Biology subjects

Vaidehi, N.

Publications and source records attributed to Vaidehi, N..

26 records · Page 2Linked to original sources

bioRxiv2023

An Ionic Lock Mediates Interdomain Interactions in G protein αi subunits and Determines Gαi Subtype Signaling Specificity

Highly homologous members of the Gi family, Gi1-3, have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gi1 effector, however, it is poorly activated by Gi2. Here, in a proteomic proximity labeling screen we observed a strong preference for Gi1 relative to Gi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gi1 into Gi2 or substitution of a single amino acid, A230 in Gi2 to the corresponding D in Gi1, largely rescues PRG activation and interactions with other Gi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gi2 relative to Gi1 and that mutation of A230s4h3.3 to D in Gi2 stabilizes HD-RLD interactions through formation of an ionic interaction with R145HD.11 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229s4h3.3 in Gi1 interacts with R144HD.11 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gi2 is unable to form the "ionic lock" to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gi1 or Gi2 by GPCRs.

pharmacology and toxicology↗

bioRxiv2022

Structural basis of odorant recognition by a human odorant receptor

Our sense of smell enables us to navigate a vast space of chemically diverse odor molecules. This task is accomplished by the combinatorial activation of approximately 400 olfactory G protein-coupled receptors (GPCRs) encoded in the human genome1-3. How odorants are recognized by olfactory receptors (ORs) remains mysterious. Here we provide mechanistic insight into how an odorant binds a human olfactory receptor. Using cryogenic electron microscopy (cryo-EM), we determined the structure of active human OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an olfactory receptor. Molecular dynamics simulations demonstrate propionate-induced conformational changes in extracellular loop 3 to activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate OR, providing insight into how this large family of GPCRs enables our olfactory sense.

biochemistry↗

bioRxiv2021

Decoding Angiotensin II Type 1 Receptor Allosteric Communication to Gq and β-arrestin

The allosteric communication between the agonist binding site and the G protein or {beta}-arrestin coupling sites in G protein-coupled receptors (GPCRs) play an important role in determining ligand efficacy towards these two signaling pathways and hence the ligand bias. Knowledge of the amino acid residue networks involved in the allosteric communication will aid understanding GPCR signaling and the design of biased ligands. Angiotensin II type I receptor (AT1R) is an ideal model GPCR to study the molecular basis of ligand bias as it has multiple {beta}-arrestin2 and Gq protein biased agonists as well as three-dimensional structures. Using Molecular Dynamics simulations, dynamic allostery analysis, and functional BRET assays, we identified a network of residues involved in allosteric communication from the angiotensin II binding site to the putative Gq coupling sites and another network to the {beta}-arrestin2 coupling sites, with 6 residues common to both pathways located in TM3, TM5 and TM6. Our findings unveil unique and common allosteric communication residue hubs for Gq and {beta}-arr2 coupling by AngII ligands and suggests that some of these residues can be targeted to design biased AT1R ligands. Finally, we show through analysis of the inter-residue distance distributions of the activation microswitches involved in class A GPCR activation for ten different agonists, that these microswitches behave like rheostats with different relative strengths of activation, which we speculate could modulate the relative efficacy of these agonists toward the two signaling pathways. Significance StatementKnowledge of the residues involved in allosteric communication from the ligand binding site to the G protein or {beta}-arrestin ({beta}-arr) coupling sites in GPCRs will aid in understanding their role in mediating ligand bias. Using a combination of molecular dynamics simulations and functional signaling assays we have identified a network of residues involved in allosteric communication from the Angiotensin II (Ang II) binding site to the Gq and {beta}-arr2 coupling sites in the Ang II type I receptor (AT1R). The residues in the allosteric network for {beta}-arr2 coupling are distributed across multiple structural regions of AT1R compared to Gq coupling. The residues in the two networks show conserved chemical properties across class A GPCRs, demonstrating the importance of allosteric communication in modulating ligand bias.

