Biology subjects

Sahoo, A. R.

Publications and source records attributed to Sahoo, A. R..

10 recordsLinked to original sources

bioRxiv2026

Host membrane cholesterol constrains constitutive signaling of the oncogenic KSHV GPCR ORF74

Cholesterol is a major structural component of the plasma membrane and a key allosteric regulator of G protein-coupled receptors (GPCRs), yet its role in controlling constitutive receptor activity remains poorly understood. Virally encoded GPCRs provide an ideal system to address this question because many exhibit constitutive signaling that promotes viral persistence and pathogenesis, although how excessive receptor activation is restrained remains unknown. Here, we identify a previously unrecognized cholesterol-dependent allosteric mechanism by which the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) GPCR ORF74 constrains its constitutive activity. CryoEM structural analysis reveals a cholesterol-binding pocket formed by transmembrane helices 3, 5, and 6 that is present only in the inactive receptor. Cholesterol binding restrains the outward movement of transmembrane helix 6, stabilizes the inactive conformation, and suppresses spontaneous receptor activation, as supported by molecular dynamics simulations. Mechanistically, replacement of the canonical DRY motif with a non-canonical VRY motif exposes the conserved R1433.50 residue, creating a membrane-facing cholesterol-binding interface that couples membrane cholesterol to receptor conformational control. Consistent with this model, cellular cholesterol depletion enhances ORF74 signaling, whereas disruption of cholesterol binding impairs receptor stabilization. Together, our findings uncover a previously unrecognized mechanism by which a viral GPCR exploits host membrane cholesterol to regulate its constitutive activity, suggesting that persistent viruses optimize constitutive signaling by coupling receptor activity to host lipid-dependent allosteric regulation.

microbiology↗

bioRxiv2026

A model for PIP2/3 and Rnd1 effects on Plexin-B1 GAP activity on Rap1b GTPase derived from molecular dynamics simulations

Plexin-B1 is a transmembrane receptor that integrates signals from Rho-family and Ras-family (Rap1b) GTPases to regulate cellular processes. While ligand simulated activation of the receptor is largely understood, the role of membrane composition and GTPase allosteric effects on plexin structure, internal protein dynamics, and function is still to be elucidated. Here, we performed multi-replica, 1 s all-atom simulations of Plexin-B1-GTPase complexes on PIP2- and PIP3-containing membranes to investigate the effects of these two signaling lipids, as well as on the GTPases. We found that both Rap1b and Rnd1 stably associate with the membrane, with PIP2 promoting broader lipid engagement and stronger Rap1b-Plexin-B1 interactions, whereas PIP3 enhances Rnd1-Plexin contacts and induces a membrane proximal orientation of Plexins juxtamembrane helix and makes contacts with a previously discovered activation switch loop. Contact map and network analyses revealed lipid-dependent shifts in allosteric communication, with PIP2 favoring Rap1b-centric hotspots and PIP3 favoring Rnd1-centric pathways. These predictions allow us to suggest a model for plexin intracellular region activation where both the identity of phosphoinositides and GTPase context synergistically stabilize Plexin-B1 membrane engagement, alter structural dynamics, and allosteric networks. Thus, we propose that the membrane is an active modulator of plexin receptor signaling.

biophysics↗

bioRxiv2025

From Dimerization to Signaling: Molecular Dynamics Insights into EphA1 and EphA2 Isoform Specificity

Ephrin receptors (Ephs) are receptor tyrosine kinases that regulate cellular growth, differentiation, and motility. EphA2, often overexpressed in cancer, is notable for its ligand-independent activation, which drives pro-oncogenic signaling distinct from the canonical, ligand-dependent pathway that restricts cell movement. While ligand binding induces extracellular clustering, kinase activation depends on dimerization within the transmembrane (TM) region. EphA1 and EphA2 differ substantially in their function, likely due to differences in their TM, but more-so in their juxtamembrane (JM), and membrane-proximal fibronectin type III (FN1/FN2) domains. How these latter two regions modulate dimerization has not yet been investigated. To address this, we performed extensive coarse-grained (CG) simulations using Martini 3 in an anionic POPC/PS/PIP2 model of the plasma membrane. Both receptors formed stable TM dimers, though EphA1 favored a symmetric AXXXGXXXG-centered interface, whereas EphA2 favoured an alternate leucine zipper interface. All protein constructs sampled multiple configurations, reflecting substantial intrinsic variability. AlphaFold3 does not yield reliable predictions and cannot account of effects of the lipid bilayer. In the CG simulations, basic residues in the JM region remained membrane-bound, and the EphA2 FN domain displayed sustained PIP2 interactions, consistent with previous observations. Notably, constructs with the FN2 domain alone restricted TM association in both receptors, whereas inclusion of the second FN1 domain restored dimerization but produced receptor-specific extracellular interfaces. These differences arise from distinct FN1-FN2 linker flexibilities, a different level sequence conservation of EphA1 compared to several other Ephs and FN-domain membrane contacts, which together shape TM geometry and lipid engagement. Our results predict how TM and TM-proximal elements cooperatively tune dimerization in Eph receptors. This work offers a molecular rationale into the divergent activation behaviors of EphA1 and EphA2 and provides testable models relevant to cancer biology and Eph-driven signaling.

