Biology subjects

Vinothkumar, K. R.

Publications and source records attributed to Vinothkumar, K. R..

7 recordsLinked to original sources

bioRxiv2026

Epistatic interaction between ribosome-associated Era GTPase and stringent response regulator RelA modulates bacterial cell growth and dormancy

Cellular homeostasis is sustained by balancing the cell growth and quiescence in response to specific environmental cues. While the mechanisms governing the transition from growth to dormancy are reasonably well understood, the processes underlying exit from dormancy remain poorly characterised. Using an integrated approach employing bacterial genetics, Cryo-EM, and molecular microbiology, we investigated this phenomenon by studying the cross-talk between two antagonistic pathways, ribosome biogenesis that promotes cell growth, and stringent response that induces dormancy. We show that the exit from dormancy requires a conserved Era GTPase whose synthesis selectively rises during onset of exponential growth phase in Escherichia coli. Era not only accelerates the maturation of head and platform regions of 30S ribosomal subunit but also dislodges RelA from 70S ribosome. This derepresses the 70S ribosome from RelA-mediated inhibition, and promotes protein synthesis driving the active cell growth. This study uncovers that temporal epistatic interaction between Era and RelA governs cellular resuscitation in bacteria. Given that the dormant bacterial populations contribute to antibiotic tolerance, understanding this regulatory axis offers new insights for resensitising the recalcitrant dormant bacteria to antibiotics.

microbiology↗

bioRxiv2024

Structures of Multiple Peptide Resistance Factor from Pseudomonas aeruginosa

The aminoacylation of lipid head group in many bacteria is carried out by bi-functional enzymes called MprF, which encode for a soluble synthase domain that typically transfers lysine or alanine from a tRNA to lipid head groups, and the modified lipid is translocated across the leaflets by a transmembrane domain. This modification of the lipids probably evolved to adapt to the environment where the microbes reside. Here, we describe the cryoEM structures of MprF enzyme from Pseudomonas aeruginosa revealing a dimeric enzyme with a distinct architecture when compared with the homologous Rhizobium enzymes and validate this arrangement with biochemical analysis. The cryoEM maps and the models in detergent micelle and nanodisc reveal a conformational change of the terminal helix of the synthase domain, highlighting the dynamic elements in the enzyme that might facilitate catalysis. Several lipid-like densities are observed in the cryoEM maps, which might indicate the path taken by the lipids and the coupling function of the two functional domains. Thus, the structure of a well-characterised PaMprF lays a platform for understanding the mechanism of amino acid transfer to a lipid head group and subsequent flipping across the leaflet that changes the property of the membrane.

biochemistry↗

bioRxiv2024

Molecular determinants of Neu5Ac binding to tripartite ATP independent periplasmic (TRAP) transporter

N-Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino-sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of the Haemophilus influenzae TRAP transport system, (Currie et al., 2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures of Fusobacterium nucleatum SiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na+ binding sites. Two of the Na+-binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.

biochemistry↗

bioRxiv2021

Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methytransferases

Methylation of specific nucleotides is integral for ribosomal biogenesis and serves as a common way to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of 30S-KsgA by cryo-EM, a state was captured, where KsgA juxtaposes between helices h44 and h45, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, factors that direct the enzyme to its cognate site with high fidelity were unearthed by creating several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27 were mutated and evaluated for their methylation efficiency. The biochemical studies delineated specificity hotspots that enable KsgA to achieve an induced fit. This study enables the identification of distal exclusive allosteric pocket and other divergent structural elements in each rMTase, which can be exploited to develop strategies to reverse methylation, mediated drug resistance.

biochemistry↗

bioRxiv2019

Breaking a Strong Amide Bond: Structure and Properties of Dimethylformamidase.

Dimethylformamidase (DMFase) breaks down the human-made synthetic solvent N,N-dimethyl formamide(DMF) used extensively in industry(1). DMF is not known to exist in nature and was first synthesized in 1893. In spite of the recent origin of DMF, certain bacterial species such as Paracoccus, Pseudomonas, and Alcaligenes have evolved pathways to breakdown DMF and use them as carbon and nitrogen source for growth(2, 3). The work presented here provides a molecular basis for the ability of DMFase from Paracoccus to function in exacting conditions of high solvent concentrations, temperature and ionic strength to catalyze the hydrolysis of a stable amide bond. The structure reveals a multimeric complex of the 2{beta}2 type or (2{beta}2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed folds and as yet of unknown evolutionary origin. The active site is made of a distinctive mononuclear iron that is coordinated by two tyrosine residues and a glutamic acid residue. The hydrolytic cleavage of the amide bond is catalyzed at the Fe3+ site with a proximal glutamate probably acting as the base. The change in the quaternary structure is salt dependent with high salt resulting in the larger oligomeric state. Kinetic characterization reveals an enzyme that shows cooperativity between subunits and the structure provides clues on the interconnection between the active sites. Significance StatementN,N-dimethyl formamide(DMF) is a commonly used industrial solvent that was first synthesized in 1893. The properties that make DMF a highly desired solvent also makes it a difficult compound to breakdown. Yet, certain bacteria have evolved to survive in environments polluted by DMF and have enzymes that breakdown DMF and use it as their carbon and nitrogen source. The molecular structure of the enzyme that breaks down the stable amide bond in these bacteria, reveals two new protein folds and a unique mononuclear iron active site. The work reported here provides the structural and biochemical framework to query the evolutionary origins of the protein, as well as in engineering this enzyme for use in bioremediation of a human made toxic solvent.

biochemistry↗

bioRxiv2019

Structural insights into filament recognition by cellular actin markers

Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here we describe the electron cryomicroscopy structures of phalloidin, lifeAct and utrophin bound to F-actin, providing the first high-resolution structures and comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce conformations and lifeAct specifically recognizes ADP-actin state, which can be used as a sensor for distinguishing different nucleotide states of F-actin. The utrophin structural model aided designing minimal utrophin, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin binding proteins. Further offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

cell biology↗

bioRxiv2017

Structure of the deactive state of mammalian respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly-active preparation of Bos taurus complex I into the biochemically-defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 [A] resolution. The deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding templates their reordering. Our structure both rationalizes biochemical data on the deactive state, and offers new insights into its physiological and cellular roles.

biochemistry↗