Conformational Plasticity of the ClpAP AAA+ Protease Couples Protein Unfolding and Proteolysis

AbstractThe ClpAP complex functions as a “bacterial proteasome” that simultaneously unfolds and degrades proteins targeted for destruction. ClpA utilizes two AAA+ domains per protomer to power substrate unfolding and translocation into the ClpP proteolytic chamber. To understand this mechanism, we determined high-resolution structures of wildtype E. coli ClpAP in distinct substrate-bound states. ClpA forms a spiral with substrate contacts across both AAA+ domains, while protomers at the seam undergo nucleotide-specific rearrangements indicating a conserved rotary mechanism. ClpA IGL loops extend flexibly to bind the planar, heptameric ClpP surface and support a large ClpA-P rotation that re-orients the translocation channel. The symmetry mismatch is maintained at the spiral seam through bind and release states of the IGL loops, which appear precisely coupled to substrate translocation. Thus, ClpA rotates around the apical surface of ClpP to processively translocate substrate into the protease.

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High resolution spectrum of NO2 loosely bound states: densities of states and long range forces

Physical Chemistry Chemical Physics ◽

10.1039/b010252k ◽

2001 ◽

Vol 3 (12) ◽

pp. 2268-2274 ◽

Cited By ~ 7

Author(s):

Sylvain Heilliette ◽

Antoine Delon ◽

Patrick Dupre´ ◽

Re´my Jost

Keyword(s):

High Resolution ◽

Long Range ◽

Bound States ◽

High Resolution Spectrum ◽

Densities Of States

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High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20001132 ◽

2020 ◽

Vol 76 (2) ◽

pp. 94-102 ◽

Cited By ~ 2

Author(s):

Maria A. Schumacher ◽

Tomoo Ohashi ◽

Lauren Corbin ◽

Harold P. Erickson

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Cell Division ◽

High Resolution ◽

Crystal Structures ◽

Bacterial Cell ◽

Model System ◽

E Coli ◽

Z Ring ◽

Main Model

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.

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High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718316115 ◽

2018 ◽

Vol 115 (6) ◽

pp. 1292-1297 ◽

Cited By ~ 56

Author(s):

Ahmet Mentes ◽

Andrew Huehn ◽

Xueqi Liu ◽

Adam Zwolak ◽

Roberto Dominguez ◽

...

Keyword(s):

High Resolution ◽

Binding Site ◽

Bound States ◽

Nucleotide Binding ◽

Structural Basis ◽

Force Sensing ◽

Dependent Manner ◽

Mechanical Loads ◽

Adp Release ◽

Pointed End

Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.

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Conformational Plasticity of the Clpap Aaa+ Protease Couples Protein Unfolding and Proteolysis

Biophysical Journal ◽

10.1016/j.bpj.2020.11.814 ◽

2021 ◽

Vol 120 (3) ◽

pp. 99a

Author(s):

Alexandrea N. Rizo ◽

Kyle Eric Lopez ◽

James Shorter ◽

Daniel R. Southworth

Keyword(s):

Protein Unfolding ◽

Conformational Plasticity

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Predictive Fluorescent Amplified-Fragment Length Polymorphism Analysis of Escherichia coli: High-Resolution Typing Method with Phylogenetic Significance

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1274-1279.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1274-1279 ◽

Cited By ~ 42

Author(s):

Catherine Arnold ◽

Lou Metherell ◽

Geraldine Willshaw ◽

Anthony Maggs ◽

John Stanley

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Amplified Fragment Length Polymorphism ◽

Polymorphism Analysis ◽

Enzyme Electrophoresis ◽

Typing Method ◽

E Coli

The fluorescent amplified-fragment length polymorphism (FAFLP) assay potentially amplifies a unique set of genome fragments from each bacterial clone. It uses stringently hybridizing primers which carry a fluorescent label. Precise fragment sizing is achieved by the inclusion of an internal size standard in every lane. Therefore, a unique genotype identifier(s) can be found in the form of fragments of precise size or sizes, and these can be generated reproducibly. In order to evaluate the potential of FAFLP as an epidemiological typing method with a valid phylogenetic basis, we applied it to 87 strains ofEscherichia coli. These comprised the EcoR collection, which has previously been classified by multilocus enzyme electrophoresis (MLEE) and which represents the genetic diversity of the species E. coli, plus 15 strains of the clinically important serogroup O157. FAFLP with an unlabelled nonselectiveEcoRI primer (Eco+0) and a labelled selectiveMseI primer (Mse+TA) gave strain-specific profiles. Fragments of identical sizes (in base pairs) were assumed to be identical, and the genetic distances between the strains were calculated. A phylogenetic tree derived from measure of distance correlated closely with the MLEE groupings of the EcoR collection and placed the verocytotoxin-producing O157 strains on an outlier branch. Our data indicate that FAFLP is suitable for epidemiological investigation of E. coli infection, providing well-defined and reproducible identifiers of genotype for each strain. Since FAFLP objectively samples the whole genome, each strain or isolate can be assigned a place within the broad context of the whole species and can also be subjected to a high-resolution comparison with closely related strains to investigate epidemiological clonality.

