Biology subjects

Sengupta, J.

Publications and source records attributed to Sengupta, J..

9 recordsLinked to original sources

bioRxiv2026

Structural investigation suggests Elongation Factor-G1 is the bona fide canonical translation factor for ribosome recycling in mycobacteria

Bacterial elongation factor G (EF-G) facilitates mRNA and tRNA translocation during peptide elongation as well as promotes splitting of ribosomal subunits during ribosome recycling in coordination with ribosome recycling factor (RRF). Two homologs of EF-G (EF-G1 and EF-G2) have been identified in some bacterial species, including mycobacteria and in mitochondria. We have previously reported that mycobacterial EF-G2 functions as a stress-specific factor. Here, we demonstrate that EF-G1 functions as the canonical elongation factor responsible for ribosome recycling in mycobacteria. In addition, our structural analyses explain why mycobacterial EF-G2 lacks recycling activity, in contrast to its mitochondrial counterpart. We report cryo-EM structures of the M. smegmatis 70S ribosome and 50S subunit in complex with RRF and/or EF-G1 at 3-4 [A] resolution, elucidating the molecular architecture of intermediate structures and mechanistic basis of ribosome recycling. Notably, GTP hydrolysis on EF-G1 is not required for ribosomal disassembly in mycobacteria, unlike in E. coli.

biochemistry↗

bioRxiv2024

Mycobacterial methionine aminopeptidase type 1c moonlights as an anti-association factor on the 30S ribosomal subunit

Methionine aminopeptidase (MetAP), an essential metalloprotease, binds the 70S ribosome near the exit site of peptide tunnel and cleaves the N-terminal methionine from the nascent polypeptide chains. While E. coli expresses a single subclass of type1 MetAP, mycobacteria possesses two subclasses, namely, MetAP1a and MetAP1c, differentiated by the presence of a 40 amino acid long N-terminal tether in the later. Here we reveal a yet unknown moonlighting role of MetAP1c in mycobacteria. We observe stationary phase-specific enhancement of expression and a unique 30S subunit affinity of MetAP1c. A 5[A] cryo-EM map of Mycobacterium smegmatis (Msm) MetAP1c-30S subunit complex manifests for the first time the binding of MetAP1c at the inter-subunit side in the vicinity of proteins S13 and S19. MetAP1c binding and resulting conformational changes make the 30S subunit unfit for association with the 50S subunit, thereby conferring an anti-association property on MetAP1c. We further constructed two N-terminal mutants to establish the pivotal role of the N-terminal extension of MetAP1c in its moonlighting function.

biochemistry↗

bioRxiv2022

Cryo-EM structures of the Mycobacterium 50S subunit reveal an intrinsic conformational dynamics of 23S rRNA helices

Pathogenic organisms encounter a broad range of stress conditions within host micro-environment and adopt variety of mechanisms to stall protein translation and protect translational machinery. Structural investigations of the ribosomes isolated from pathogenic and non-pathogenic Mycobacterium species have identified several mycobacteria-specific structural features of ribosomal RNA and proteins. Here, we report a growth phase-dependent conformational switch of domain III and IV helices (H54a and H67-H71) of the mycobacterium 23S rRNA. Cryo-electron microscopy (cryo-EM) structures ([~]3-4 [A]) of the M. smegmatis (Msm) 50S ribosomal subunit of log-phase manifested that, while H68 possesses the usual stretched conformation in one of the maps, another one exhibits an unprecedented conformation of H68 curling onto a differently oriented H69, indicating an intrinsic dynamic nature of H68. Remarkably, a 2.8[A] cryo-EM map of the Msm stationary-state 50S subunit unveiled that H68 preferably acquires folded conformation in this state (closely mimicking dormant state). Formation of a bulge-out structure by H68 at the inter-subunit surface of the stationary-state 50S subunit due to the rRNA conformational changes prevents association with 30S subunit and keeps an inactive pool of the 50S subunit representing a ribosome-protection mechanism during dormancy. Evidently, this dynamic nature of H68 is an integral part of the cellular functions of mycobacterium ribosome, and irreversibly arresting H68 flexible motion would stall ribosome function. Thus, this conformational change may be exploited to develop anti-mycobacterium drug molecules. Significant statementBacteria utilize several mechanisms to reprogram the protein synthesis machinery so that their metabolism is reduced in the dormant state. Mycobacteria are capable of hiding themselves in a dormant state during physiological stresses. Our study identified a hitherto-unknown folded conformation of the helix 68 (H68) of domain IV of mycobacterial 23S rRNA, which is predominantly present in the stationary state (closely mimicking latency). Our results suggest that this conformational transition is instrumental in keeping an inactive pool of the 50S subunit in the stationary state. Irreversibly arresting such conformational dynamics would lead to protein synthesis shutdown in mycobacteria during dormancy. Thus, this folded conformation of H68 offers an excellent therapeutic intervention site to treat mycobacterial latent infection. HighlightsO_LIIdentification of a hitherto-unknown folded conformation of the helix 68 of mycobacterial 23S rRNA C_LIO_LIH68 conformation transition represents a new ribosome protection mechanism in dormant mycobacteria C_LIO_LIThe conformational switch of mycobacterial H68 offers an excellent therapeutic intervention site C_LI

