteins before degradation. Understanding how these enzymes choose the "right" protein substrates at the "right" time is key to understanding their biological function. Recently, proteomic approaches have identified numerous substrates for some bacterial enzymes and the sequence motifs responsible for recognition. Advances have also been made in elucidating the mechanism and impact of adaptor proteins in regulating substrate choice. Finally, recent biochemical dissection of the ATPase cycle and its coupling to protein unfolding has revealed fundamental operating principles of this important, ubiquitous family of molecular machines.

ATP-Dependent Proteases↗

PubMed2006

Engineering controllable protein degradation.

Complex biological networks are regulated via alterations in protein expression, degradation, and function. Synthetic control of these processes allows dissection of natural systems and the design of new networks. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. To engineer controlled degradation, we have designed a series of modified ssrA tags that have weakened interactions with ClpXP. When SspB is present, ClpXP degrades purified substrates bearing these engineered peptide tags 100-fold more efficiently. Importantly, substrates bearing these tags are stable in the absence of SspB in vivo but are rapidly degraded upon SspB induction. Our studies supply a conceptual foundation and working components for controllable degradation, improve mechanistic understanding of adaptor-mediated proteolysis, and demonstrate that the relative importance of adaptor proteins in degradation is correlated with the strength of protease-substrate contacts.

Amino Acid Sequence↗

PubMed2006

Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon.

ClpXP, a bacterial AAA+ protease, controls intracellular levels of many stress-response proteins. To investigate substrate profile changes caused by a specific environmental stress, quantitative mass spectrometry (SILAC) was used to analyze proteins trapped by ClpXP(trap) before and after DNA damage. The abundance of half of the trapped proteins changed more than 3-fold after damage. Overrepresented substrates included the DNA-repair proteins RecN and UvrA. Among SOS-response proteins, 25% were ClpXP substrates and, importantly, nearly all of the highly induced regulon members were rapidly degraded. Other proteins, including the stress regulator sigma(S), were underrepresented in ClpXP(trap) after DNA damage; overproduction experiments suggest that simple substrate competition does not account for this reduced recognition. We conclude that damage-response proteins are an unusually rapidly degraded family and that ClpXP has substantial capacity to process the influx of newly synthesized substrates while maintaining the ability to degrade its other substrates in an environmentally responsive manner.

Adenosine Triphosphatases↗

PubMed2005

Sequence determinants of a conformational switch in a protein structure.

The Arc repressor of bacteriophage P22 is a dimeric member of the ribbon-helix-helix family of transcription factors. Residues 9-14 of each wild-type Arc subunit pair to form two antiparallel beta-strands and have the alternating pattern of polar and nonpolar residues expected for a beta-ribbon with one solvent-exposed face and one face that forms part of the hydrophobic core. Simultaneously switching Asn-11 to Leu and Leu-12 to Asn changes the local binary sequence pattern to that of an amphipathic helix. Previous studies have shown that this double mutation results in replacement of the wild-type beta-ribbon by two right-handed 3(10)-helices. Moreover, an Arc variant bearing just the Asn-11 --> Leu mutation has an ambiguous binary pattern and can form either the ribbon or the helical structures, which interchange rapidly. Here, we study Arc mutants in which position 11 is occupied by Gly, Ala, Val, Ile, Leu, Met, Phe, or Tyr. These mutants adopt the wild-type beta-ribbon structure in a sequence context that stabilizes this fold, but they assume the alternative helical structure in a sequence background in which the wild-type fold is precluded by negative design. In an otherwise wild-type sequence background, the detailed chemical properties of the position 11 side chain dictate which of the two competing conformational folds is preferred.

Amino Acid Sequence↗

PubMed2005

Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines.

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.

Adenosine Triphosphatases↗

PubMed2005

Specificity versus stability in computational protein design.

Protein-protein interactions can be designed computationally by using positive strategies that maximize the stability of the desired structure and/or by negative strategies that seek to destabilize competing states. Here, we compare the efficacy of these methods in reengineering a protein homodimer into a heterodimer. The stability-design protein (positive design only) was experimentally more stable than the specificity-design heterodimer (positive and negative design). By contrast, only the specificity-design protein assembled as a homogenous heterodimer in solution, whereas the stability-design protein formed a mixture of homodimer and heterodimer species. The experimental stabilities of the engineered proteins correlated roughly with their calculated stabilities, and the crystal structure of the specificity-design heterodimer showed most of the predicted side-chain packing interactions and a main-chain conformation indistinguishable from the wild-type structure. These results indicate that the design simulations capture important features of both stability and structure and demonstrate that negative design can be critical for attaining specificity when competing states are close in structure space.

