Biology subjects

Ramakumar, S.

Publications and source records attributed to Ramakumar, S..

5 recordsLinked to original sources

bioRxiv2023

Mycobacterium tuberculosis methyltransferase perturbs host epigenetic programming to promote bacterial survival

Mycobacterium tuberculosis (Mtb) has evolved several mechanisms to counter host defense arsenal for its proliferation. We show that Mtb employs multi-pronged approach to modify host epigenetic machinery for its survival upon infection. It secretes methyltransferase (MTase) Rv2067c into macrophages, trimethylating K79 of histone H3 in non-nucleosomal context. Rv2067c downregulates host MTase DOT1L, decreasing its H3K79me3 mark added nucleosomally on pro-inflammatory response genes. Consequent inhibition of caspase8 dependent apoptosis and enhancement of RIPK3 mediated necrosis results in increased pathogenesis. In parallel, Rv2067c enhances the expression of SESTRIN3, NLRC3 and TMTC1 enabling the pathogen to overcome host inflammatory and oxidative response. We provide structural basis for differential methylation of H3 by Rv2067c and DOT1L. The structure of Rv2067c and DOT1L explain how their action on H3K79 is temporally and spatially separated enabling Rv2067c to effectively intercept the host epigenetic circuit and downstream signalling.

microbiology↗

bioRxiv2021

Targeted redesign of suramin analogs for novel antimicrobial lead development

The emergence of new viral infections and drug resistant bacteria urgently necessitates expedient therapeutic development. Repurposing and redesign of existing drugs against different targets is one potential way in which to accelerate this process. Suramin was initially developed as a successful anti-parasitic drug but has also shown promising antiviral and antibacterial activities. However, due to its high conformational flexibility and negative charge, suramin is considered quite promiscuous towards positively charged sites within nucleic acid binding proteins. Although some suramin analogs have been developed against specific targets, only limited structure activity relationship (SAR) studies were performed, and virtual screening has yet to be used to identify more specific inhibitor(s) based on its scaffold. Using available structures, we investigated suramins target diversity, confirming that suramin preferentially binds to protein pockets which are both positively charged and enriched in aromatic or leucine residues. Further, suramins high conformational flexibility allows adaptation to structurally diverse binding surfaces. From this platform, we developed a framework for structure- and docking-guided elaboration of suramin analog scaffolds using virtual screening of suramin and heparin analogs against a panel of diverse therapeutically relevant viral and bacterial protein targets. Use of this new framework to design potentially specific suramin analogs is exemplified using the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and nucleocapsid protein, identifying leads that might inhibit a wide range of coronaviruses. The approach presented here establishes a computational framework for designing suramin analogs against different bacterial and viral targets and repurposing existing drugs for more specific inhibitory activity. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/444489v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@324301org.highwire.dtl.DTLVardef@1cd0c4borg.highwire.dtl.DTLVardef@e235c7org.highwire.dtl.DTLVardef@138b6f0_HPS_FORMAT_FIGEXP M_FIG Table of Contents Graphic C_FIG

microbiology↗

bioRxiv2020

Phylogenetic Studies and Inhibitor Design Targeting Protein Interacting Interface of Nucleoid-Associated Protein HU

The formations of nucleoprotein structures by promiscuous DNA binding proteins like HU are assisted with their protein protein interaction capability with other proteins. In E. coli Gal repressosome assembly, GalR piggybacks HU to the critical position on the DNA (hbs site) through a specific GalR-HU interaction using an interface at the bottom of alpha helical region, which we termed as HUpb interface. Similarly, MtbHU also interact with Topoisomerase I with the same interface to enhance its relaxation activity. In an earlier study, we determined the crystal structure of MtbHU, inhibited it using stilbene derivatives which inhibited the cell growth. It motivated us to understand the evolutionary and structural characteristics of the HUpb interface, which has not been investigated previously for HU or for any other NAPs. Our analyses found residue positions corresponding to MtbHU Thr11 to Gln20 form the interface while Ala23 serves the pocket lining residue. Due to ancestral mutations in the duplication event before the HU and IHF split, physicochemical properties of the interface vary among clades. Thus, this interface could engage different proteins in different HU oligomeric states in Proteobacteria. Protein-protein interfaces are usually a challenging target due to its flatter surface. In case of MtbHUpb interface, we observed that due to the presence of a partially hydrophobic pocket, small molecule scaffolds could fit into it, while the ligand can be further designed to interact with D17, which is the crucial residue for Topoisomerase I interaction. We used a two-step virtual screening protocol with known drug like molecules as starting set to an aim to re-purpose drugs. Our docking results showed compounds like Maltotetraose, Valrubicin, Iodixanol, Enalkiren, indinavir, Carfilzomib, oxytetracycline, quinalizarin, Raltitrexed, Epigallocatechin and their analogues exhibit high scoring binding at MtbHUpb interface. Our present report gives a model example of an evolutionary study of an interface of nucleoid associated protein, which is used to computationally design inhibitors. This strategy could be in general useful for designing inhibitors for various types of protein-protein interfaces using evolutionary guided design.

