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Robert T Sauer

Publications and source records attributed to Robert T Sauer.

At least 37 records · Page 2Linked to original sources
PubMed2004

SspB delivery of substrates for ClpXP proteolysis probed by the design of improved degradation tags.

The ssrA-degradation tag sequence contains contiguous binding sites for the SspB adaptor and the ClpX component of the ClpXP protease. Although SspB normally enhances ClpXP degradation of ssrA-tagged substrates, it inhibits proteolysis under conditions that prevent tethering to ClpX. By increasing the spacing between the protease and adaptor-binding determinants in the ssrA tag, substrates were obtained that displayed improved SspB-mediated binding to and degradation by ClpXP. These extended-tag substrates also showed significantly reduced conditional inhibition but bound SspB normally. Both wild-type and mutant tags showed highly dynamic SspB interactions. Together, these results strongly support delivery models in which SspB and ClpX bind concurrently to the ssrA tag, but also suggest that clashes between SspB and ClpX weaken simultaneous binding. During substrate delivery, this signal masking is overcome by tethering SspB to ClpX, which ensures local concentrations high enough to drive tag engagement. This obstruct-then-stimulate mechanism may have evolved to allow additional levels of regulation and could be a common trait of adaptor-mediated protein degradation.

Adenosine Triphosphatases↗
PubMed2004

Computational and experimental probes of symmetry mismatches in the Arc repressor-DNA complex.

Arc repressor from bacteriophage P22 is a homodimeric member of the ribbon-helix-helix family of transcription factors. Two dimers bind cooperatively to adjacent sites on operator DNA with the antiparallel beta-sheet of each Arc dimer contacting the major groove. Despite the 2-fold symmetry of the dimer, the sequence of the DNA half-site is not symmetric, and thus there is an inherent symmetry mismatch in the complex. Here, using a combination of computational and experimental methods, we further characterize this symmetry mismatch. Continuum electrostatic calculations show that the DNA-contacting side-chains of Gln9, Asn11 and Arg13 each make better electrostatic interactions in one subunit of the Arc dimer than in the other. Based on the computational results, one or both Gln9 side-chains were substituted with Arg or His and Asn11 was similarly substituted with Ile in the context of a single-chain variant of Arc repressor. Although the double mutants that maintain the symmetry of the protein bind much more weakly than scArc, mutants with single Q9R or N11I substitutions bind operator DNA with affinities close to wild-type. Whereas the effects are nearly additive for the mutations at position 9, the effects of single N11I mutations are non-additive. The calculations thus enabled the identification of functionally tolerated mutations in the DNA-binding surface of Arc repressor, which in turn revealed energetic consequences of asymmetric environments in this protein-DNA interaction.

Base Pair Mismatch↗
PubMed2004

Communication between ClpX and ClpP during substrate processing and degradation.

In the ClpXP compartmental protease, ring hexamers of the AAA(+) ClpX ATPase bind, denature and then translocate protein substrates into the degradation chamber of the double-ring ClpP(14) peptidase. A key question is the extent to which functional communication between ClpX and ClpP occurs and is regulated during substrate processing. Here, we show that ClpX-ClpP affinity varies with the protein-processing task of ClpX and with the catalytic engagement of the active sites of ClpP. Functional communication between symmetry-mismatched ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes in its IGF loops, which contact ClpP. A conserved arginine in the sensor II helix of ClpX links the nucleotide state of ClpX to the binding of ClpP and protein substrates. A simple model explains the observed relationships between ATP binding, ATP hydrolysis and functional interactions between ClpX, protein substrates and ClpP.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Role of the processing pore of the ClpX AAA+ ATPase in the recognition and engagement of specific protein substrates.

ClpX binds substrates bearing specific classes of peptide signals, denatures these proteins, and translocates them through a central pore into ClpP for degradation. ClpX with the V154F po e mutation is severely defective in binding substrates bearing C-motif 1 degradation signals and is also impaired in a subsequent step of substrate engagement. In contrast, this mutant efficiently processes substrates with other classes of recognition signals both in vitro and in vivo. These results demonstrate that the ClpX pore functions in the recognition and catalytic engagement of specific substrates, and that ClpX recognizes different substrate classes in at least two distinct fashions.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Bivalent tethering of SspB to ClpXP is required for efficient substrate delivery: a protein-design study.

SspB homodimers deliver ssrA-tagged substrates to ClpXP for degradation. SspB consists of a substrate binding domain and an unstructured tail with a ClpX binding module (XB). Using computational design, we engineered an SspB heterodimer whose subunits did not form homodimers. Experiments with the designed molecule and variants lacking one or two tails demonstrate that both XB modules are required for strong binding and efficient substrate delivery to ClpXP. Assembly of stable SspB-substrate-ClpX delivery complexes requires the coupling of weak tethering interactions between ClpX and the SspB XB modules as well as interactions between ClpX and the substrate degradation tag. The ClpX hexamer contains three XB binding sites, one per N domain dimer, and thus binds strongly to just one SspB dimer at a time. Because different adaptor proteins use the same tethering sites in ClpX, those which employ bivalent tethering, like SspB, will compete more effectively for substrate delivery to ClpXP.