Bacterial Proteins↗

PubMed2005

Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine.

ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.

Adenosine Diphosphate↗

PubMed2005

Versatile modes of peptide recognition by the AAA+ adaptor protein SspB.

Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we report the crystal structure of a complex between SspB and its recognition peptide in RseA. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery.

Amino Acid Sequence↗

PubMed2005

Nucleotide-dependent substrate recognition by the AAA+ HslUV protease.

ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg(2+). Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.

Adenosine Triphosphatases↗

PubMed2005

Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure.

We designed a single-chain variant of the Arc repressor homodimer in which the beta strands that contact operator DNA are connected by a hairpin turn and the alpha helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker. The permuted protein binds operator DNA with nanomolar affinity, refolds on the sub-millisecond time scale, and is as stable as Arc-L1-Arc. The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Noncyclic rearrangement of secondary structure may allow grouping of critical active-site residues in other proteins and could be a useful tool for protein design and minimization.

Bacteriophage P22↗

PubMed2005

Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing.

Energy-dependent proteases, such as ClpXP, are responsible for the regulated destruction of proteins in all cells. AAA+ ATPases in these proteases bind protein substrates and power their mechanical denaturation and subsequent translocation into a secluded degradation chamber where polypeptide cleavage occurs. Here, we show that model unfolded substrates are engaged rapidly by ClpXP and are then spooled into the degradation chamber at a rate proportional to their length. Degradation and competition studies indicate that ClpXP initially binds native and unfolded substrates similarly. However, stable native substrates then partition between frequent release and infrequent denaturation, with only the latter step resulting in committed degradation. During degradation of a fusion protein with three tandem native domains, partially degraded species with one and two intact domains accumulated. These processed proteins were not bound to the enzyme, showing that release can occur even after translocation and degradation of a substrate have commenced. The release of stable substrates and committed engagement of denatured or unstable native molecules ensures that ClpXP degrades less stable substrates in a population preferentially. This mechanism prevents trapping of the enzyme in futile degradation attempts and ensures that the energy of ATP hydrolysis is used efficiently for protein degradation.

Binding Sites↗

PubMed2005

Cytoplasmic degradation of ssrA-tagged proteins.

Degradation of ssrA-tagged proteins is a central feature of protein-quality control in all bacteria. In Escherichia coli, the ATP-dependent ClpXP and ClpAP proteases are thought to participate in this process, but their relative contributions to degradation of ssrA-tagged proteins in vivo have been uncertain because two adaptor proteins, ClpS and SspB, can modulate proteolysis of these substrates. Here, intracellular levels of these protease components and adaptors were determined during exponential growth and as cells entered early stationary phase. Levels of ClpA and ClpP increased about threefold during this transition, whereas ClpX, ClpS and SspB levels remained nearly constant. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. Both ClpXP and ClpAP degraded GFP-ssrA in the cell, demonstrating that wild-type levels of SspB and ClpS do not inhibit ClpAP completely. Upon entry into stationary phase, increased levels of ClpAP resulted in increased degradation of ssrA-tagged substrates. As measured by maximum turnover rates, ClpXP degradation of GFP-ssrA in vivo was significantly more efficient than in vitro. Surprisingly, ClpX-dependent ClpP-independent degradation of GFP-ssrA was also observed. Thus, unfolding of this substrate by ClpX appears to enhance intracellular degradation by other proteases.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2005

Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coli.

When protein synthesis stalls in bacteria, tmRNA acts first as a surrogate tRNA and then as an mRNA in a series of reactions that append a peptide tag to the nascent polypeptide and 'rescue' the ribosome. The peptide tag encoded by wild-type tmRNA promotes rapid degradation of rescued proteins. Using a mutant tmRNA that encodes a tag that does not lead to degradation, we demonstrate that the synthesis of approximately 0.4% of all proteins terminates with tagging and ribosome rescue during normal exponential growth of Escherichia coli. The frequency of tagging was not significantly increased in cells expressing very high levels of tmRNA and its binding protein SmpB, suggesting that recognition of 'stalled' ribosomes does not involve competition between tmRNA and other translation factors for A-sites that are unoccupied transiently during protein synthesis. When the demand for ribosome rescue was increased artificially by overproduction of a non-stop mRNA, tmRNA levels did not increase but tmRNA-mediated tagging increased substantially. Thus, the ribosome-rescue system usually operates well below capacity.

