Biomedical subjects

Robert T Sauer

Publications and source records attributed to Robert T Sauer.

48 records · Page 3Linked to original sources

PubMed2003

Flexible linkers leash the substrate binding domain of SspB to a peptide module that stabilizes delivery complexes with the AAA+ ClpXP protease.

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2003

Cleavage of the A site mRNA codon during ribosome pausing provides a mechanism for translational quality control.

Cells employ many mechanisms to ensure quality control during protein biosynthesis. Here, we show that, during the pausing of a bacterial ribosome, the mRNA being translated is cleaved at a site within or immediately adjacent to the A site codon. The extent of this A site mRNA cleavage is correlated with the extent of ribosome pausing as assayed by tmRNA-mediated tagging of the nascent polypeptide. Cleavage does not require tmRNA, the ribosomal alarmone (p)ppGpp, or bacterial toxins such as RelE which have been shown to stimulate a similar activity. Translation is required for cleavage, suggesting that the ribosome participates in the reaction in some fashion. When normal protein synthesis is compromised, A site mRNA cleavage and the tmRNA system provide a mechanism for reducing translational errors and the production of aberrant and potentially harmful polypeptides.

Bacterial Toxins↗

PubMed2003

Nickel coordination is regulated by the DNA-bound state of NikR.

The uptake of nickel in Escherichia coli and other microorganisms is transcriptionally regulated by the NikR repressor or its homologs. Here we report the structure of the high-affinity nickel-binding site in NikR and show that it responds dramatically to DNA binding. X-ray absorption spectroscopy reveals that nickel in the holo-NikR protein is bound in a novel four-coordinate planar site consisting of two histidines, one additional O- or N-donor ligand and one S-donor ligand. Site-directed mutation of His87, His89, Cys95 or Glu97 in NikR to alanine eliminates high-affinity nickel binding and abolishes DNA binding but maintains stable protein folding. An unanticipated feature of the NikR structure is that the nickel coordination responds to DNA binding. A six-coordinate nickel site composed of O- or N-donor ligands, but lacking cysteine, forms when NikR binds to operator DNA. Because nickel binding and DNA binding are mediated by different domains within NikR, a communication link between the two domains is implicated, consistent with the finding that the nickel-binding site in a fragment corresponding to the C-terminal domain of NikR is structurally distinct from that found in holo-NikR.

Amino Acid Motifs↗

PubMed2003

C-terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding.

ClpX mediates ATP-dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + family members, ClpX contains an alphabeta ATPase domain and an alpha-helical C-terminal domain. ClpX proteins with mutations in the C-terminal domain were constructed and screened for disassembly activity in vivo. Seven mutant enzymes with defective phenotypes were purified and characterized. Three of these proteins (L381K, D382K and Y385A) had low activity in disassembly or unfolding assays in vitro. In contrast to wild-type ClpX, substrate binding to these mutants inhibited ATP hydrolysis instead of increasing it. These mutants appear to be defective in a reaction step that engages bound substrate proteins and is required both for enhancement of ATP hydrolysis and for unfolding/disassembly. Some of these side chains form part of the interface between the C-terminal domain of one ClpX subunit and the ATPase domain of an adjacent subunit in the hexamer and appear to be required for communication between adjacent nucleotide binding sites.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2003

Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing.

ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATPgammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATPgammaS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. K(M) and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximately 15 micro M on K(D) for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS ( approximately 2 micro M) and ADP ( approximately 3 micro M) under the reaction conditions used for steady-state kinetics. In the absence of Mg(2+), where hydrolysis does not occur, the inhibition constant for ATP ( approximately 55 micro M) was weaker but very similar to the value for ATPgammaS ( approximately 45 micro M). Compared with ATP, ATPgammaS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2002

Mutational studies of protein stability and folding of the hyperstable MYL Arc repressor variant.

The Arc-MYL variant is hyperstable and has an earlier transition state than wild type as a consequence of replacing the wild-type salt-bridge triad formed by R31, E36 and R40 with hydrophobic interactions formed by M31, Y36 and L40. Amino acid substitution mutations were constructed at 16 positions in the Arc-MYL background and the equilibrium stabilities of the corresponding mutant proteins were determined. At three positions, mutations were found to be less destabilizing in MYL than in wild-type Arc, and, at one position, the opposite result was obtained. The kinetics of refolding and unfolding were determined for a subset of the Arc-MYL core mutants. Three mutations--VA18, LA19 and LA40--had their major energetic effects on the refolding rate. The interactions perturbed by these mutations appear to be substantially formed in the transition state. V18 and L19 are in the N-terminal turn of helix A and L40 is in the center of helix B. The remaining mutations--VA22, MA31, VA33, YA36, VA41, MA42 and FA45--had some effects on refolding but exerted their major effects on the unfolding rate. Approximately 30% of the energetic interactions mediated by these latter side chains seem to be present in the transition state of Arc-MYL.

Kinetics↗

PubMed2002

Proline residues at the C terminus of nascent chains induce SsrA tagging during translation termination.