biophysics↗

bioRxiv2020

A Universal Allosteric Mechanism for G Protein Activation

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote GTP binding and the dissociation of G from the G{beta}{gamma} subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the G{beta}{gamma} dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation. HIGHLIGHTSO_LIReceptors promote GTP-GDP exchange and dissociation of G protein and {beta}{gamma} subunits C_LIO_LIWe find an allosteric network linking the {gamma} phosphate of GTP with release of G{beta}{gamma} C_LIO_LIThe network consists of a conserved Gly-Arg-Glu "activation triad" C_LIO_LITriad mutations prevent subunit dissociation, regardless of agonist or GTP binding C_LIO_LITriad mutations are responsible for human endocrine and neurological disorders C_LI

biochemistry↗

bioRxiv2020

Activation microswitches in GPCRs function as rheostats in cell membrane

Although multiple components of the cell membrane modulate the stability and activation of G protein coupled receptors (GPCRs), the activation mechanism comes from detergent studies, since it is challenging to study activation in multi-component lipid bilayer. Using the multi-scale molecular dynamics simulations(50s), our comparative study between cell membrane and detergents shows that: the changes in inter-residue distances, known as activation microswitches, show an ensemble of states in the extent of activation in cell membrane. We forward a rheostat model of GPCR activation rather than a binary switch model. Phosphatidylinositol bisphosphate (PIP2) and calcium ions, through a tug of war, maintain a balance between the GPCR stability and activity in cell membrane. Due to the lack of receptor stiffening effects by PIP2, detergents promote more transitions among conformational states than cell membrane. These findings connect the chemistry of cell membrane lipids to receptor activity useful to design detergents mimicking cell membrane.

biophysics↗

bioRxiv2019

Machine Learning for Prioritization of Thermostabilizing Mutations for G-protein Coupled Receptors

Although the three-dimensional structures of G-protein-coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence, structure and dynamics based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available dataset. A blind prediction for thermostable mutations of the Complement factor C5a Receptor retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.\n\nStatement Of SignifiganceG-protein-coupled receptors (GPCRs), the largest superfamily of membrane proteins play a vital role in cellular physiology and are targets to blockbuster drugs. Hence it is imperative to solve the three dimensional structures of GPCRs in various conformational states with different types of ligands bound. To reduce the experimental burden in identifying thermostable GPCR mutants, we report a computational framework using machine learning algorithms trained on thermostability data for 1231 mutants and features calculated from analysis of GPCR sequences, structure and dynamics to predict thermostable mutations ahead of experiments. This work represents a significant advancement in the development, validation and testing of a computational framework that can be extended to other class A GPCRs and helical membrane proteins.

biophysics↗

bioRxiv2019

Divergence from conserved residues underlies intracellular retention of mammalian odorant receptors

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in non-olfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here, we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing "consensus" odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations. Significance StatementOdor detection in mammals depends on the largest family of G protein-coupled receptors, the odorant receptors, which represent [~]2% of our protein-coding genes. The vast majority of odorant receptors are trapped within the cell when expressed in non-olfactory cells. The underlying causes of why odorant receptors cannot be functionally expressed in non-olfactory cells have remained enigmatic for over 20 years. Our study points to divergence from a consensus sequence as a key factor in a receptors inability to function in non-olfactory cells, which in turn, helps explain odorant receptors exceptional functional diversity and rapid evolution. We also show the success of protein engineering strategies for promoting odorant receptor cell surface expression.

cell biology↗

bioRxiv2019

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell Engaging antibodies) for cellular immunotherapy

Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. Here we show that an intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (db BiTES, 300 kDa). The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of the 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES maintain high in vivo tumor targeting as demonstrated by PET imaging, and redirect dbBiTE coated T-cells (1 microgram/10 million cells) to kill CEA+ target cells both in vitro, and in vivo in CEA transgenic mice.

cancer biology↗