ATP hydrolysis inactivating Walker B mutation perturbs E. coli ClpA self-assembly energetics in the absence of nucleotide

Duran, Elizabeth C.; Lucius, Aaron L.

doi:10.1016/j.bpc.2018.08.005

Cited by 3 publications

(6 citation statements)

References 38 publications

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“…As for S. aureus, oxidative stress was a key theme of the DEPs identified for E. coli. Subunit A (ClpA) of the ATP-dependent protease (ClpAP), which mediates unfolding and translocation of abnormal proteins 35 , had decreased significantly in the presence of NW-850-5. Oxidative stress is known to inactivate such chaperone proteins 36 .…”

Section: Characterisation Of Tio 2 Nanopillars On Titanium Substratementioning

confidence: 99%

Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration and induction of oxidative stress

et al. 2020

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Some insects, such as dragonflies, have evolved nanoprotrusions on their wings that rupture bacteria on contact. This has inspired the design of antibacterial implant surfaces with insectwing mimetic nanopillars made of synthetic materials. Here, we characterise the physiological and morphological effects of mimetic titanium nanopillars on bacteria. The nanopillars induce deformation and penetration of the Gram-positive and Gram-negative bacterial cell envelope, but do not rupture or lyse bacteria. They can also inhibit bacterial cell division, and trigger production of reactive oxygen species and increased abundance of oxidative stress proteins. Our results indicate that nanopillars' antibacterial activities may be mediated by oxidative stress, and do not necessarily require bacterial lysis.

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Section: Characterisation Of Tio 2 Nanopillars On Titanium Substratementioning

confidence: 99%

Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration and induction of oxidative stress

et al. 2020

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“…The L n ,0 terms were constrained in the NLLS analysis of L n ,app ATP‐binding isotherms. The self‐association equilibrium constants, L 2,0 , L 4,0 , and L 6,0 were recently determined for ClpA E286A/E565A in the absence of nucleotide . In the absence of nucleotide, we do not observe ClpA E286A/E565A dodecamers and therefore could not experimentally measure L 12,0 .…”

Section: Resultsmentioning

confidence: 67%

“…We recently examined the self‐assembly mechanism and hydrodynamic properties of ClpA E286A/E565A in the absence of nucleotide. We observe that in the absence of nucleotide, ClpA E286A/E565A resides in a distribution of monomers, dimers, tetramers, and hexamers . As a next step, here we sought to quantify the ATP‐linked self‐association reaction of ClpA E286A/E565A using sedimentation velocity experiments.…”

Section: Resultsmentioning

confidence: 99%

“…ClpA E286A/E565A was overexpressed in E. coli ΔClpA WT ‐BL21(DE3) cells as previously described . After overexpression, the protein was isolated following the same isolation protocol as that used to isolate ClpA WT , previously described .…”

Section: Methodsmentioning

confidence: 99%

“…Time corrected sedimentation velocity was subjected to c(s) analysis in SedFit (Peter Schuck, NIH) or difference curve analysis SedAnal, as previously described . Sedimentation coefficient values expressed in terms of s 20, w reflect standard conditions of infinite dilution in water at 20 °C as defined in Eq.…”

Section: Methodsmentioning

confidence: 99%

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Examination of the nucleotide‐linked assembly mechanism of E. coli ClpA

Duran

Lucius

2019

Protein Science

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Escherichia coli ClpA is a AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins targeted for degradation by a protease, ClpP. ClpA hexamers associate with one or both ends of ClpP tetradecamers to form ClpAP complexes. Each ClpA protomer contains two nucleotide-binding sites, NBD1 and NBD2, and self-assembly into hexamers is thermodynamically linked to nucleotide binding. Despite a number of studies aimed at characterizing ClpA and ClpAP-catalyzed substrate unfolding and degradation, respectively, to date the field is unable to quantify the concentration of ClpA hexamers available to interact with ClpP for any given nucleotide and total ClpA concentration. In this work, sedimentation velocity studies are used to quantitatively examine the self-assembly of a ClpA Walker B variant in the presence of ATP. In addition to the hexamerization, we observe the formation of a previously unreported ClpA dodecamer in the presence of ATP. Further, we report apparent equilibrium constants for the formation of each ClpA oligomer obtained from direct boundary modeling of the sedimentation velocity data. The energetics of nucleotide binding to NBD1 and NBD2 are revealed by examining the dependence of the apparent association equilibrium constants on free nucleotide concentration.

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E. coliClpB is a Robust and Processive Protein Unfoldase

Banwait,

Islam,

Lucius

2024

Preprint

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Motor proteins of the AAA+ (ATPases Associated with Various Cellular Activity) superfamily are responsible for maintenance of the protein crowded environment of the cell. E. coli ClpB is a AAA+ molecular motor that, in collaboration with co-chaperones, couples the energy from ATP hydrolysis to resolving protein aggregates that form during heat shock or stress. ClpB assembles into hexameric rings and, based on structural comparison to known protein translocases or unfoldases, the motor has been proposed to processively unfold proteins and translocate the polypeptide through its axial channel. However, since the translocated polypeptide both enters and leaves the reaction without covalent modification it has proven difficult to test and quantify this model of function. Here we have developed a sequential mixing stopped-flow method that reports on processive protein unfolding catalyzed by ClpB. Using this approach, we show that ClpB processively and sequentially unfolds up to three tandem repeats of the stably folded Titin I27 domain with a maximum length of 362 amino acids. We report that ClpB unfolds ~60 amino acids between two rate limiting steps at a rate of ~0.9 aa s-1 in the presence of a 1:1 mixture of ATP:ATPγS. In contrast to what was previously reported by others, in the absence of ATP, we show that ClpB efficiently couples ATPγS hydrolysis to processive unfolding at a rate of ~0.09 aa s-1. Our approach solves the problem of needing covalent modification of the substrate to detect translocation. Thus, we have opened the door to addressing many questions regarding AAA+ protein function that is desperately needed to complement and test the wealth of new structural information emerging from cryo-EM studies on these motors.

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ATP hydrolysis inactivating Walker B mutation perturbs E. coli ClpA self-assembly energetics in the absence of nucleotide

Cited by 3 publications

References 38 publications

Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration and induction of oxidative stress

Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration and induction of oxidative stress

Examination of the nucleotide‐linked assembly mechanism of E. coli ClpA

E. coliClpB is a Robust and Processive Protein Unfoldase

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