Biology subjects

Madhu, M. K.

Publications and source records attributed to Madhu, M. K..

5 recordsLinked to original sources

bioRxiv2025

Mechanistic basis for GPCR phosphorylation-dependent allosteric signaling specificity of β-arrestin 1 and 2

{beta}-arrestins ({beta}arr1 and {beta}arr2) are key transducers of G protein-coupled receptor (GPCR) signaling, orchestrating both shared and isoform-specific intracellular pathways. Phosphorylation of the receptor C-terminal tail by GPCR kinases encodes regulatory "barcodes" that modulate {beta}-arrestin conformations and interactions with downstream effectors. However, how distinct phosphorylation patterns shape {beta}-arrestin structure and function remains poorly understood. Here, we combine all-atom molecular dynamics simulations with machine learning and graph neural networks to systematically map the barcode-dependent conformational landscapes of {beta}-arrestins bound to the phosphorylated vasopressin receptor 2 tail (V2Rpp). We find that V2Rpp engages {beta}arr1 more stably than {beta}arr2, mediated by isoform-specific residue contacts that trigger distinct allosteric responses. These include differential interdomain rotations and rearrangements in key structural motifs, potentially facilitating selective effector protein engagement. Furthermore, we identify critical residue networks that transmit phosphorylation signals to effector-binding interfaces in a barcode- and isoform-specific manner. Notably, {beta}arr1 exhibits stronger allosteric coupling between V2Rpp and c-edge loop 2 compared to {beta}arr2, which is consistent with its enhanced membrane association. Together, these findings advance our understanding of the molecular mechanisms by which {beta}-arrestins interpret GPCR phosphorylation signatures, offering a framework that could aid in designing pathway-selective therapeutics.

biophysics↗

bioRxiv2024

Mechanistic Insights into G-protein Activation via Phosphorylation Mediated Non-Canonical Pathway

Activation of heterotrimeric G-proteins (G{beta}{gamma}) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of G that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce G activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the Gi3, a subtype of G-protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. The additional negative charges introduced by phosphorylations rewired the inter-residue interactions and significantly weakened the salt bridges at the domain interface, contributing to the increased separation of the Ras-like and helical domains of G-protein. Furthermore, the unfolding of helix F resulted in greater flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated Gi3. The release of GDP in the phosphorylated G-protein occurred at a faster rate compared to the unphosphorylated state, caused by increased fluctuations in conserved regions of P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.

biophysics↗

bioRxiv2023

Biased Signaling in Mutated Variants of β2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations

The molecular basis of receptor bias in G protein-coupled receptors (GPCRs) caused by mutations that preferentially activate specific intracellular transducers over others remains poorly understood. Two experimentally identified biased variants of {beta}2-adrenergic receptors ({beta}2AR), a prototypical GPCR, are a triple mutant (T68F, Y132A, and Y219A) and a single mutant (Y219A); the former bias the receptor towards the {beta}-arrestin pathway by disfavoring G protein engagement, while the latter induces G protein signaling explicitly due to selection against GPCR kinases (GRKs) that phosphorylate the receptor as a prerequisite of {beta}-arrestin binding. Though rigorous characterizations have revealed functional implications of these mutations, the atomistic origin of the observed transducer selectivity is not clear. In this study, we investigate the allosteric mechanism of receptor bias in {beta}2AR using microseconds of all-atom Gaussian accelerated molecular dynamics (GaMD) simulations. Our observations reveal distinct rearrangements in transmembrane helices, intracellular loop 3, and critical residues R1313.50 and Y3267.53 in the conserved motifs D(E)RY and NPxxY for the mutant receptors, leading to their specific transducer interactions. The reorganization of allosteric communications from the extracellular agonist BI-167107 to the intracellular receptor-transducer interfaces drives the conformational rearrangements responsible for receptor bias in the single and triple mutants. The molecular insights into receptor bias of {beta}2AR presented here could improve the understanding of biased signaling in GPCRs, potentially opening new avenues for designing novel therapeutics with fewer side effects and superior efficacy.

biophysics↗

bioRxiv2021

Molecular insights into phosphorylation-induced allosteric conformational changes in β2-adrenergic receptor

The large conformational flexibility of G protein-coupled receptors (GPCRs) has been a puzzle in structural and pharmacological studies for the past few decades. Apart from structural rearrangements induced by ligands, enzymatic phosphorylations by GPCR kinases (GRKs) at the carboxy-terminal tail (C-tail) of a GPCR also makes conformational alterations to the transmembrane helices and facilitates the binding of one of its transducer proteins named {beta}-arrestin. Phosphorylation-induced conformational transition of the receptor that causes specific binding to {beta}-arrestin but prevents the association of other transducers such as G proteins lacks atomistic understanding and is elusive to experimental studies. Using microseconds of all-atom conventional and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigate the allosteric mechanism of phosphorylation induced-conformational changes in {beta}2-adrenergic receptor, a well-characterized GPCR model system. Free energy profiles reveal that the phosphorylated receptor samples a new conformational state in addition to the canonical active state corroborating with recent nuclear magnetic resonance experimental findings. The new state has a smaller intracellular cavity that is likely to accommodate {beta}-arrestin better than G protein. Using contact map and inter-residue interaction energy calculations, we found the phosphorylated C-tail adheres to the cytosolic surface of the transmembrane domain of the receptor. Transfer entropy calculations show that the C-tail residues drive the correlated motions of TM residues, and the allosteric signal is relayed via several residues at the cytosolic surface. Our results also illustrate how the redistribution of inter-residue nonbonding interaction couples with the allosteric communication from the phosphorylated C-tail to the transmembrane. Atomistic insight into phosphorylation-induced {beta}-arrestin specific conformation is therapeutically important to design drugs with higher efficacy and fewer side effects. Our results therefore open novel opportunities to fine-tune {beta}-arrestin bias in GPCR signaling.

biophysics↗

bioRxiv2018

Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph Methylococcus capsulatus str. Bath

The increase in greenhouse gases with high global warming potential such as methane is a matter of concern and requires multifaceted efforts to reduce its emission and increase its mitigation from the environment. Microbes such as methanotrophs can assist in methane mitigation. To understand the metabolic capabilities of methanotrophs, a complete genome-scale metabolic model of an obligate methanotroph, Methylococcus capsulatus str. Bath was reconstructed. The model contains 535 genes, 898 reactions and 784 unique metabolites and is named iMC535. The predictive potential of the model was validated using previously-reported experimental data. The model predicted the Entner-Duodoroff (ED) pathway to be essential for the growth of this bacterium, whereas the Embden-Meyerhof-Parnas (EMP) pathway was found non-essential. The performance of the model was simulated on various carbon and nitrogen sources and found that M. capsulatus can grow on amino acids. The analysis of network topology of the model identified that six amino acids were in the top-ranked metabolic hubs. Using flux balance analysis (FBA), 29% of the metabolic genes were predicted to be essential, and 76 double knockout combinations involving 92 unique genes were predicted to be lethal. In conclusion, we have reconstructed a genome-scale metabolic model of a unique methanotroph Methylococcus capsulatus str. Bath. The model will serve as a knowledge-base for deeper understanding, as a platform for exploring the metabolic potential, and as a tool to engineer this bacterium for methane mitigation and industrial applications.

bioengineering↗