Biology subjects

Rajakumara, E.

Publications and source records attributed to Rajakumara, E..

6 recordsLinked to original sources

bioRxiv2026

Direct engagement of mycobacterial RadA by c-di-AMP at a non-catalytic site suppresses its DNA-stimulated ATPase activity

Cyclic di-adenosine monophosphate couples DNA damage sensing to genome maintenance in mycobacteria, but how it regulates homologous recombination machinery beyond RecA remains unclear. Using Mycobacterium smegmatis RadA (MsRadA) as a model, we show by isothermal titration calorimetry that c-di-AMP binds to MsRadA, providing biophysical evidence for an interaction previously inferred only from genetic studies. C-di-AMP binding is independent of magnesium ions and is not affected by prior occupancy of the ATP-binding site by the ATP analogue {beta},{gamma}-methylene adenosine 5'-triphosphate, indicating that c-di-AMP binds at a site distinct from the canonical ATPase pocket. Functionally, c-di-AMP reduces MsRadA DNA-dependent ATPase activity. Structural modelling and molecular dynamics (MD) simulations show that c-di-AMP is stably bound in the central channel of the RadA hexamer, non-covalently engaged with the DNA-binding residues Arg197, Lys226, and Arg234, which are contributed by two adjacent subunit interfaces of the hexamer. In the modelled RadA-ATP-c-di-AMP-DNA quaternary complex, c-di-AMP was predicted at a peripheral site near, but distinct from, the ATP-binding pocket. Together, these findings identify RadA as a direct physical target of c-di-AMP within the homologous recombination pathway and provide a framework for testing how c-di-AMP regulates RadA during DNA damage.

biophysics↗

bioRxiv2026

Evolutionary plasticity of regulatory--catalytic coupling in mono- and poly-ADP-ribosyl transferases

The evolution of modular proteins enables biological systems to diversify regulatory functions while preserving conserved catalytic activities. ADP-ribosyltransferases (ARTs), including mono-ADP-ribosyltransferases (MARylating enzymes) and poly-ADP-ribosyltransferases (PARylating enzymes), provide an ideal model for investigating how regulatory and catalytic domains co-evolve. Although ART catalytic domains are highly conserved, their regulatory architectures vary extensively, raising the question of whether regulatory domains determine catalytic activation, allosteric communication, and enzymatic output. Here, we systematically examined regulatory--catalytic interoperability by reconstituting evolutionarily distant ARTs from mammals, plants, and bacteria. We show that the regulatory domain of the MARylating enzyme human PARP3 (hPARP3) functionally complements the catalytic domains from the PARylating enzymes human PARP1 (hPARP1), human PARP2 (hPARP2), and Arabidopsis PARP2 (atPARP2), restoring robust DNA-break-dependent allosteric coupling for catalytic activation in response to both phosphorylated double-strand and single-strand DNA breaks. Complementation stimulates NAD+ consumption, promotes cis- and trans-poly(ADP-ribosyl)ation, and is accompanied by direct physical interactions between heterologous regulatory and catalytic domains. Unexpectedly, the strongest activation was observed between the hPARP3 regulatory domain and the atPARP2 catalytic domain, demonstrating conservation of productive allosteric communication across both enzymatic classes and species boundaries. In contrast, regulatory domains from PARylating ARTs failed to activate the catalytic domain of MARylating hPARP3, revealing a striking asymmetry in regulatory--catalytic compatibility. These findings identify conserved and specialised modes of allosteric communication within the ART superfamily and establish the concepts of general and specific combining ability as a framework for understanding modular protein evolution. More broadly, our results demonstrate that conserved regulatory--catalytic signalling networks can be functionally transferred between divergent enzymes, providing a foundation for mapping allosteric communication across ARTs and for engineering synthetic ADP-ribosylation systems with programmable regulatory inputs and catalytic outputs.

biochemistry↗

bioRxiv2026

Conformational Diversity and Interaction Signatures of NADH across protein families

Nicotinamide adenine dinucleotide (NADH) is a ubiquitous redox cofactor that participates in a wide range of enzymatic and regulatory processes. These include metabolism, signalling, and diseases such as cancer and neurodegeneration. Despite the abundance of NADH-protein complex structures, general principles governing how proteins shape NADH conformation and interaction modes remain unclear, which limits our ability to rationally interpret cofactor specificity, catalytic efficiency, and off-target effects in inhibitors. Here, we present a comprehensive structural analysis of NADH recognition across protein families using 345 NADH-bound crystal structures from the Protein Data Bank. We adopted a descriptor-driven strategy that quantitatively captures the internal geometry of NADH using angles, dihedrals, and interatomic distances, enabling direct comparison of cofactor shapes independent of protein fold. These studies reveal that 65% of structures preferred conformers with a conserved adenine- nicotinamide separation while allowing limited flexibility in the pyrophosphate. The interaction profiles demonstrate that NADH recognition is dominated by hydrogen bonding and electrostatic interactions involving nearly all heteroatoms, while most carbon positions remain non-interacting. Residue and moiety level analyses further show that the nicotinamide region acts as the primary interaction hotspot across enzyme classes, while only a handful of structures exhibit adenine-centric recognition. Together, this study establishes a unified biophysical framework that links NADH shape, interaction signatures, and protein context, providing rational insights for cofactor engineering and the design of NADH-targeted inhibitors.