Adenosine Triphosphatases↗
PubMed2003

Distinct peptide signals in the UmuD and UmuD' subunits of UmuD/D' mediate tethering and substrate processing by the ClpXP protease.

The Escherichia coli UmuD' protein is a component of DNA polymerase V, an error-prone polymerase that carries out translesion synthesis on damaged DNA templates. The intracellular concentration of UmuD' is strictly controlled by regulated transcription, by posttranslational processing of UmuD to UmuD', and by ClpXP degradation. UmuD' is a substrate for the ClpXP protease but must form a heterodimer with its unabbreviated precursor, UmuD, for efficient degradation to occur. Here, we show that UmuD functions as a UmuD' delivery protein for ClpXP. UmuD can also deliver a UmuD partner for degradation. UmuD resembles SspB, a well-characterized substrate-delivery protein for ClpX, in that both proteins use related peptide motifs to bind to the N-terminal domain of ClpX, thereby tethering substrate complexes to ClpXP. The combined use of a weak substrate recognition signal and a delivery factor that tethers the substrate to the protease allows regulated proteolysis of UmuD/D' in the cell. Dual recognition strategies of this type may be a relatively common feature of intracellular protein turnover.

Adenosine Triphosphatases↗
PubMed2003

Crystal structure of the nickel-responsive transcription factor NikR.

NikR is a metal-responsive transcription factor that controls nickel uptake in Escherichia coli by regulating expression of a nickel-specific ATP-binding cassette (ABC) transporter. We have determined the first two structures of NikR: the full-length apo repressor at a resolution of 2.3 A and the nickel-bound C-terminal regulatory domain at a resolution of 1.4 A. NikR is the only known metal-responsive member of the ribbon-helix-helix family of transcription factors, and its structure has a quaternary arrangement consisting of two dimeric DNA-binding domains separated by a tetrameric regulatory domain that binds nickel. The position of the C-terminal regulatory domain enforces a large spacing between the contacts that each NikR DNA-binding domain can make with the nik operator. The regulatory domain of NikR contains four nickel-binding sites at the tetramer interface, each exhibiting a novel square-planar coordination by three histidines and one cysteine side chain.

Amino Acid Sequence↗
PubMed2003

Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine.

Proteolytic machines powered by ATP hydrolysis bind proteins with specific peptide tags, denature these substrates, and translocate them into a sequestered compartment for degradation. To determine how ATP is used during individual reaction steps, we assayed ClpXP degradation of ssrA-tagged titin variants with different stabilities in native and denatured forms. The rate of ATP turnover was 4-fold slower during denaturation than translocation. Importantly, this reduced turnover rate was constant during denaturation of native variants with different stabilities, but total ATP consumption increased with substrate stability, suggesting an iterative application of a uniform, mechanical unfolding force. Destabilization of substrate structure near the degradation tag accelerated degradation and dramatically reduced ATP consumption, revealing an important role for local protein stability in resisting denaturation. The ability to denature more stable proteins simply by using more ATP endows ClpX with a robust unfolding activity required for its biological roles in degradation and complex disassembly.

Adenosine Triphosphatases↗
PubMed2003

Latent ClpX-recognition signals ensure LexA destruction after DNA damage.

The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly. Here, both fragments of LexA are shown to be substrates for the ClpXP protease. ClpXP recognizes these fragments using sequence motifs that flank the auto-cleavage site but are dormant in intact LexA. Furthermore, ClpXP degradation of the LexA-DNA-binding fragment is important to cell survival after DNA damage. These results demonstrate how one protein-processing event can activate latent protease recognition signals, triggering a cascade of protein turnover in response to environmental stress.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2003

OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain.

Transmembrane signaling between intracellular compartments is often controlled by regulated proteolysis. Escherichia coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sigma(E)-dependent transcription of stress genes in the cytoplasm. This process requires a proteolytic cascade initiated by the DegS protease, which destroys a transmembrane protein (RseA) that normally binds to and inhibits sigma(E). Here, we show that peptides ending with OMP-like C-terminal sequences bind the DegS PDZ domain, activate DegS cleavage of RseA, and induce sigma(E)-dependent transcription. These results suggest that DegS acts as a sensor of envelope stress by binding unassembled OMPs. DegS activation involves relief of inhibitory interactions between its PDZ and protease domains. Peptide binding to inhibitory PDZ domains in proteases related to DegS, including DegP/HtrA, may also regulate the degradation of specific substrates by these enzymes.

