3 research outputs found
Design, Synthesis, and Experimental Validation of Peptide Ligands Targeting Mycobacterium tuberculosis sigma Factors
Transcription in prokaryotes is a multistep process and is :primarily regulated at the initiation stage. sigma factors are involved in promoter recognition and thus govern prokaryotic gene expression. Mycobacterium tuberculosis (MO) sigma factors have been previously suggested as important drug targets through large-scale genome analyses. Here we demonstrate the feasibility of specific targeting of Mtb sigma factors using designed peptides. A peptide library was generated using three-dimensional structural features corresponding to the interface regions of sigma factors and the RNA polymerase. In silico optimization of the peptides, employing structural as well as sequence features, aided specific targeting of sigma(A) and sigma(B). We synthesized and characterized the best hit peptide from the peptide library along with other control peptides and studied the, interaction of these peptides with;sigma(B) using biolayer interferometry. The experimental data validate; the design strategy. These studies suggest the feasibility of designing specific peptides via in silico methods that bind sigma(B) with nanomolar affinity. We note that this strategy can be broadly applied to modulate prokaryotic transcription by designed peptides, thereby providing a tool for studying bacterial adaptation as well as host pathogen interactions in infectious bacteria
Determination of Redox Sensitivity in Structurally Similar Biological Redox Sensors
Redox
stimuli govern a variety of biological processes. The relative
sensitivity of redox sensors plays an important role in providing
a calibrated response to environmental stimuli and cellular homeostasis.
This cellular machinery plays a crucial role in the human pathoge<i>n Mycobacterium tuberculosis</i> as it encounters diverse microenvironments
in the host. The redox sensory mechanism in <i>M. tuberculosis</i> is governed by two component and one-component systems, alongside
a class of transcription factors called the extra cytoplasmic function
(ECF) σ factors. ECF σ factors that govern the cellular
response to redox stimuli are negatively regulated by forming a complex
with proteins called zinc associated anti-σ factors (ZAS). ZAS
proteins release their cognate σ factor in response to oxidative
stress. The relative sensitivity of the ZAS sensors to redox processes
dictate the concentration of free ECF σ factors in the cell.
However, factors governing the redox threshold of these sensors remain
unclear. We describe here, the molecular characterization of three
σ factor/ZAS pairsσ<sup>L</sup>/RslA, σ<sup>E</sup>/RseA, and σ<sup>H</sup>/RshAusing a combination
of biophysical and electrochemical techniques. This study reveals,
conclusively, the differences in redox sensitivity in these proteins
despite apparent structural similarity and rationalizes the hierarchy
in the activation of the cognate ECF σ factors. Put together,
the study provides a basis for examining sequence and conformational
features that modulate redox sensitivity within the confines of a
conserved structural scaffold. The findings provide the guiding principles
for the design of intracellular redox sensors with tailored sensitivity
and predictable redox thresholds, providing a much needed biochemical
tool for understanding host–pathogen interaction
Design, Synthesis, and Experimental Validation of Peptide Ligands Targeting <i>Mycobacterium tuberculosis</i> σ Factors
Transcription
in prokaryotes is a multistep process and is primarily
regulated at the initiation stage. σ factors are involved in
promoter recognition and thus govern prokaryotic gene expression. <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) σ
factors have been previously suggested as important drug targets through
large-scale genome analyses. Here we demonstrate the feasibility of
specific targeting of <i>Mtb</i> σ factors using designed
peptides. A peptide library was generated using three-dimensional
structural features corresponding to the interface regions of σ
factors and the RNA polymerase. <i>In silico</i> optimization
of the peptides, employing structural as well as sequence features,
aided specific targeting of σ<sup>A</sup> and σ<sup>B</sup>. We synthesized and characterized the best hit peptide from the
peptide library along with other control peptides and studied the
interaction of these peptides with σ<sup>B</sup> using biolayer
interferometry. The experimental data validate the design strategy.
These studies suggest the feasibility of designing specific peptides
via <i>in silico</i> methods that bind σ<sup>B</sup> with nanomolar affinity. We note that this strategy can be broadly
applied to modulate prokaryotic transcription by designed peptides,
thereby providing a tool for studying bacterial adaptation as well
as host–pathogen interactions in infectious bacteria