biochemistry↗

bioRxiv2025

Allosteric activation of PARP2 self-PARylation by SUMO constrains DNA break-dependent catalytic Function

Poly(ADP-ribose)polymerase 2 (PARP2) is a key player in sensing DNA breaks and initiating DNA damage repair by catalysing the transfer of ADP-ribose units from NAD+ to target proteins, a process known as Poly(ADP-ribosyl)ation (PARylation). Post-translational modifications (PTMs) such as phosphorylation, ubiquitylation, SUMOylation, and PARylation are intricately linked to the DNA damage response (DDR) and repair. However, it is often overlooked that physical interactions between these enzymes and PTMs lead to DNA damage detection, DDR, and DNA repair. SUMOylation plays a vital role in DDR and DNA repair through covalent modification and non-covalent interactions. Here, we report new insight that Small ubiquitin like modifier (SUMO) binds with human PARP2 through non-covalent interactions, predominantly mediated by the N-terminal region (NTR) of PARP2. Surprisingly, SUMO stimulated PARP2 self-PARylation activity but hampered the DNA-dependent stimulation. Further competition binding studies suggest that SUMO binding promotes DNA release from PARP2. Altogether, our work uncovers a novel mechanism of SUMO-mediated allosteric regulation of PARP2 function, providing new insights into the possible interplay between SUMOylation and PARylation in DDR and DNA repair.

biochemistry↗

bioRxiv2021

PAR recognition by multiple reader domains of PARP1 allosterically regulates the DNA-dependent activities and independently stimulates the catalytic activity of PARP1

Poly(ADP-ribosyl)ation is predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single strand (SSB) and double strand (DSB) DNA breaks are bonafide stimulators of PARP1 activity. However, PAR mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT and WGR, hitherto uncharacterized, as PAR reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared to PAR but bind with weak or no affinity to DNA breaks as standalone domains. Conversely, ZnF1 and ZnF2 of PARP1 recognize DNA breaks but weakly to PAR. In addition, PAR reader domains, together, exhibit a synergy to recognize PAR or PARylated protein. Further competition binding studies suggest that PAR binding releases DNA from PARP1, and WGR domain facilitates the DNA release. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampers the DNA-dependent stimulation. Altogether, our work discovers dedicated high-affinity PAR reader domains of PARP1 and uncovers a novel mechanism of allosteric stimulation, but retardation of DNA-dependent activities of PARP1 by its catalytic product PAR. Therefore, our studies can be used as a model to understand the effect of one or more allosteric activators on the regulation of receptors or modular enzyme activities by another allosteric activator.

biophysics↗

bioRxiv2019

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4.

TDP-43 is an RNA/DNA-binding protein of versatile physiological functions and it is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease in addition to several other implicated proteins such as mutant SOD1 and FUS etc. Cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation of TDP-43 are suggested to be important TDP-43-toxicity causing mechanisms for the ALS manifestation. So far, therapeutic options for ALS are extremely minimal and ineffective therefore, multi-faceted approaches such as treating the oxidative stress and inhibiting the TDP-43s aggregation are being actively pursued. In our recent study, an acridine imidazolium derivative compound, AIM4, has been identified to have anti-TDP-43 aggregation propensity however, its mechanism of inhibition is not deciphered. In this study, we have utilized computational methods to examine binding site(s) of AIM4 in the TDP-43 structure and have also compared its binding efficiency with several other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic core region of amino acids aa: 288-319, which coincides with one of the key residue motifs that could potentially mediate liquid-liquid phase separation (LLPS) of TDP-43. Importantly, alike to the previously reported effects exerted by RNA molecules, we found that AIM4 could also inhibit the in vitro LLPS of a recombinantly purified C-terminal fragment TDP-432C bearing an A315T familial mutation. Antagonistic effects of AIM4 towards LLPS which is believed as the precursor process to the TDP-43s aggregation and the in silico prediction of a binding site of AIM4 on TDP-43 occurring in the same region, assert that AIM4 could be an important molecule for further investigations on TDP-43s anti-aggregation effects with relevance to the ALS pathogenesis.

bioinformatics↗