biochemistry↗

bioRxiv2020

Transmission of allosteric response within the homotrimer of SARS-CoV-2 spike upon recognition of ACE2 receptor by the receptor-binding domain

The pathogenesis of novel SARS-CoV-2 virus initiates through recognition of ACE2 receptor (Angiotensin-converting enzyme 2) of the host cells by the receptor-binding domain (RBD) located at spikes of the virus. Following receptor-recognition, proteolytic cleavage between S1 and S2 subunits of the spike protein occurs with subsequent release of fusion peptide. Here, we report our study on allosteric communication within RBD that propagates the signal from ACE2-binding site towards allosteric site for the post-binding activation of proteolytic cleavage. Using MD simulations, we have demonstrated allosteric crosstalk within RBD in apo- and receptor-bound states where dynamic correlated motions and electrostatic energy perturbations contribute. While allostery, based on correlated motions, dominates inherent distal communication in apo-RBD, electrostatic energy perturbations determine favorable crosstalk within RBD upon binding to ACE2. Notably, allosteric path is constituted with evolutionarily conserved residues pointing towards their biological relevance. As revealed from recent structures, in the trimeric arrangement of spike, RBD of one copy interacts with S2 domain of another copy. Interestingly, the allosteric site identified is in direct contact (H-bonded) with a region in RBD that corresponds to the interacting region of RBD of one copy with S2 of another copy in trimeric constitution. Apparently, inter-monomer allosteric communication orchestrates concerted action of the trimer. Based on our results, we propose the allosteric loop of RBD as a potential drug target.

biophysics↗

bioRxiv2019

Heme minimizes Parkinson’s disease-associated toxicity by inducing a conformational distortion in the oligomers of alpha-Synuclein

Aggregation of the intrinsically disordered protein alpha-Synuclein (-Syn) into insoluble fibrils with a cross-{beta} sheet amyloid structure plays a key role in the neuronal pathology of Parkinsons disease (PD). The fibrillation pathway of -Syn encompasses a multitude of transient oligomeric forms differing in size, secondary structure, hydrophobic exposure and toxicity. According to a recent solid state NMR study, the fibrillating unit of -Syn contains the core residues of the protein arranged into in-register parallel {beta} sheets with a unique Greek key topology. Here, we have shown that the physiologically available small molecule heme (hemin chloride) when added at sub-stoichiometric ratios to either monomeric or aggregated -Syn, arrests its aggregation in an oligomeric state, which is minimally toxic. Using cryo-EM, we observed that these heme-induced oligomers are mace-shaped and consist of approximately four monomers. However, the presence of a crucial twist or contortion in their Greek key structural architecture prevents further hierarchical appending into annular oligomers and protofilament formation. We confirm using a His50Gln mutant that the binding of heme onto His50 is crucial in inflicting the structural distortion and is responsible for the stabilization of the non-toxic and off-pathway -Syn oligomers. We believe that this study provides a novel strategy of developing a therapeutic solution of PD, which has been elusive so far.

biophysics↗

bioRxiv2019

Resveratrol prevents amyloid fibrillation of insulin by arresting it in a bioactive oligomeric form

