Papers by Swastik Phulera
Structure, 2024
Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activi... more Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. To elucidate the mechanism of action of LqhαIT, we determined a 3.9 Å cryoelectron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in an “S4 down” conformation. The functionally essential C-terminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrates the functional importance of this toxin-glycan interaction. These findings establish a structural basis for the specificity achieved by scorpion α-toxins and reveal the conserved glycan as an essential component of the toxin-binding epitope.
bioRxiv (Cold Spring Harbor Laboratory), Jan 30, 2024
Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activi... more Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. Numerous protein toxins have been identified that modulate Nav gating, and structures of Nav channels in complex with these toxins helped elucidate the molecular mechanisms of voltage-dependent channel gating. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. Biochemical studies show that LqhαIT features a functionally essential epitope at its C-terminus that is not shared with related scorpion α-toxins. To elucidate the mechanism of action of LqhαIT, we determined a 3.9 Å cryo-electron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in a "S4 down" conformation to stabilize the open state. To promote binding, the functionally essential Cterminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrate the functional importance of this toxin-glycan interaction. These findings help establish a structural basis for the specificity achieved by scorpion α-toxins and provide crucial insights for the development and optimization of new Nav channel modulators. .
FEBS Letters, Apr 26, 2019
The transcription factor Rv0081 of Mycobacterium tuberculosis controls hypoxic gene expression an... more The transcription factor Rv0081 of Mycobacterium tuberculosis controls hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis infection. We report here the crystal structure of Rv0081 at 2.9 Å resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. However, unlike other members in this family, Rv0081 has neither a metal binding domain nor does it possess Cys residues, suggesting an alternate mechanism of gene regulation. Our structural and biochemical analyses suggest the molecular basis for the recognition of self-regulatory DNA sequences and a plausible mechanism of regulation of Rv0081 in the latent phase of tuberculosis infection.
Cancers
BEND3 is a transcription factor that plays a critical role in the regulation of gene expression i... more BEND3 is a transcription factor that plays a critical role in the regulation of gene expression in mammals. While there is limited research on the role of BEND3 as a tumor suppressor or an oncogene and its potential role in cancer therapy is still emerging, several studies suggest that it may be involved in both the processes. Its interaction and regulation with multiple other factors via p21 have already been reported to play a significant role in cancer development, which serves as an indication of its potential role in oncogenesis. Its interaction with chromatin modifiers such as NuRD and NoRC and its role in the recruitment of polycomb repressive complex 2 (PRC2) are some of the additional events indicative of its potential role in cancer development. Moreover, a few recent studies indicate BEND3 as a potential target for cancer therapy. Since the specific mechanisms by which BEND3 may contribute to cancer progression are not yet fully elucidated, in this review, we have discuss...
Members of the NrdH family of redox proteins, which consists of small glutaredoxin-like proteins ... more Members of the NrdH family of redox proteins, which consists of small glutaredoxin-like proteins with thioredoxin-like activity, serve as the reducing partners of class Ib ribonucleotide reductases. Here, we report the crystal structure of NrdH from <i>Mycobacterium tuberculosis</i>, refined to a crystallographic <i>R</i> factor of 14.02% (<i>R</i><sub>free</sub> = 15.53%) at 0.87 Å resolution. The tertiary structure of <i>M. tuberculosis</i> NrdH has a typical thioredoxin fold as expected. The extremely high resolution of the structure allows us to dissect the functionality of the protein in great depth. Structural superimposition of <i>M. tuberculosis</i> NrdH and thioredoxin reductase over the <i>Escherichia coli</i> thioredoxin reductase–thioredoxin complex suggests the ability of NrdH to accept electrons from <i>M. tuberculosis</i> thioredoxin reductase. This raises the important que...
The transcription factor Rv0081 of M. tuberculosis controls the hypoxic gene expression and acts ... more The transcription factor Rv0081 of M. tuberculosis controls the hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis infection. We report here the crystal structure of Rv0081 at 3.3 Å resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. ArsR/SmtB family transcriptional repressors exert gene regulation by reversible metal binding. Hypoxia in general is sensed by bacterial transcriptional regulators via metals or Cys-mediated thiol switches. Oxygen sensing typically leads to transcriptional repressor changing its conformational state with altered DNA-binding property under different oxygen levels. Surprisingly Rv0081 neither has a metal binding domain nor does it possess Cys residues suggesting an alternate mechanism of gene regulation. Our structural analysis identified Ser 48, Ser 49, Ser 52 and Gln 53 as potential residues of Rv0081 involved in DNA binding. We probed DNA-binding of Rv0081 with electrophoretic mobility...
Evolution of life took place in a reducing atmosphere. As a reminiscence of this, cytoplasm of al... more Evolution of life took place in a reducing atmosphere. As a reminiscence of this, cytoplasm of all living organisms is reducing in nature. Furthermore most biochemical reactions like energy production etc. are redox in nature requiring either an electron supplier or acceptor. The maintenance of redox state thus becomes extremely crucial for any cell. Cells typically devote considerable resources for the maintenance of their redox state. M. tuberculosis successfully evades an array of host generated redox stresses. For this M. tuberculosis employs a large number of redox sensors and effectors. In our laboratory we have been studying some of these central players with a hope to be able to gain insights into the pathogen. We shall try to discuss the relevance of redox and our efforts to understand the same in this review.
Knotted peptides present a wealth of structurally diverse, biologically active molecules, with th... more Knotted peptides present a wealth of structurally diverse, biologically active molecules, with the inhibitor cystine knot/knottin class among the most ecologically common ones. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. Such compounds are often produced in low concentrations by intractable organisms, making structural and biological characterization challenging, which is frequently overcome by various expression strategies. Here, we sought to test a biosynthetic route for the expression and study of knotted peptides. We screened expression constructs for a biosynthesized knotted peptide to determine the most influential parameters for successful disulfide folding and used NMR spectroscopic fingerprinting to validate topological structures. We performed pharmacokinetic characterization, which indicated that the interlocking disulfide structure minimizes liabilities of linear peptide sequences, and propose a mechanism by which knotted peptides are cleared. We then developed an assay to monitor solution folding in real time, providing a strategy for studying the folding process during maturation, which provided direct evidence for the importance of backbone organization as the driving force for topology formation.
eLife, Jul 25, 2018
Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABA rec... more Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric α1β1γ2S GABA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the α1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pa...
Journal of virology, Oct 19, 2017
Poxviruses display species tropism - variola virus is a human-specific virus, while vaccinia viru... more Poxviruses display species tropism - variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE exhibit selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP display selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species-specificity of VCP and SPICE towards the classical pathway (CP). We observed that VCP is ∼43-fold superior in inhibiting bovine CP than SPICE. Further, functional assays revealed that increased inhibitory activity of VCP towards bovine CP is solely due to its enhanced cofactor activity with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino acid substitution mutants wherein one or more of the 11 variant VCP residues were substituted onto the SPICE template. Exami...
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Papers by Swastik Phulera