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Interfacial nanomechanical heterogeneity of the E. coli biofilm matrix

Nanoscale ◽

10.1039/d0nr03646c ◽

2020 ◽

Vol 12 (32) ◽

pp. 16819-16830

Author(s):

Christian Titus Kreis ◽

Ruby May A. Sullan

Keyword(s):

High Resolution ◽

Quantitative Imaging ◽

E Coli ◽

High Resolution Structure ◽

Biofilm Matrix ◽

Resolution Structure

Quantitative imaging correlates high-resolution structure and nanomechanics of the biofilm interface.

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High resolution characterisation of E. coli proliferation profiles in livestock faeces

Waste Management ◽

10.1016/j.wasman.2019.02.037 ◽

2019 ◽

Vol 87 ◽

pp. 537-545 ◽

Cited By ~ 1

Author(s):

Kenneth D.H. Porter ◽

Richard S. Quilliam ◽

Sim M. Reaney ◽

David M. Oliver

Keyword(s):

High Resolution ◽

E Coli

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High Resolution Melt Assays to Detect and Identify Vibrio parahaemolyticus, Bacillus cereus, Escherichia coli, and Clostridioides difficile Bacteria

Microorganisms ◽

10.3390/microorganisms8040561 ◽

2020 ◽

Vol 8 (4) ◽

pp. 561

Author(s):

Allison C. Bender ◽

Jessica A. Faulkner ◽

Katherine Tulimieri ◽

Thomas H. Boise ◽

Kelly M. Elkins

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Bacillus Cereus ◽

Vibrio Parahaemolyticus ◽

Bacterial Species ◽

Human Pathogens ◽

High Resolution Melt ◽

E Coli ◽

Clostridioides Difficile ◽

Sensitivity Specificity

Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus are found inside of the human body and B. cereus and E. coli are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from C. difficile, E. coli, B. cereus, and V. parahaemolyticus melted at 80.37 ± 0.45 °C, 82.15 ± 0.37 °C, 84.43 ± 0.50 °C, and 86.74 ± 0.65 °C, respectively. A triplex PCR assay was developed to simultaneously detect and identify E. coli, B. cereus, and V. parahaemolyticus, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.

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Probing the vibrational spectroscopy of the deprotonated thymine radical by photodetachment and state-selective autodetachment photoelectron spectroscopy via dipole-bound states

Chemical Science ◽

10.1039/c5sc00704f ◽

2015 ◽

Vol 6 (5) ◽

pp. 3129-3138 ◽

Cited By ~ 22

Author(s):

Dao-Ling Huang ◽

Hong-Tao Liu ◽

Chuan-Gang Ning ◽

Guo-Zhu Zhu ◽

Lai-Sheng Wang

Keyword(s):

High Resolution ◽

Vibrational Spectroscopy ◽

Photoelectron Spectroscopy ◽

Bound States

High-resolution state-selective autodetachment photoelectron spectroscopy via dipole-bound states and photodetachment spectroscopy of cryogenically cooled deprotonated thymine anions are reported.

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Conformational Plasticity of the Active Site Entrance in E. coli Aspartate Transcarbamoylase and Its Implication in Feedback Regulation

International Journal of Molecular Sciences ◽

10.3390/ijms21010320 ◽

2020 ◽

Vol 21 (1) ◽

pp. 320

Author(s):

Zhen Lei ◽

Nan Wang ◽

Hongwei Tan ◽

Jimin Zheng ◽

Zongchao Jia

Keyword(s):

Active Site ◽

Feedback Regulation ◽

Aspartate Transcarbamoylase ◽

Regulatory Subunit ◽

Dynamic Simulations ◽

E Coli ◽

Open Conformation ◽

Conformational Plasticity ◽

The Stability ◽

Remote Feedback

Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a “textbook example” for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its remote feedback regulation. Herein, we determined a structure of E. coli ATCase in which a key residue located (Arg167) at the entrance of the active site adopted an uncommon open conformation, representing the first wild-type apo-form E. coli ATCase holoenzyme that features this state. Based on the structure and our results of enzymatic characterization, as well as molecular dynamic simulations, we provide new insights into the feedback regulation of E. coli ATCase. We speculate that the binding of pyrimidines or purines would affect the hydrogen bond network at the interface of the catalytic and regulatory subunit, which would further influence the stability of the open conformation of Arg167 and the enzymatic activity of ATCase. Our results not only revealed the importance of the previously unappreciated open conformation of Arg167 in the active site, but also helped to provide rationalization for the mechanism of the remote feedback regulation of ATCase.

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