Biology subjects

Panjikar, S.

Publications and source records attributed to Panjikar, S..

10 recordsLinked to original sources

bioRxiv2026

Extraction of directional electron-density features from diffraction data using spherical-harmonic decomposition

Directional anisotropy in electron density provides key information about chemical bonding that is not readily accessible from conventional electron-density maps. Here, a model-independent framework is presented for decomposing experimental structure factors into angular components using spherical harmonics. Reciprocal-space projection onto spherical harmonics followed by standard Fourier synthesis yields angularly filtered density maps. The{ell} = 0 component captures the isotropic part of the density, while the{ell} = 1 components resemble px, py and pz-like dipolar functions that highlight directional electronic structure. Applications to high-resolution datasets, including urea, the Gly-Ala dipeptide and a 0.97 [A]{beta}-lactamase structure, reveal chemically interpretable dipolar features associated with carbonyl and amide bonds, N-H interactions and aromatic{pi} systems. Quantitative analysis using bond-centred sampling demonstrates stable dipolar signatures that remain detectable under moderate resolution truncation. These results establish spherical-harmonic angular decomposition as a practical framework for extracting directional electronic information from crystallographic electron-density maps. SynopsisAngular decomposition of experimental structure factors reveals dipolar anisotropy and directional electron-density features that are directly meaningful for chemical interpretation.

biophysics↗

bioRxiv2025

Mechanism of NanR transcriptional activation of sialic acid metabolism in Streptococcus pneumoniae.

In Streptococcus pneumoniae, the RpiR transcriptional regulator NanR (SpNanR) senses sialic acid in the environment and upregulates transcription of the nan and siaA operons to increase uptake and metabolism of sialic acid. The molecular basis of this activation is unknown. Here, we demonstrate that SpNanR binds N-acetylmannosamine-6-phosphate, a metabolite of sialic acid catabolism. SpNanR exists in a dimer-tetramer equilibrium, and N-acetylmannosamine-6-phosphate binding strongly stabilizes the tetramer. Crystal structures and site-specific substitutions demonstrate that N-acetylmannosamine-6-phosphate bridges and stabilizes the SpNanR tetramer. SpNanR binds its DNA recognition sequence with nanomolar affinity. Notably, the effector N-acetylmannosamine-6-phosphate does not affect the affinity of SpNanR for DNA. The DNA binding domains are not structurally coupled to the sugar isomerase domains, explaining why N-acetylmannosamine-6-phosphate binding does not affect DNA binding. Structural analysis reveals that sequence specificity arises through distortion of B-DNA and an unusual {pi}-stack formed by two arginine residues in the minor groove, while affinity is driven by backbone contacts. We propose a mechanism by which S. pneumoniae regulates sialic acid metabolism, consistent with our biophysical experiments and in vivo regulatory behavior. These findings define a unique activation mechanism for an RpiR regulator and provide new insights into carbohydrate-responsive gene regulation in pneumococci.

molecular biology↗

bioRxiv2025

Natural product-mediated reaction hijacking mechanism validates Plasmodium aspartyl-tRNA synthetase as an antimalarial drug target

Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5'-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2 phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition.

biochemistry↗

bioRxiv2024

On the function of TRAP substrate-binding proteins: conformational variation of the sialic acid binding protein SiaP

Tripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding proteins to scavenge metabolites (e.g., N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAP N-acetylneuraminate substrate-binding protein from Aggregatibacter actinomycetemcomitans (AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, where AaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at how N-acetylneuraminate binding stabilises a fully closed state. AaSiaP preferentially binds N-acetylneuraminate (KD = 0.4 {micro}M) compared to N-glycolylneuraminate (KD = 4.4 {micro}M), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest the AaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, it is likely that the open unliganded conformation encompasses multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.

biochemistry↗

bioRxiv2022

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families

CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by [~]150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

biochemistry↗

bioRxiv2022

Phenol Sensing in Nature Modulated via a Conformational Switch Governed by Dynamic Allostery

NtrC family of proteins sense external stimuli and accordingly stimulate stress and virulence pathways via activation of associated {sigma}54-dependent RNA polymerases. Here, we establish that MopR, an NtrC protein, harbors a dynamic bi-directional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allo-steric-linker". While G-hinge influences the entry of phenol, the allosteric-linker passes the signal to the downstream ATPase domain. Phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery, and it was demonstrated that perturbation of the core relay residues results in a complete loss of ATPase stimulation. A mutation of the G-hinge,[~]20[A] from the phenol pocket, demonstrated altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. A global analysis illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species.

biochemistry↗

bioRxiv2022

From bacteria to plants: a repurposing strategy in the pursuit for novel herbicides

Herbicide resistance represents one of the biggest threats to our natural environment and agricultural sector. Thus, new herbicides are urgently needed to tackle the rise in herbicideresistant weeds. Here, we employed a novel strategy to repurpose a failed antibiotic into a new and target-specific herbicidal compound. Specifically, we identified an inhibitor of bacterial dihydrodipicolinate reductase (DHDPR), an enzyme involved in lysine biosynthesis in plants and bacteria, that exhibited no antibacterial activity but severely attenuated germination of the plant Arabidopsis thaliana. We confirmed that the inhibitor targets plant DHDPR orthologues in vitro, and exhibits no toxic effects against human cell lines. A series of analogues were then synthesised with improved efficacy in germination assays and against soil-grown A. thaliana plants. We also showed that our lead compound is the first lysine biosynthesis inhibitor with herbicidal activity against a weed species, providing proof-of-concept that DHDPR inhibition may represent a much-needed new herbicide mode of action. Furthermore, this study exemplifies the untapped potential of repurposing failed antibiotic scaffolds to fast-track the development of herbicide candidates targeting the respective plant enzymes to combat the global rise in herbicide-resistant weeds.

biochemistry↗

bioRxiv2021

Towards Novel Herbicide Modes of Action by Inhibiting Lysine Biosynthesis in Plants

Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. Using a high throughput chemical screen, we identified the first class of plant DHDPS inhibitors that have micromolar potency against Arabidopsis thaliana DHDPS isoforms. Employing X-ray crystallography, we determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. We also demonstrated that the inhibitors attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide resistant weeds.

biochemistry↗

bioRxiv2020

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Plantacyclin B21AG is a circular bacteriocin produced by Lactobacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 17 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Truncation of an N-terminal extension abolishes cooperative binding. The effector, N-acetylneuraminate, binds NanR and attenuates DNA binding. Crystal structure data show that N-acetylneuraminate binding to NanR causes a domain rearrangement that locks the protein in a conformation that prevents DNA binding. Single-particle cryo-electron microscopy structures of NanR bound to DNA reveal the DNA binding domain is reorganized to engage DNA, while the three dimers assemble in close proximity across the (GGTATA)3-repeat operator allowing protein-protein interactions to form via the N-terminal extensions. Our data provides a molecular basis for the regulation of bacterial sialic acid metabolism.

molecular biology↗