6 research outputs found
Thermodynamics of HIVā1 Reverse Transcriptase in Action Elucidates the Mechanism of Action of Non-Nucleoside Inhibitors
HIV-1 reverse transcriptase (RT)
is a heterodimeric enzyme that
converts the genomic viral RNA into proviral DNA. Despite intensive
biochemical and structural studies, direct thermodynamic data regarding
RT interactions with its substrates are still lacking. Here we addressed
the mechanism of action of RT and of non-nucleoside RT inhibitors
(NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC
approach, a step-by-step thermodynamic dissection of the RT polymerization
activity showed that most of the driving force for DNA synthesis is
provided by initial dNTP binding. Surprisingly, thermodynamic and
kinetic data led to a reinterpretation of the mechanism of inhibition
of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered
by a kinetic barrier and NNRTIs mostly interact with free RT. Once
formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent
orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA
complexes being unable to bind the incoming nucleotide substrate
Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp
Peptide-based covalent
inhibitors targeted to nucleophilic protein
residues have recently emerged as new modalities to target proteināprotein
interactions (PPIs) as they may provide some benefits over more classic
competitive inhibitors. Covalent inhibitors are generally targeted
to cysteine, the most intrinsically reactive amino acid residue, and
to lysine, which is more abundant at the surface of proteins but much
less frequently to histidine. Herein, we report the structure-guided
design of targeted covalent inhibitors (TCIs) able to bind covalently
and selectively to the bacterial sliding clamp (SC), by reacting with
a well-conserved histidine residue located on the edge of the peptide-binding
pocket. SC is an essential component of the bacterial DNA replication
machinery, identified as a promising target for the development of
new antibacterial compounds. Thermodynamic and kinetic analyses of
ligands bearing different mild electrophilic warheads confirmed the
higher efficiency of the chloroacetamide compared to Michael acceptors.
Two high-resolution X-ray structures of covalent inhibitorāSC
adducts were obtained, revealing the canonical orientation of the
ligand and details of covalent bond formation with histidine. Proteomic
studies were consistent with a selective SC engagement by the chloroacetamide-based
TCI. Finally, the TCI of SC was substantially more active than the
parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis
assay, validating the potential of the approach to design covalent
inhibitors of proteināprotein interactions targeted to histidine
Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp
Peptide-based covalent
inhibitors targeted to nucleophilic protein
residues have recently emerged as new modalities to target proteināprotein
interactions (PPIs) as they may provide some benefits over more classic
competitive inhibitors. Covalent inhibitors are generally targeted
to cysteine, the most intrinsically reactive amino acid residue, and
to lysine, which is more abundant at the surface of proteins but much
less frequently to histidine. Herein, we report the structure-guided
design of targeted covalent inhibitors (TCIs) able to bind covalently
and selectively to the bacterial sliding clamp (SC), by reacting with
a well-conserved histidine residue located on the edge of the peptide-binding
pocket. SC is an essential component of the bacterial DNA replication
machinery, identified as a promising target for the development of
new antibacterial compounds. Thermodynamic and kinetic analyses of
ligands bearing different mild electrophilic warheads confirmed the
higher efficiency of the chloroacetamide compared to Michael acceptors.
Two high-resolution X-ray structures of covalent inhibitorāSC
adducts were obtained, revealing the canonical orientation of the
ligand and details of covalent bond formation with histidine. Proteomic
studies were consistent with a selective SC engagement by the chloroacetamide-based
TCI. Finally, the TCI of SC was substantially more active than the
parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis
assay, validating the potential of the approach to design covalent
inhibitors of proteināprotein interactions targeted to histidine
NucleosāID: A New Search Engine Enabling the Untargeted Identification of RNA Post-transcriptional Modifications from Tandem Mass Spectrometry Analyses of Nucleosides
As RNA post-transcriptional modifications are of growing
interest,
several methods were developed for their characterization. One of
them established for their identification, at the nucleosidic level,
is the hyphenation of separation methods, such as liquid chromatography
or capillary electrophoresis, to tandem mass spectrometry. However,
to our knowledge, no software is yet available for the untargeted
identification of RNA post-transcriptional modifications from MS/MS
data-dependent acquisitions. Thus, very long and tedious manual data
interpretations are required. To meet the need of easier and faster
data interpretation, a new user-friendly search engine, called NucleosāID,
was developed for CE-MS/MS and LCāMS/MS users. Performances
of this new software were evaluated on CE-MS/MS data from nucleoside
analyses of already well-described Saccharomyces cerevisiae transfer RNA and Bos taurus total
tRNA extract. All samples showed great true positive, true negative,
and false discovery rates considering the database size containing
all modified and unmodified nucleosides referenced in the literature.
The true positive and true negative rates obtained were above 0.94,
while the false discovery rates were between 0.09 and 0.17. To increase
the level of sample complexity, untargeted identification of several
RNA modifications from Pseudomonas aeruginosa 70S ribosome was achieved by the NucleosāID search following
CE-MS/MS analysis
The C-terminal p6 domain of the HIV-1 Pr55<sup>Gag</sup> precursor is required for specific binding to the genomic RNA
<p>The Pr55<sup>Gag</sup> precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55<sup>Gag</sup> interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gagāp6 is a truncated form of Pr55<sup>Gag</sup> lacking the p6 domain usually used as a default surrogate for wild type Pr55<sup>Gag</sup> for <i>in vitro</i> analysis. With recent advance in production of full-length recombinant Pr55<sup>Gag</sup>, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55<sup>Gag</sup> by systematically comparing binding of Pr55<sup>Gag</sup> and Gagāp6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55<sup>Gag</sup> to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gagāp6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55<sup>Gag</sup> and Gagāp6 uniformly increased the Kd values of the RNA-protein complexes by ~Ā 2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55<sup>Gag</sup>-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation.</p
Differential Modes of Peptide Binding onto Replicative Sliding Clamps from Various Bacterial Origins
Bacterial sliding clamps are molecular
hubs that interact with
many proteins involved in DNA metabolism through their binding, via
a conserved peptidic sequence, into a universally conserved pocket.
This interacting pocket is acknowledged as a potential molecular target
for the development of new antibiotics. We previously designed short
peptides with an improved affinity for the Escherichia
coli binding pocket. Here we show that these peptides
differentially interact with other bacterial clamps, despite the fact
that all pockets are structurally similar. Thermodynamic and modeling
analyses of the interactions differentiate between two categories
of clamps: group I clamps interact efficiently with our designed peptides
and assemble the Escherichia coli and
related orthologs clamps, whereas group II clamps poorly interact
with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that
the peptide binding process could occur via different mechanisms,
which depend on the type of clamp