biophysics↗

bioRxiv2025

Tyrosine phosphorylation and the inhibitory C-terminal SAM domain moderately affect transient interactions in a EphA2 cytoplasmic fragment in solution: A combined experimental and molecular modeling study.

Eph receptors, the largest family of receptor tyrosine kinases (RTKs), orchestrate diverse developmental processes, including axon guidance, tissue patterning, and cell positioning, while also contributing to adult functions such as synaptogenesis and tissue homeostasis. Proteolytic cleavage of Eph receptors by neurodegeneration-associated proteases generates a membrane-detached intracellular region (ICR) fragment whose structural organization and signaling remain poorly understood. Here, we characterize a model for such a fragment from EphA2 and discover that the juxtamembrane region, the kinase domain, and the C-terminally linked sterile alpha motif (SAM) domain comprise a configurational ensemble of domain contacts and orientations which inhibit the intermolecular interactions and tyrosine cross-phosphorylation. Recombinant EphA2 ICR expressed in E. coli retains catalytic activity and undergoes autophosphorylation at multiple tyrosine residues which have previously been identified in eukaryotic systems. Phosphorylation weakens intramolecular interactions, as measured by microscale thermophoresis, indicating a phosphorylation-dependent remodeling of the ICR. While AlphaFold2-Multimer (AF2M) and Alphafold3 (AF3) predictions as well as available crystal structures provide only limited insight into these configurational states, coarse-grained molecular dynamics simulations using the Martini 3 force field reveal highly dynamic and predominantly transient interactions between the kinase and SAM domains that cluster at two discrete regions on the kinase surface. NMR spectroscopy of isotopically labeled EphA2 SAM suggests non-canonical intramolecular contacts that preserve the canonical SAM interaction interface for binding of partner proteins. Addition of the SHIP2 SAM domain promotes formation of the canonical EphA2 and SHIP2 SAM complex while also revealing previously unrecognized interactions of the kinase domain on the SHIP2 SAM domain. Collectively, these findings establish the EphA2 ICR as a dynamic, phosphorylation-responsive signaling module in which transient intramolecular interactions regulate accessibility of several interfaces for binding. Thus, the proteolytically released EphA2 intracellular fragment is likely to have distinct characteristics in cancer and neurodegenerative disease, compared to the membrane bound form in the full receptor.

biophysics↗

bioRxiv2025

Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier.

The opportunistic and neuroinvasive fungus, Cryptococcus neoformans (Cn), causes a life-threatening brain infection that despite treatment can cause long-term cognitive deficiencies. Studies have shown that Cn can infiltrate the central nervous system (CNS) through a transcellular route across the brain endothelium, however, the molecular process that drives brain endothelial cells to internalize Cn remains poorly defined. Here we examine the molecular interactions between fungal cells and the brain endothelium by utilizing a human 3D organoid model of the blood-brain barrier (BBB). We show that Cn exploits the process of macropinocytosis as the mechanism of endocytosis into brain endothelial cells by recruiting CD44 and EphA2 as a molecular complex. We identified two predicted binding sites on EphA2 for CD44, suggesting that the two structurally distinct regions may provide a molecular basis for cooperative signaling in brain endothelial cells that stimulate macropinocytosis as the mode of entry for Cn.

molecular biology↗

bioRxiv2025

Computation model predicts Rho GTPase function with the Plexin Transmembrane receptor GAP activity on Rap1b via dynamic allosteric changes

