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1.
Elife ; 122023 07 03.
Article in English | MEDLINE | ID: mdl-37395731

ABSTRACT

Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the cryo-EM structure of the lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.


Subject(s)
Cysteine Loop Ligand-Gated Ion Channel Receptors , Animals , Humans , Cysteine Loop Ligand-Gated Ion Channel Receptors/genetics , Cysteine Loop Ligand-Gated Ion Channel Receptors/chemistry , Cysteine Loop Ligand-Gated Ion Channel Receptors/metabolism , Ligands , Invertebrates , Binding Sites , Sterols
2.
Sci Adv ; 4(3): eaap9714, 2018 03.
Article in English | MEDLINE | ID: mdl-29546243

ABSTRACT

Bacterial protein synthesis is intricately connected to metabolic rate. One of the ways in which bacteria respond to environmental stress is through posttranslational modifications of translation factors. Translation elongation factor Tu (EF-Tu) is methylated and phosphorylated in response to nutrient starvation upon entering stationary phase, and its phosphorylation is a crucial step in the pathway toward sporulation. We analyze how phosphorylation leads to inactivation of Escherichia coli EF-Tu. We provide structural and biophysical evidence that phosphorylation of EF-Tu at T382 acts as an efficient switch that turns off protein synthesis by decoupling nucleotide binding from the EF-Tu conformational cycle. Direct modifications of the EF-Tu switch I region or modifications in other regions stabilizing the ß-hairpin state of switch I result in an effective allosteric trap that restricts the normal dynamics of EF-Tu and enables the evasion of the control exerted by nucleotides on G proteins. These results highlight stabilization of a phosphorylation-induced conformational trap as an essential mechanism for phosphoregulation of bacterial translation and metabolism. We propose that this mechanism may lead to the multisite phosphorylation state observed during dormancy and stationary phase.


Subject(s)
Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/metabolism , Protein Biosynthesis , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Diphosphate/metabolism , Models, Molecular , Nucleotides/metabolism , Phosphorylation , Phosphothreonine/metabolism , Protein Binding , Protein Conformation , Thermodynamics
3.
Nat Chem Biol ; 12(7): 490-6, 2016 07.
Article in English | MEDLINE | ID: mdl-27159580

ABSTRACT

Conditional cooperativity is a common mechanism involved in transcriptional regulation of prokaryotic type II toxin-antitoxin operons and is intricately related to bacterial persistence. It allows the toxin component of a toxin-antitoxin module to act as a co-repressor at low doses of toxin as compared to antitoxin. When toxin level exceeds a certain threshold, however, the toxin becomes a de-repressor. Most antitoxins contain an intrinsically disordered region (IDR) that typically is involved in toxin neutralization and repressor complex formation. To address how the antitoxin IDR is involved in transcription regulation, we studied the phd-doc operon from bacteriophage P1. We provide evidence that the IDR of Phd provides an entropic barrier precluding full operon repression in the absence of Doc. Binding of Doc results in a cooperativity switch and consequent strong operon repression, enabling context-specific modulation of the regulatory process. Variations of this theme are likely to be a common mechanism in the autoregulation of bacterial operons that involve intrinsically disordered regions.


Subject(s)
Antitoxins/metabolism , Entropy , Allosteric Regulation , Antitoxins/genetics , Bacteriophage P1/genetics , Bacteriophage P1/metabolism , Operon/genetics
4.
Protein Expr Purif ; 108: 30-40, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25582764

ABSTRACT

Toxin-antitoxin (TA) modules are stress response elements that are ubiquitous in the genomes of bacteria and archaea. Production and subsequent purification of individual TA proteins is anything but straightforward as over-expression of the toxin gene is lethal to bacterial and eukaryotic cells and over-production of the antitoxin leads to its proteolytic degradation because of its inherently unstructured nature. Here we describe an effective production and purification strategy centered on an on-column denaturant-induced dissociation of the toxin-antitoxin complex. The success of the method is demonstrated by its application on four different TA families, encoding proteins with distinct activities and folds. A series of biophysical and in vitro activity tests show that the purified proteins are of high quality and suitable for structural studies.


Subject(s)
Archaeal Proteins , Bacterial Proteins , Multiprotein Complexes , Archaeal Proteins/biosynthesis , Archaeal Proteins/chemistry , Archaeal Proteins/isolation & purification , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Multiprotein Complexes/biosynthesis , Multiprotein Complexes/chemistry , Multiprotein Complexes/isolation & purification
5.
Nucleic Acids Res ; 42(20): 12928-38, 2014 Nov 10.
Article in English | MEDLINE | ID: mdl-25324313

ABSTRACT

The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds 'structural' mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations.


Subject(s)
Single-Domain Antibodies/pharmacology , Transcriptional Activation/drug effects , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/immunology , Cell Line , Humans , Models, Molecular , Promoter Regions, Genetic , Protein Structure, Tertiary , Single-Domain Antibodies/immunology , Tumor Suppressor Protein p53/antagonists & inhibitors
6.
J Biol Chem ; 289(49): 34013-23, 2014 Dec 05.
Article in English | MEDLINE | ID: mdl-25326388

ABSTRACT

The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site.


Subject(s)
Bacteriophage P1/genetics , Escherichia coli/virology , Gene Expression Regulation, Viral , Molecular Chaperones/chemistry , Viral Proteins/chemistry , Bacteriophage P1/chemistry , Bacteriophage P1/metabolism , Crystallography, X-Ray , Models, Molecular , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Folding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Thermodynamics , Viral Proteins/genetics , Viral Proteins/metabolism
7.
Biomol NMR Assign ; 8(1): 145-8, 2014 Apr.
Article in English | MEDLINE | ID: mdl-23420131

ABSTRACT

Toxin-antitoxin (TA) modules in bacteria are involved in pathogenesis, antibiotic stress response, persister formation and programmed cell death. The toxin Doc, from the phd/doc module, blocks protein synthesis by targeting the translation machinery. Despite a large wealth of biophysical and biochemical data on the regulatory aspects of the operon transcription and role of Doc co-activator and co-repressor, little is still know on the molecular basis of Doc toxicity. Structural information about this toxin is only available for its inhibited state bound to the antitoxin Phd. Here we report the (1)H, (15)N and (13)C backbone and side chain chemical shift assignments of the toxin Doc from of bacteriophage P1 (the model protein from this family of TA modules) in its free state. The BMRB accession number is 18899.


Subject(s)
Bacteriophage P1/metabolism , Nuclear Magnetic Resonance, Biomolecular , Viral Proteins/chemistry , Amino Acid Sequence , Carbon Isotopes , Hydrogen , Molecular Sequence Data , Nitrogen Isotopes , Protein Structure, Secondary
8.
Nat Chem Biol ; 9(12): 811-7, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24141193

ABSTRACT

Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.


Subject(s)
Escherichia coli Proteins/metabolism , Nucleotidyltransferases/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Guanosine Triphosphate/genetics , Guanosine Triphosphate/metabolism , Models, Molecular , Mutation , Nucleotidyltransferases/genetics , Peptide Elongation Factor Tu/genetics , Peptide Elongation Factor Tu/metabolism , Phosphorylation , Protein Folding , RNA, Transfer/genetics , RNA, Transfer/metabolism
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