Biology subjects

Das, C. K.

Publications and source records attributed to Das, C. K..

8 recordsLinked to original sources

bioRxiv2026

How the Azadithiolate Ligand Impacts O2-Stability of Group B -Hydrogenase ToHydA

[FeFe]-hydrogenases are metalloenzymes that catalyze the reversible oxidation and production of H2, making them potential candidates for sustainable energy solutions. However, their practical application is restricted by their extreme O2 sensitivity, which leads to irreversible active site degradation. A newly characterized Group B hydrogenase, ToHydA from Thermosediminibacter oceani, has exhibited exceptional O2-stability even after longtime exposure to air. In ToHydA, the highly conserved proton-transporting cysteine (C212) safeguards the H-cluster from O2-induced degradation by formation of the Hinact state. In this study, we investigate the effects of replacing the azadithiolate (ADT) ligand of [2Fe]H with propanedithiolate (PDT), revealing that this substitution prevents the formation of the Hinact and Htrans states observed in ToHydA WT (bearing the ADT ligand). By combining ATR-FTIR spectroscopy and molecular dynamics (MD) simulations, we show that a hydrogen bond between the nitrogen bridgehead of the ADT ligand and the C212 sidechain is crucial for stabilizing these states. The absence of this interaction in ToHydAPDT (bearing the PDT ligand) prevents the C212 sidechain from approaching the Fed center of [2Fe]H, thereby reducing Hinact accumulation. Moreover, as-isolated ToHydAPDT predominantly exhibits the Hhyd state, which is unusual for [FeFe]-hydrogenases with bound PDT ligand. These findings demonstrate how ligand substitution at the [2Fe]H site of ToHydA affects the structural dynamics, offering detailed molecular insights into the ligand-dependent modulation of [FeFe]-hydrogenases.

biophysics↗

bioRxiv2026

AlphaFold 3 captures oligomeric states and interaction dynamics of MLO ion channels

Mildew resistance Locus O (MLO) proteins have been originally identified as susceptibility factors for the fungal powdery mildew disease. Beyond immunity, they function in polarized secretion, including root and root hair elongation, trichome development, and fertilization. Moreover, MLO proteins mediate Ca{superscript 2} influx, either indirectly by recruiting Ca{superscript 2}-permeable channels to the plasma membrane or by acting as ion channels themselves. The latter raises the question of whether MLO proteins oligomerize to mediate ion transport across membranes. Here, we present an AlphaFold 3-based modeling pipeline for the reproducible assessment of MLO-containing protein complexes using AlphaFolds built-in confidence metrics together with structural and dynamic analyses. The resulting predictions for homo-oligomers of the prototypic barley Mlo support dimeric and trimeric assemblies, with the trimer forming a central membrane-spanning pore. Notably, AlphaFold 3 captured discrete conformational states of this trimer, as reflected by the clustering of confidence metrics. Computational structural analyses indicated that higher-confidence models adopt a closed pore conformation, whereas lower-confidence predictions reflect progressively expanding pore diameters. Molecular dynamics simulations further showed Ca{superscript 2} permeability of the putative open models. Our pipeline similarly predicts trimeric assemblies for MLO variants from Arabidopsis thaliana and Marchantia polymorpha, suggesting a conserved MLO structural scaffold within the land plant lineage. Additional Molecular Dynamics simulations revealed that closed models of barley Mlo and A. thaliana MLO2 open under simulated membrane tension, supporting the notion that MLO proteins are mechanosensitive ion channels. Moreover, predictions of MLO proteins with its known interactors, EF-hand proteins and exocyst complex subunit EXO70 proteins, suggest a mechanism for feedback inhibition of MLO-mediated ion flux and provide comprehensive experimental support for AlphaFold 3-predicted protein interfaces. Altogether, our results provide a structural framework for MLO channel architecture and regulation, while our prediction, modeling, and simulation pipeline should be useful beyond the study of this specific protein family. One-sentence summaryThis article describes AlphaFold 3-based analyses of MLO proteins, revealing the predicted structure of MLO membrane pores, their dynamic opening and closing, and their association with interacting proteins, including calmodulin and calmodulin-like calcium sensor proteins and exocyst complex subunit EXO70 proteins.

plant biology↗

bioRxiv2025

Tuning the H2 Production Activity of ToHydA by Molecular Simulation-Informed Protein Engineering

In the context of bio-hydrogen production, the O2-stable Group B [FeFe]-hydrogenase from Thermosediminibacter oceani has attracted significant interest due to its distinctive "TSCCCP" motif near the active site, which contains an additional (third) cysteine residue that is absent in the "TSCCP" motif of standard Group A hydrogenases. The precise role of the additional cysteine residue in H2 production has remained an open question. In this study, we sought to contribute to the understanding of this cysteines role in H2 production by combining molecular dynamics (MD) simulations, site-directed mutagenesis, biochemical assays, and Fourier-transform infrared (FTIR) spectroscopy. Remarkably, a cysteine-to-serine exchange variant (TSSCCP) demonstrated enhanced H2 production activity without compromising the O2-stability of ToHydA, offering new insights into its functional dynamics.