Insulin fibrillation is a limiting factor for its long-term storage because of considerably reduced bioavailable moieties. Deposition of fibrillated insulin can also cause subcutaneous insulin amyloidoma. Toxic phenolic compounds along with Zinc are used during commercial preparation of insulin to stabilize it in a hexameric form. Designed or repurposed natural small molecules with anti-amyloidogenic properties could thus be attractive agents for preventing insulin fibrillation. Natural polyphenolic compounds which have been shown to serve as anti-amyloid agents for proteins associated with neurodegenerative diseases are potential candidates for such function.\n\nIn this study we have demonstrated that resveratrol, a natural polyphenol, can not only prevent insulin fibrillation but can also preserve insulin in a bioactive oligomeric form even at high temperature. While investigating the influence of some natural polyphenols on human insulin (hINS) in a condition inductive to amyloid fibrillation at physiological pH, we found attenuation, to different extents, of insulin fibril formation. However, visualization of polyphenol-treated hINS revealed that resveratrol in particular has the unique ability to arrest hINS before the onset of fibrillation growth in soluble oligomeric forms with discrete spherical morphology. Importantly, insulin treated with resveratrol retains its full biological activity in vivo and exerts no toxicity towards cell lines. Structural characterization of the major population of resveratrol-induced insulin oligomers by cryo-EM and single particle 3D reconstruction revealed its morphology that resembled crystal structure of insulin hexamer formulated with phenolic compounds. Thus, our study suggests that resveratrol can be an effective nontoxic substituent of phenolic compounds for insulin preservation.

biochemistry↗

bioRxiv2018

Cryo-EM structures reveal dynamic interplay of nascent chain-processing factors on the ribosome

During protein biosynthesis in bacteria, one of the earliest phenomena that a nascent polypeptide chain experiences is the co-translational enzymatic processing. The event includes two enzymatic pathways, deformylation of the N-terminal methionine followed by methionine excision catalyzed by peptide deformylase (PDF) and methionine aminopeptidase (MetAP). The ribosome tunnel exit serves as the podium for recruiting proteins involved in maturation processes of the nascent chain. During the process, the emerging nascent protein likely remains shielded by the chaperone trigger factor (TF).\n\nHere, we present the first cryo-EM structures of E. coli ribosome in complex with the nascent chain processing proteins. The structures reveal overlapping binding sites for PDF and MetAP when they bind individually at the tunnel exit site, where proteins L22 and L32 are identified as primary anchoring sites for both proteins. Interestingly however, MetAP has a remarkable ability of repositioning itself to adjacent locations in the presence of PDF and TF at the tunnel exit. Thus, our results disclose an unexpected scanning mechanism that MetAP adopts for context-specific ribosome association.

biochemistry↗

bioRxiv2017

Electron microscopy reveals unique nano forms of bacterial spores

Endospore formation under environmental stress conditions is a well-established phenomenon for members of bacterial phylum Firmicutes, among which the most well studied ones belong to genus Bacillus and Clostridium. So far, known sizes of the spores are all larger than 500 nm. Nano-forms of bacteria have been reported but the notion still remains controversial. In this study, we provide visual evidences of living nano-entities (named here as nano-spores) formed by a bacterial species Bacillus cereus under prolonged stress, which are capable of escaping though standard sterile filtration procedure. The existence of nano-forms of bacteria was initially identified in a yeast ribosome preparation. We further demonstrate the transformation of the nano-spores into mature cells upon nutrient supply. Our study not only demonstrates the ability of bacteria to get transformed into yet-unknown form in order to survive under harsh environment, but also brings to light the existence of the smallest possible form of life.

microbiology↗

bioRxiv2017

Allosteric Activation of p300 Autoacetylation by the Tumor Suppressor p53

The transcriptional coactivator p300 is essential for p53 transactivation, although its precise mechanism remains unclear. We report that, p53 allosterically activates the acetyltransferase activity of p300 through the enhancement of p300 autoacetylation. Cryo-electron microscopy revealed that the domain organization of p300 is substantially altered upon binding of p53, suggesting that a structural switch may underpin the activation. Acetylated p300 accumulates near the transcription start sites accompanied by a similar enrichment of activating histone marks near those sites. Disruption of p53-p300 interaction by a site-directed peptide inhibitor abolished autoacetylated p300-mediated enhanced histone acetylation, suggesting a crucial role played by the allosteric activation in p53-mediated gene regulation. Gain-of-function mutant p53, known to impart aggressive proliferative properties in tumor cells, also activate p300 autoacetylation. The same peptide abolished many of the gain-of-functions of mutant p53 as well. We conclude that allosteric activation of p300 by p53 underpins gene regulation by p53. Reversal of gain-of-function properties of mutant p53 suggests that molecules targeting the p53-p300 interface may be good candidates for anti-tumor drugs.

molecular biology↗