Plexin-semaphorin signaling regulates key processes such as cell migration, neuronal development, angiogenesis, and immune responses. Plexins stand out because they can directly bind with both Rho- and Ras-family small GTPases through their intracellular domains when these GTPases are in their active, GTP-bound states. This binding occurs via intracellular regions which include a Rho-GTPase Binding Domain (RBD) and a GTPase Activating Protein (GAP) segment. Studies have shown that Rho and Ras GTPases play vital roles in plexin signaling and activation. However, the structural dynamics of plexins and GTPases and how these conformational changes affect interactions when plexin is bound with both Ras and Rho-GTPases or bound to only one specific GTPase has remained unclear. In this study, we conducted molecular dynamics (MD) simulations on six distinct plexin-GTPase bound systems to investigate the differences in conformations and dynamics between Plexin-B1 and three GTPases: Rap1b, Rnd1, and Rac1. Our analysis revealed that dynamics with Rac1 are more altered, compared to Rnd1 depending on whether plexins GAP domain is bound or unbound to Rap1b. In addition, we further investigated alterations in network centralities and compared the network dynamics of the Plexin-GTPases complexes, focusing on the differences when Plexin is bound to both Ras (Rap1b) and Rho-GTPases (Rnd1/Rac1) versus when it is bound to only one GTPase. Our study revealed that Rnd1 exhibits stronger and more stable interactions with Plexin-B1 in the absence of Rap1b, while Rac1 shows fewer and less stable connections in comparison. These computational models have features that broadly agree with experimental results from hydrogen-deuterium exchange detected by mass spectrometry (HDX-MS). Such insights provide a better understanding of the molecular mechanisms underlying Plexin-GTPase interactions and the complexities of signaling mechanisms involving GTPases in general.

biophysics↗

bioRxiv2025

Cholesterol-Dependent Dimerization and Conformational Dynamics of EphA2 Receptors: Insights from Coarse-Grained and All-Atom Simulations

The EphA2 transmembrane receptor regulates cellular growth, differentiation, and motility, and its overexpression in various cancers makes it a potential biomarker for clinical cancer management. EphA2 signaling occurs through ligand-induced dimerization, where the transmembrane (TM) and juxtamembrane (JM) domains play crucial roles in stabilizing the dimer conformations and thereby facilitating signal transduction. Electrostatic interactions between basic JM residues and signaling lipids (PIP2 and PIP3) regulate phosphorylation while Cholesterols potential role in modulating EphA2 activation remains unclear. To investigate this, we modeled the TM-full JM peptide of EphA2 and employed coarse-grain and all-atom simulations to investigate its dimerization in cholesterol-rich and cholesterol-deficient membranes. Our findings reveal that cholesterol stabilizes specific TM dimers and TM-JM interactions with PIP2, highlighting the importance of membrane composition in EphA2 dimerization, oligomerization, and clustering. These insights enhance our understanding of lipid-mediated regulation of EphA2 and its implications in receptor signaling and cancer progression.

biophysics↗

bioRxiv2024

Cholesterol inhibits oncogenic EphA2 activation

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

biochemistry↗

bioRxiv2022

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anti-cancer therapeutics. Current therapeutics target EGFRs kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFRs ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

cell biology↗

bioRxiv2021

Predicting Transmembrane (TM) Domain Dimer Structures using Martini 3

Determination of the structure and dynamics of transmembrane (TM) regions of single-transmembrane receptors is key to understanding their mechanism of signal transduction across the plasma membrane. Although many studies have been performed on isolated soluble extra- and intracellular receptor domains in aqueous solutions, limited knowledge exists on the lipid embedded TM domain. In this study, we examine the assembly of configurations of receptor TM region dimers using the Martini 3 force field for coarse-grain (CG) molecular dynamics simulations. This recently published version of Martini has new bead types and sizes, which allows more accurate predictions of molecular interactions compared to the previous versions. At first glance our results with Martini 3 simulations show only a reasonable agreement with ab initio predictions using PREDDIMER (for TM domains only), AlphaFold2 Multimer and with available NMR derived structures for TM helix dimers. Specifically, 6 of 11 CG TM structures are similar to the NMR structures (within < 3.5 [A] mainchain RMSD) compared to 10 of 11 and 9 of 11 using PREDDIMER and Alphafold, respectively (7 structures of the latter are within 1.5 [A]) Surprisingly, AlphaFold2 predictions are more comparable with NMR structures when the database of 2001 (mainly composed of soluble proteins) instead of 2020 PDB structures are used. While there are some differences in the conditions used, the CG simulations primarily reveal that alternate configurations of the TM dimers that are sampled, which readily interconvert with a predominant population. The implications of these findings for our understanding of the signalling mechanism of TM receptors are discussed, including opportunities for the development of new pharmaceuticals, some of which are peptide based.

biophysics↗