bioinformatics↗

bioRxiv2019

A short commentary on indents and edges of β-sheets

{beta}-sheets in proteins are formed by extended polypeptide chains, called {beta}-strands. While there is a general consensus on two types of {beta}-strands, viz. edge strands (or edges) and inner strands (or central strands), the possibility of distinguishing between different regions of inner strands remains less explored. In this paper, we address the portions of inner strands of {beta}-sheets that stick out on either or both sides. We call these portions the indent strands or indents because they give the typical indented appearance to {beta}-sheets. Similar to the edge strands, the indent strands also have {beta}-bridge partner residues on one side while the other side is still open for backbone hydrogen bonds. Despite this similarity, the indent strands differ from the edge strands in terms of various properties such as {beta}-bulges and amino acid composition due to their localization within {beta}-sheets and therefore within folded proteins to certain extent. The localization of indents and edges within folded proteins seems to govern the strategies deployed to deter unhindered {beta}-sheet propagation through {beta}-strand stacking interactions. Our findings suggest that, edges and indents differ in their strategies to avoid further {beta}-strand stacking. Short length itself is a good strategy to avoid stacking and a majority of indents are two residue or shorter in length. Edge strands on the other hand are overall longer. While long edges are known to use various negative design strategies like {beta}-bulges, prolines, strategically placed charges, inward-pointing charged side chains and loop coverage to avoid further {beta}-strand stacking, long indents seem to favor mechanisms such as enrichment in flexible residues with high solvation potential and depletion in hydrophobic residues in response to their less solvent exposed nature. Such subtle differences between indents and edges could be leveraged for designing novel {beta}-sheet architectures.

bioinformatics↗

bioRxiv2019

Structural excavation of cryptic sites in proteins using glycol decoys: implications in drug-design

Cryptic pockets are visible in ligand-bound protein structures but are occluded in unbound structures. Utilizing these pockets in fragment-based drug-design provides an attractive option for proteins not tractable by classical binding sites. However, owing to their hidden nature, they are difficult to identify. Here, we show that small glycols find cryptic pockets on diverse set of proteins. Initial crystallography experiments serendipitously revealed the ability of ethylene glycol, a small glycol, to identify a cryptic pocket on W6A mutant of RBSX protein (RBSX-W6A). Explicit-solvent molecular dynamics (MD) simulations of RBSX-W6A with exposed-state of the cryptic pocket (ethylene glycol removed) revealed closure of the pocket reiterating that cryptic pockets in general prefer to stay in closed-state in absence of the ligands. Also, no change in the pocket was observed for simulations of RBSX-W6A with occluded-state of the cryptic pocket, suggesting that water molecules are not able to open the cryptic pocket. "Cryptic-pocket finding" potential of small glycols was then supported and generalized through additional crystallography experiments, explicit-cosolvent MD simulations, protein dataset construction and analysis. The cryptic pocket on RBSX-W6A was found again upon repeating the crystallography experiments with another small glycol, propylene glycol. Use of ethylene glycol as probe molecule in cosolvent MD simulations led to the enhanced sampling of the exposed-state of experimentally observed cryptic sites on test set of two proteins (Niemann-Pick C2, Interleukin-2). Further, analyses of protein structures with validated cryptic sites showed that ethylene glycol molecules binds to sites on proteins (G-actin, Myosin II, Bcl-xL, Glutamate receptor 2) which become apparent upon binding of biologically relevant ligands. Our study thus suggests potential application of the small glycols in experimental and computational fragment-based approaches to identify cryptic pockets in apparently undruggable and/or difficult targets, making these proteins amenable to drug-design strategies.

biophysics↗