The SsrA or tmRNA quality control system relieves ribosome stalling and directs the addition of a degradation tag to the C terminus of the nascent chain. In some instances, SsrA tagging of otherwise full-length proteins occurs when the ribosome pauses at stop codons during normal translation termination. Here, the identities of the C-terminal residues of the nascent chain are shown to play an important role in full-length protein tagging. Specifically, a subset of C-terminal Xaa-Pro sequences caused SsrA tagging of the full-length YbeL protein from Escherichia coli. This tagging increased when a less efficient stop codon was used, increased in cells lacking protein release factor-3, and decreased when protein release factor-1 was overexpressed. Incorporation of the analog azetidine-2-carboxylic acid in place of proline suppressed tagging, whereas incorporation of 3,4-dehydroproline increased SsrA tagging of full-length YbeL. These results suggest that the detailed chemical or conformational properties of the C-terminal residues of the nascent polypeptide can affect the rate of translation termination, thereby influencing ribosome pausing and SsrA tagging at stop codons.

Codon, Terminator↗

PubMed2002

Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in Escherichia coli.

The SsrA or tmRNA quality control system intervenes when ribosomes stall on mRNAs and directs the addition of a C-terminal peptide tag that targets the modified polypeptide for degradation. Although hundreds of SsrA-tagged proteins can be detected in cells when degradation is prevented, most of these species have not been identified. Consequently, the mRNA sequence determinants that cause ribosome stalling and SsrA tagging are poorly understood. SsrA tagging of Escherichia coli ribokinase occurs at three specific sites at or near the C terminus of this protein. The sites of tagging correspond to ribosome stalling at the termination codon and at rare AGG codons encoding Arg-307 and Arg-309, the antepenultimate and C-terminal residues of E. coli ribokinase. Mutational analyses and studies of the effects of overexpressing the tRNA that decodes AGG reveal that the combination of a rare arginine codon at the C terminus and the adjacent inefficient UGA termination codon act to recruit the SsrA-tagging system, presumably by slowing the rate of translation elongation and termination.

Amino Acid Sequence↗

PubMed2002

NikR repressor: high-affinity nickel binding to the C-terminal domain regulates binding to operator DNA.

E. coli NikR repressor binds operator DNA in a nickel-dependent fashion. The pM affinity of NikR for nickel is mediated by its C-terminal 86 residues. Nickel binding induced additional secondary structure, decreased the compactness, and increased the stability of NikR. Tetramer formation by the C-terminal domain and intact NikR did not require nickel. High-affinity nickel binding decreased the NikR concentration needed to half maximally protect operator DNA from undetectable levels to 30 nM. The intracellular concentration of NikR in E. coli is high enough that saturation of the high-affinity nickel sites should lead to substantial occupancy of the nik operator. Nickel binding to a set of low-affinity NikR sites resulted in an additional large increase in operator affinity and substantially increased the size of the NikR footprint on the operator.

Circular Dichroism↗

PubMed2002

Characterization of a specificity factor for an AAA+ ATPase: assembly of SspB dimers with ssrA-tagged proteins and the ClpX hexamer.

SspB, a specificity factor for the ATP-dependent ClpXP protease, stimulates proteolysis of protein substrates bearing the ssrA degradation tag. The SspB protein is shown here to form a stable homodimer with two independent binding sites for ssrA-tagged proteins or peptides. SspB by itself binds to ClpX and stimulates the ATPase activity of this enzyme. In the presence of ATPgammaS, a ternary complex of SspB, GFP-ssrA, and the ClpX ATPase was sufficiently stable to isolate by gel-filtration or ion-exchange chromatography. This complex consists of one SspB dimer, two molecules of GFP-ssrA, and one ClpX hexamer. SspB dimers do not commit bound substrates to ClpXP degradation but increase the affinity and cooperativity of binding of ssrA-tagged substrates to ClpX, facilitating enhanced degradation at low substrate concentrations.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2002

Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects.

Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.

Acid Anhydride Hydrolases↗

PubMedDate not supplied

Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation.

ClpX and related AAA+ ATPases of the Clp/Hsp100 family are able to denature native proteins. Here, we explore the role of protein stability in ClpX denaturation and subsequent ClpP degradation of model substrates bearing ssrA degradation tags at different positions. ClpXP degraded T. thermophilus RNase-H* with a C-terminal ssrA tag very efficiently, despite the very high global stability of this thermophilic protein. In fact, global thermodynamic stability appears to play little role in susceptibility to degradation, as a far less stable RNase-H*-ssrA mutant was degraded more slowly than wild type by ClpXP and a completely unfolded mutant variant was degraded less than twice as fast as the wild-type parent. When ssrA peptide tags were covalently linked to surface cysteines at positions 114 or 140 of RNase-H*, the conjugates were proteolyzed very slowly. This resistance to degradation was not caused by inaccessibility of the ssrA tag or an inability of ClpXP to degrade proteins with side-chain linked ssrA tags. Our results support a model in which ClpX denatures proteins by initially unfolding structural elements attached to the degradation tag, suggest an important role for the position of the degradation tag and direction of force application, and correlate well with the mapping of local protein stability within RNase-H* by native-state hydrogen exchange.

Adenosine Triphosphatases↗