Biomedical subjects

Tania A Baker

Publications and source records attributed to Tania A Baker.

At least 37 records · Page 2Linked to original sources

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

MuA transposase separates DNA sequence recognition from catalysis.

Confronted with thousands of potential DNA substrates, a site-specific enzyme must restrict itself to the correct DNA sequence. The MuA transposase protein performs site-specific DNA cleavage and joining reactions, resulting in DNA transposition-a specialized form of genetic recombination. To determine how sequence information is used to restrict transposition to the proper DNA sites, we performed kinetic analyses of transposition with DNA substrates containing either wild-type transposon sequences or sequences carrying mutations in specific DNA recognition modules. As expected, mutations near the DNA cleavage site reduce the rate of cleavage; the observed effect is about 10-fold. In contrast, mutations within the MuA recognition sequences do not directly affect the DNA cleavage or joining steps of transposition. It is well established that the recognition sequences are necessary for assembly of stable, multimeric MuA-DNA complexes, and we find that recognition site mutations severely reduce both the extent and the rate of this assembly process. Yet if the MuA-DNA complexes are preassembled, the first-order rate constants for both DNA cleavage and DNA strand transfer (the joining reaction) are unaffected by the mutations. Furthermore, most of the mutant DNA molecules that are cleaved also complete DNA strand transfer. We conclude that the sequence-specific contacts within the recognition sites contribute energetically to complex assembly, but not directly to catalysis. These results contrast with studies of more orthodox enzymes, such as EcoRI and some other type II restriction enzymes. We propose that the strategy employed by MuA may serve as an example for how recombinases and modular restriction enzymes solve the DNA specificity problem, in that they, too, may separate substrate recognition from catalysis.

Bacteriophage mu↗

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

Reorganization of the Mu transpososome active sites during a cooperative transition between DNA cleavage and joining.

Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 '-flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 '-flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.

Bacteriophage mu↗

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

The terminal nucleotide of the Mu genome controls catalysis of DNA strand transfer.

Members of the transposase/retroviral-integrase superfamily use a single active site to perform at least two reactions during transposition of a DNA transposon or a retroviral cDNA. They hydrolyze a DNA sequence at the end of the mobile DNA and then join this DNA end to a target DNA (a reaction called DNA strand transfer). Critical to understanding the mechanism of recombination is elucidating how these distinct reactions are orchestrated by the same active site. Here we find that DNA substrates terminating in a dideoxynucleotide allow Mu transposase to hydrolyze a target DNA, combining aspects of both natural reactions. Analyses of the sequence preferences for target hydrolysis and of the structure of the cleaved product indicate that this reaction is promoted by the active site in the conformation that normally promotes DNA strand transfer. Dissecting the DNA requirements for target hydrolysis reveals that the ribose of the last nucleotide of the Mu DNA activates transposase's catalytic potential, even when this residue is not a direct chemical participant. These findings provide insight into the molecular mechanism insuring that DNA strand transfer ordinarily occurs rather than inappropriate DNA cleavage. The required presence of the terminal nucleotide in the transposase active site creates a great advantage for the attached 3'OH to serve as nucleophile.

Bacteriophage mu↗

PubMed2003

Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase.

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.

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

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

Mu transpososome architecture ensures that unfolding by ClpX or proteolysis by ClpXP remodels but does not destroy the complex.

The Clp/Hsp100 ATPases are protein unfoldases that both alter protein conformation and target proteins for degradation. An unresolved question has been how such seemingly destructive enzymes can "remodel" some protein substrates rather than destroy them. Here, we investigate the products of ClpX-mediated remodeling of a hyper-stable protein-DNA complex, the Mu transpososome. We find that although an oligomeric complex is maintained, release of some subunits accompanies ClpX action. Replacement of transposase's endogenous ClpX-recognition sequence with an exogenous signal reveals that the mechanism of remodeling is independent of both the recognition signal and the identity of the unfoldase. Finally, examination of the transposase-DNA contacts reveals only a localized region that is altered during remodeling. These results provide a framework for protein remodeling, wherein the physical attributes of a complex can limit the unfolding activity of its remodeler.

ATPases Associated with Diverse Cellular Activitie↗

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↗

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

DNA gyrase requirements distinguish the alternate pathways of Mu transposition.

The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non-replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA-DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.

Bacteriolysis↗

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

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↗