Biology subjects

Venkatramani, R.

Publications and source records attributed to Venkatramani, R..

5 recordsLinked to original sources

bioRxiv2026

High Resolution Solvated Models Reveal Mechanisms of Allosteric Activation of mTORC1 by RHEB

The mechanistic target of rapamycin complex 1 (mTORC1) is a [~]1.2 MDa dimeric assembly comprising mTOR, mLST8, and RAPTOR that integrates nutrient, energy, and stress signals to regulate cell growth. While Cryo-EM structures have provided insights into allosteric activation of the complex by the small GTPase RHEB, their limited resolution has constrained a full mechanistic understanding. Here, we combine deep learning-based AlphaFold-3 models with Molecular Dynamics Flexible Fitting and simulations to generate refined, atomistic solvated models of mTORC1{+/-}ATP{+/-}RHEB. Simulations reveal a global remodelling of the complex by RHEB, which strengthens mTOR-RAPTOR interactions while weakening mTOR-mLST8 contacts. These drive the reorganization of Kinase N- and C-lobes into a catalytically competent state in which ATP binding is stabilized enthalpically with improved Mg{superscript 2} coordination. Our studies present structural, energetic and dynamic changes induced by RHEB binding which collectively cause allosteric preorganization of mTORC1 for catalysis prior to substrate binding.

bioinformatics↗

bioRxiv2024

mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

In metazoans, epithelia perform functions of absorption, diffusion, and secretion. The actin-based apical projections on the epithelial cells, contribute to these functions and are formed via cell-autonomous mechanisms that control cell polarity, intracellular transport, and the cytoskeleton. However, the cues that function upstream of these cell-autonomous regulators remain poorly known. Using microridges on zebrafish epithelial cells as a paradigm, we show that mTOR, a metabolic sensor, regulates the formation of apical projections. Mechanistically, mTORC1 controls the RhoA-ROCK activity via S6K1 to prevent the overactivation of Non-muscle Myosin II (NMII) to restrict microridge elongation. Furthermore, genetic, biochemical, and molecular dynamics simulation analyses reveal that mTORC2 regulates microridge pattern by modulating the activity of aPKC via its differential phosphorylation at two conserved sites. We propose that mTOR integrates the developmental and/or metabolic status of epithelial cells with cell autonomously acting RhoA and aPKC to regulate tissue wide formation of apical projections.

cell biology↗

bioRxiv2024

A Cell-Permeable Fluorescent Probe Reveals Temporally Diverse PI(4,5)P2 Dynamics Evoked by Distinct GPCR Agonists in Neurons

Lipids, key constituents of cell-membranes, are the first responders to cell signals. At the crux of spatiotemporal dynamics of lipid-signaling response are phosphoinositides. Indeed, phosphoinositides like phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), present in the inner-leaflet of eukaryotic cell-membranes, form the link between signal reception and downstream signal-transmission. In this backdrop, reversible fluorescent probes that can track live PI(4,5)P2 dynamics on a seconds time-scale will afford key insights into lipid-mediated signaling. However, realizing cell-permeable PI(4,5)P2-selective sensors for imaging dynamics remains a challenge due to the presence of structurally similar lipids and low levels of PI(4,5)P2. We report a computationally-designed, rapid-response, reversible, photo-stable, fluorescent sensor that permeates living cells, neurons, and a multicellular organism within few min of direct incubation and distinctly visualizes PI(4,5)P2 pools. We used the sensor to interrogate the role of PI(4,5)P2 in driving heterogeneity of signaling responses and contrasting behavioral effects that ensue upon binding of distinct ligands to the same G protein-coupled receptor. Specifically, we asked whether probing PI(4,5)P2 dynamics using our novel sensor could uncover the earliest of signaling differences evoked by hallucinogenic versus non-hallucinogenic ligands at the serotonin2A (5-HT2A) receptor. Our results reveal that a hallucinogenic ligand at the 5-HT2A receptor leads to a slower rate of PI(4,5)P2-depletion when compared to a non-hallucinogenic ligand, within the initial seconds of ligand addition, but has a sustained effect. The ability of our designer chemical probe in timing early seconds-minute timescale lipid-dynamics in living cells opens avenues for tracking early time-point molecular events in neuronal response to chemical and physical stimuli.

biochemistry↗

bioRxiv2024

ATP-Binding Free Energy Simulations Reveal an Allosteric Link Between the Enzyme Active Site and Multiple Functional Protein-Protein Interaction Interfaces in Cyclin Dependent Kinase-1

The ATP dependent phosphorylation activity of Cyclin Dependent Kinase 1 (CDK1), an essential enzyme for cell cycle progression, is intrinsically dependent upon interactions with Cyclin-B, substrate, and Cks proteins. A recent joint experimental-computational study from our group showed intriguingly that acetylation at the active site abrogated the binding of CDK with Cyclin-B. These results posit the possibility of a bi-directional communication between the catalytic site and the protein-protein interface(s). Now, we present evidence for a general allosteric link between the CDK1 active site and all three of its protein-protein interaction (PPI) interfaces through atomistic molecular dynamics simulations (MD). Specifically, we examined ATP binding free energies to CDK1 in native non-acetylated (K33wt) and acetylated (K33Ac) forms as well as in two mutant forms of CDK1, the acetyl-mimic K33Q and the acetyl-null K33R which are accessible in-vitro. We find, in agreement with experiments, that ATP binding is energetically more favorable in K33wt relative to all other states wherein the active site lysine is perturbed (K33Ac, K33Q, and K33R). We also develop an entropy decomposition scheme which reveals, in addition to expected local changes in entropy, significant non-local entropy responses to ATP binding/perturbation of K33 from the -helix, activation loop (A-loop), and the -H loop segments in CDK1 which interface with Cyclin-B, substrate, and Cks proteins. Statistical analyses over a large set of MD trajectories reveal that while the local and non-local entropic responses to active site perturbations are on average correlated with dynamical changes in the associated protein segments, such correlations may be lost in about 9-48 % of the dataset depending on the segment. Besides, proving the bi-directional communication between the active site and CDK1:Cyclin-B interface, our study provides new insights into the regulation of ATP binding by multiple PPI interfaces in CDK1.

biophysics↗

bioRxiv2018

Deacetylation of catalytic lysine in CDK1 is essential for Cyclin-B binding and cell cycle

Cyclin-dependent-kinases (CDKs) are essential for cell cycle progression. While dependence of CDK activity on Cyclin levels is established, molecular mechanisms that regulate their binding are less studied. Here, we show that CDKl:Cyclin-B interactions are regulated by acetylation, which was hitherto unknown. We demonstrate that cell cycle dependent acetylation of the evolutionarily conserved catalytic lysine in CDK1 or eliminating its charge state abrogates Cyclin-B binding. Opposing activities of SIRT1 and P300 regulate acetylation, which marks a reserved pool of CDK1. Our high resolution structural analyses into the formation of kinase competent CDK1: Cyclin-B complex have unveiled long-range effects of catalytic lysine in configuring the CDK1 interface for Cyclin-B binding. Cells expressing acetylation mimic mutant of Cdc2 in yeast are arrested in G2 and fail to divide. Thus, by illustrating cell cycle dependent deacetylation as a determinant of CDK1:Cyclin-B interaction, our results redefine the current model of CDK1 activation and cell cycle progression.

cell biology↗