Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria.

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

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High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122964 ◽

1992 ◽

Vol 50 (1) ◽

pp. 512-513

Author(s):

J. Jakana ◽

M.F. Schmid ◽

P. Matsudaira ◽

W. Chiu

Keyword(s):

High Resolution ◽

Binding Proteins ◽

Actin Binding ◽

Eukaryotic Cells ◽

Dimensional Structure ◽

Structural Basis ◽

Actin Bundle ◽

Actin Bundles ◽

3 Dimensional ◽

Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽

10.3390/cells8111325 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1325 ◽

Cited By ~ 1

Author(s):

Ke Yue ◽

Tran Nam Trung ◽

Yiyong Zhu ◽

Ralf Kaldenhoff ◽

Lei Kai

Keyword(s):

Functional Analysis ◽

Membrane Proteins ◽

Fluorescent Protein ◽

Water Channel ◽

New Approach ◽

E Coli ◽

Straightforward Method ◽

Lipid Environment ◽

Green Fluorescent ◽

Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

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Structural basis for the tight binding inhibition of E. coli CTP synthase inhibition by gemcitabine and its analogues

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s0108767320099183 ◽

2020 ◽

Vol 76 (a1) ◽

pp. a81-a81

Author(s):

Matthew McLeod ◽

Todd Holyoak

Keyword(s):

Tight Binding ◽

Structural Basis ◽

E Coli ◽

Ctp Synthase ◽

Binding Inhibition

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Bacterial SSB interactome: Structural basis of interaction between E. coli Exonuclease I and SSB and exploitation of this interaction to identify novel anti‐bacterial compounds

The FASEB Journal ◽

10.1096/fasebj.23.1_supplement.836.3 ◽

2009 ◽

Vol 23 (S1) ◽

Author(s):

Duo Lu ◽

Douglas Bernstein ◽

James Keck

Keyword(s):

Structural Basis ◽

E Coli ◽

Exonuclease I

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Structural basis for transcription inhibition by E. coli SspA

Nucleic Acids Research ◽

10.1093/nar/gkaa672 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9931-9942 ◽

Cited By ~ 1

Author(s):

Fulin Wang ◽

Jing Shi ◽

Dingwei He ◽

Bei Tong ◽

Chao Zhang ◽

...

Keyword(s):

Structural Information ◽

Protein A ◽

Acid Tolerance ◽

In Vitro Transcription ◽

Nucleotide Metabolism ◽

Structural Basis ◽

E Coli ◽

Promoter Escape ◽

Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

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Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate

Scientific Reports ◽

10.1038/s41598-019-53301-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Jolanta Krucinska ◽

Michael N. Lombardo ◽

Heidi Erlandsen ◽

Akram Hazeen ◽

Searle S. Duay ◽

...

Keyword(s):

Bacterial Infections ◽

Antibacterial Properties ◽

High Energy ◽

Structural Basis ◽

Gram Negative Bacteria ◽

E Coli ◽

Promising Target ◽

Antibiotic Discovery ◽

Growth And Reproduction

AbstractMany years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

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Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision

Scientific Reports ◽

10.1038/srep45863 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 15

Author(s):

Julie-Anne Gandier ◽

David N. Langelaan ◽

Amy Won ◽

Kylie O’Donnell ◽

Julie L. Grondin ◽

...

Keyword(s):

Class I ◽

Structural Basis ◽

High Similarity ◽

Similarity Class

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Subcellular Microanatomy by 3D Deconvolution Brightfield Microscopy: Method and Analysis Using Human Chromatin in the Interphase Nucleus

Anatomy Research International ◽

10.1155/2012/848707 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7

Author(s):

Paul Joseph Tadrous

Keyword(s):

Plasma Cell ◽

Interphase Nucleus ◽

Light And Electron Microscopy ◽

3D Structure ◽

Cell Types ◽

Structural Basis ◽

3 Dimensional ◽

First Results ◽

Brightfield Microscopy ◽

Subcellular Resolution

Anatomy has advanced using 3-dimensional (3D) studies at macroscopic (e.g., dissection, injection moulding of vessels, radiology) and microscopic (e.g., serial section reconstruction with light and electron microscopy) levels. This paper presents the first results in human cells of a new method of subcellular 3D brightfield microscopy. Unlike traditional 3D deconvolution and confocal techniques, this method is suitable for general application to brightfield microscopy. Unlike brightfield serial sectioning it has subcellular resolution. Results are presented of the 3D structure of chromatin in the interphase nucleus of two human cell types, hepatocyte and plasma cell. I show how the freedom to examine these structures in 3D allows greater morphological discrimination between and within cell types and the 3D structural basis for the classical “clock-face” motif of the plasma cell nucleus is revealed. Potential for further applications discussed.

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A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.245-251.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 245-251 ◽

Cited By ~ 6

Author(s):

Ju-Yuan Zhang ◽

Jie Zou ◽

Qiyu Bao ◽

Wen-Li Chen ◽

Li Wang ◽

...

Keyword(s):

Arthrospira Platensis ◽

Biochemical Properties ◽

Salt Sensitivity ◽

E Coli ◽

Multiple Origins ◽

Manic Depressive ◽

Sodium Toxicity ◽

Function Relationship ◽

Eukaryotic Organisms

ABSTRACT 3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg2+ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li+ (50% inhibitory concentration [IC50] = 3.6 mM) but only slightly sensitive to Na+ (IC50 = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li+ and Na+, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li+ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.

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