Escherichia coli↗

PubMed2004

Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA+ ClpXP protease.

The SspB adaptor enhances ClpXP degradation by binding the ssrA degradation tag of substrates and the AAA+ ClpX unfoldase. To probe the mechanism of substrate delivery, we engineered a disulfide bond between the ssrA tag and SspB and demonstrated otherwise normal interactions by solving the crystal structure. Although the covalent link prevents adaptor.substrate dissociation, ClpXP degraded GFP-ssrA that was disulfide bonded to the adaptor. Thus, crosslinked substrate must be handed directly from SspB to ClpX. The ssrA tag in the covalent adaptor complex interacted with ClpX.ATPgammaS but not ClpX.ADP, suggesting that handoff occurs in the ATP bound enzyme. By contrast, SspB alone bound ClpX in both nucleotide states. Similar handoff mechanisms will undoubtedly be used by many AAA+ adaptors and enzymes, allowing assembly of delivery complexes in either nucleotide state, engagement of the recognition tag in the ATP state, and application of an unfolding force to the attached protein following hydrolysis.

Adenosine Diphosphate↗

PubMed2004

Sculpting the proteome with AAA(+) proteases and disassembly machines.

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Modulating substrate choice: the SspB adaptor delivers a regulator of the extracytoplasmic-stress response to the AAA+ protease ClpXP for degradation.

Adaptor proteins help proteases modulate substrate choice, ensuring that appropriate proteins are degraded at the proper time and place. SspB is an adaptor that delivers ssrA-tagged proteins to the AAA+ protease ClpXP for degradation. To identify new SspB-regulated substrates, we examined proteins captured by ClpXP(trap) in sspB(+) but not sspB(-) strains. RseA(1-108), a fragment of a transmembrane protein that regulates the extracytoplasmic-stress response, fits this criterion. In response to stress, RseA is cleaved on each side of the membrane and is released as a cytoplasmic fragment that remains bound in an inhibitory complex with the sigma(E) transcription factor. Trapping experiments together with biochemical studies show that ClpXP functions in concert with SspB to efficiently recognize and degrade RseA(1-108), and thereby releases sigma(E). Genetic studies confirm that ClpX and SspB participate in induction of the sigma(E) regulon in vivo, acting at the final step of an activating proteolytic cascade. Surprisingly, the SspB-recognition sequence in RseA(1-108) is unrelated to its binding sequence in the ssrA tag. Thus, these experiments elucidate the final steps in induction of the extracytoplasmic stress response and reveal that SspB delivers a broader spectrum of substrates to ClpXP than has been recognized.

Adenosine Triphosphatases↗

PubMed2004

Ribosomal protein S1 binds mRNA and tmRNA similarly but plays distinct roles in translation of these molecules.

Ribosomes stalled during protein synthesis can be rescued by tmRNA, which acts first as a tRNA and then as an mRNA to direct addition of a C-terminal degradation tag to the nascent polypeptide. Ribosomal protein S1 binds tmRNA, but its functional role in tmRNA-mediated tagging is uncertain. To probe interactions between S1 and tmRNA, truncated variants missing one or more of the six contiguous S1 domains were studied. The third S1 domain (R1) plays a critical role in binding tmRNA and mRNA but requires additional N- or C-terminal S1 domains. The binding of S1 and its fragments to tmRNA and mRNA is positively cooperative, and the essential role of the R1 domain may be to mediate protein-protein interactions. Overproduction of N-terminal fragments of S1 in Escherichia coli displaces endogenous S1 from ribosomes, inhibits general protein synthesis, and slows growth but causes little if any disruption of tmRNA-mediated tagging. Moreover, tagging of proteins translated from model mRNAs with either no or an increased requirement for S1 is indistinguishable. These results raise the possibility that S1 plays little or no role in tmRNA-mediated tagging.

Escherichia coli↗