Bacterial Proteins↗
PubMed2003

Solution structure of switch Arc, a mutant with 3(10) helices replacing a wild-type beta-ribbon.

Adjacent N11L and L12N mutations in the antiparallel beta-ribbon of Arc repressor result in dramatic changes in local structure in which each beta-strand is replaced by a right-handed helix. The full solution structure of this "switch" Arc mutant shows that irregular 3(10) helices compose the new secondary structure. This structural metamorphosis conserves the number of main-chain and side-chain to main-chain hydrogen bonds and the number of fully buried core residues. Apart from a slight widening of the interhelical angle between alpha-helices A and B and changes in side-chain conformation of a few core residues in Arc, no large-scale structural adjustments in the remainder of the protein are necessary to accommodate the ribbon-to-helix change. Nevertheless, some changes in hydrogen-exchange rates are observed, even in regions that have very similar structures in the two proteins. The surface of switch Arc is packed poorly compared to wild-type, leading to approximately 1000A(2) of additional solvent-accessible surface area, and the N termini of the 3(10) helices make unfavorable head-to-head electrostatic interactions. These structural features account for the positive m value and salt dependence of the ribbon-to-helix transition in Arc-N11L, a variant that can adopt either the mutant or wild-type structures. The tertiary fold is capped in different ways in switch and wild-type Arc, showing how stepwise evolutionary transformations can arise through small changes in amino acid sequence.

Circular Dichroism↗
PubMed2003

Toxin-antitoxin pairs in bacteria: killers or stress regulators?

Plasmid toxin-antitoxin systems, which kill daughter cells that fail to inherit the plasmid genome, have chromosomal homologs in eubacteria and archaea. In this issue of Cell, Pederson et al. show that the E. coli RelE toxin cleaves mRNA in the ribosomal A site, potentially allowing it to function as a stress regulator during amino acid starvation.

Antitoxins↗
PubMed2003

Structure of a delivery protein for an AAA+ protease in complex with a peptide degradation tag.

Substrate selection by AAA+ ATPases that function to unfold proteins or alter protein conformation is often regulated by delivery or adaptor proteins. SspB is a protein dimer that binds to the ssrA degradation tag and delivers proteins bearing this tag to ClpXP, an AAA+ protease, for degradation. Here, we describe the structure of the peptide binding domain of H. influenzae SspB in complex with an ssrA peptide at 1.6 A resolution. The ssrA peptides are bound in well-defined clefts located at the extreme ends of the SspB homodimer. SspB contacts residues within the N-terminal and central regions of the 11 residue ssrA tag but leaves the C-terminal residues exposed and positioned to dock with ClpX. This structure, taken together with biochemical analysis of SspB, suggests mechanisms by which proteins like SspB escort substrates to AAA+ ATPases and enhance the specificity and affinity of target recognition.

Adenosine Triphosphatases↗
PubMed2003

Expression of N-formylated proteins in Escherichia coli.

In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal.

Amidohydrolases↗
PubMed2003

Role of an N(cap) residue in determining the stability and operator-binding affinity of Arc repressor.

The Arc repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of transcription factors. Ser32 is a solvent-exposed position that serves a structural role as the N(cap) residue of alpha-helix B of Arc, but also serves a functional role because its side chain is packed close to the sugar-phosphate DNA backbone in the repressor-operator complex. The tolerance of this N(cap) position to amino-acid substitutions was probed by determining the repressor activity in vivo, the thermal stability and the operator-binding activity in vitro of a set of 13 mutant proteins. The stability of position-32 Arc variants, except for Cys32, correlated well with the frequencies observed for the corresponding residues at N(cap) positions in alpha-helices of other proteins. Cysteine was quite stabilizing at the helix-B N(cap) position in Arc, but surprisingly was the least frequent N(cap) residue in the protein database. This latter finding may reflect a hyper-reactivity of N(cap) cysteines, which makes them prone to chemical modification. In general, only Arc variants with small, uncharged residues at position 32 were active in vivo or showed strong operator binding in vitro. Based upon the results presented here, revised sequence alignments of the MetJ and NikR subfamilies with Arc and other ribbon-helix-helix proteins are proposed.

Amino Acid Sequence↗
PubMed2003

Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.

ClpXP is a protease involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. To date, however, only a handful of ClpXP substrates have been identified. Using a tagged and inactive variant of ClpP, substrates of E. coli ClpXP were trapped in vivo, purified, and identified by mass spectrometry. The more than 50 trapped proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. Analysis of the sequences of the trapped proteins revealed five recurring motifs: two located at the C terminus of proteins, and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations established that sequences from each motif class targeted proteins for degradation by ClpXP. These results represent a description of general rules governing substrate recognition by a AAA+ family ATPase and suggest strategies for regulation of protein degradation.

ATPases Associated with Diverse Cellular Activitie↗