biophysics↗

bioRxiv2025

Protein dynamics affect O2-stability of Group B -hydrogenase from Thermosediminibacter oceani

In the pursuit of sustainable green energy generation, [FeFe]-hydrogenases have attracted significant attention due to their ability to catalyze hydrogen production. However, the sensitivity of these enzymes to O2 is a major obstacle for their application as biocatalysts in energy conversion technologies. In the search for an O2-stable [FeFe]-hydrogenase, we identified the hydrogenase ToHydA from Thermosediminibacter oceani that belongs to the rarely characterized Group B (M2a) [FeFe]-hydrogenases. Our findings demonstrate that ToHydA exhibits remarkable O2-stability, even under prolonged O2 exposure. By characterizing site-directed mutagenesis variants, we found that the highly conserved proton-transporting cysteine protects H-cluster from O2-induced degradation by forming Hinact state. The additional cysteine residue in the TSCCCP motif of ToHydA, a feature unique to Group B (M2a) [FeFe]-hydrogenases, enhances the flexibility of that motif and facilitates the formation of the Hinact state. Moreover, ToHydA possesses unique features, including the formation of an unusual Hinact resting state that distinguishes the enzyme from other [FeFe]-hydrogenases. Our atomistic molecular dynamics simulations reveal a previously unrecognized cluster of hydrophobic residues centered around the proton-transporting cysteine-bearing loop. This structural feature appears to be a common molecular characteristic in hydrogenases that form the O2-protected Hinact state. By exploiting these molecular features of ToHydA, future research can aim to rationally design hydrogenases that combine high catalytic activity with enhanced O2 stability, to develop more efficient and durable catalysts. Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/643706v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@96b4aorg.highwire.dtl.DTLVardef@62f367org.highwire.dtl.DTLVardef@59f170org.highwire.dtl.DTLVardef@fa2fd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis

The androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here, we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 expression is abnormally gained in prostate cancer cells and its functional inhibition impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites, which is greater than 65% of the AR tumor cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and instead contain the canonical palindromic motifs. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in the physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Concordantly, a dual NSD1/2 PROTAC degrader, called LLC0150, was preferentially cytotoxic in AR-dependent prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the AR neo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 as viable paralog co-targets in advanced prostate cancer.

cancer biology↗

bioRxiv2024

Methyl substituted β-lactam framework based antibiotics and β-lactamase inhibitors: Proof of concept by computations

Increasing the life-time of the acyl-enzyme complex formed between an inhibitor or drug molecule and the {beta}-lactamase through chemical modifications of existing drug molecules is an important strategy towards developing inhibitors. In this direction, our group proposed a methyl-substituted {beta}-lactam framework for the design of inhibitors for {beta}-lactamases (J. Phys. Chem. B. 2018, 122, 4299). This unconventional design was guided by the transition state structure of the deacylation reaction of the acyl-enzyme complex. Here, we present a proof of principle study of this concept through detailed molecular simulations and free energy calculations. In particular, we improve the antimicrobial activity of the first-generation cephalosporin antibiotic, cephalothin, through C6-methylation. The proposed molecule, (6R,7R)-3-(acetyloxymethyl)-6-methyl-8-oxo-7-[(2-thiophen-2-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate) slows down the deacylation of the acyl-enzyme complex 109-fold with no apparent effect on its binding to class-C {beta}-lactamase and formation of the acyl-enzyme intermediate. The design strategy presented in this work can be further extended to all {beta}-lactam antibiotics, like monobactams, carbapenems, cephalosporins, and penicillins.

biochemistry↗

bioRxiv2022

Epigenetic CpG Duplex Marks Probed by an Evolved DNA Reader via a Well-Tempered Conformational Plasticity

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel "CpG duplex marks" could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.

genomics↗

bioRxiv2021

The CTPase activity of ParB acts as a timing mechanism to control the dynamics and function of prokaryotic DNA partition complexes

DNA partitioning CTPases of the ParB family mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromeric region of target DNA molecules and then spread laterally to form large nucleoprotein complexes that serve as docking points for the DNA segregation machinery. Here, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. Moreover, we solve crystal structures of ParB in the pre- and post-hydrolysis state and provide insights into the catalytic mechanism underlying nucleotide hydrolysis. The characterization of CTPase-deficient ParB variants reveals that CTP hydrolysis serves as a timing mechanism to control the sliding time of ParB. Hyperstable clamps are trapped on the DNA, leading to excessing spreading and severe chromosome segregation defects in vivo. These findings clarify the role of the ParB CTPase cycle in partition complex dynamics and function and thus complete our understanding of this prototypic CTP-dependent molecular switch.

microbiology↗