Biology subjects

Bahar, I.

Publications and source records attributed to Bahar, I..

At least 19 recordsLinked to original sources

bioRxiv2026

Environment-dependent landscapes of coding variant impacts on coproporphyrinogen oxidase

Hereditary coproporphyria (HCP) -- caused by variants in coproporphyrinogen oxidase (CPOX) -- can be diagnosed via genome sequencing. However, 74% of clinically-reported CPOX missense variants are classified as variants of uncertain significance (VUS) due to lack of evidence. CPOX variant classification is further complicated by environment-dependence: For example, the CPOX variant p.Asn272His (c.814A>C) is classified as benign yet has been associated with HCP-like symptoms in the context of mercury exposure. Here we measured the functional impact of nearly all possible CPOX amino acid substitutions in both the presence and absence of mercury. The resulting CPOX variant effect maps reflect known protein structure and mutational tolerance patterns while also offering new sequence-structure-function insights. Scores from this atlas not only distinguish pathogenic from benign variants but also identify mercury-dependent variant impacts, thus informing our clinical, structural, and functional understanding of CPOX deficiency and illustrating the value of systematic context-dependent multiplexed assays of genetic variant effects.

molecular biology↗

bioRxiv2026

Structure and Dynamics of a Long-Acting Insulin Analog in Hexameric and Dihexameric States

Elucidating the structure and dynamics of insulin and its analogs has been of broad interest, while presenting challenges due to the unique structural dynamics of insulin (composed itself of two multiply cross-linked peptides A and B) and its ability to assemble in a variety of oligomeric structures under physiological conditions. Here, we present two distinct X-ray crystallographic structures of the long-acting human insulin analog detemir (INSD) resolved in hexameric and dodecameric (or dihexameric) states at 2.85 [A] and 2.70 [A] resolution, respectively, using diffraction data collected under ambient temperature conditions. Characterization of the collective dynamics of these oligomers using the Gaussian Network Model (GNM) reveals several key features: (i) Oligomerization imparts high cooperativity in structural dynamics evidenced by dissection of the cross-correlations at various hierarchical levels; (ii) detemir monomers conformational flexibility is highly suppressed within oligomeric constructs, the effect being particularly strong in the dihexamer due to the asymmetric packing of the hexamers and the presence of myristoyl groups at B peptides termini whose interactions imparts further heterogeneities; and (iii) a number of key residues retain, however, their intrinsic dynamics, to be deployed upon release from the oligomers. We distinguish in particular residues serving as hinge sites that mediates the conformational dynamics of the asymmetric units (dimers) and monomers (I2A-V3A and Y19 A -C20A, and L11B-L15B and Y26B of the respective peptides A and B), or as anchors supporting structural stability (disulfide-bridge forming cysteines, plus selected residues such as L16A, G8B and R22B-F24B. Overall, this study provides a structural-dynamic framework for gaining new insights into the dynamics of long-acting analog INSD and helps identify actionable sites for modulating insulin (analogs) dynamics toward designing more effective therapeutics.

biophysics↗

bioRxiv2026

Generation of Pathway Signatures by Combining Normal Modes with Weighted Ensemble Simulations

Molecular dynamics simulations provide a "computational microscope" by which molecular phenomena can be studied at atomic resolution. However, such simulations are often expensive, usually due to a combination of system size and timescale. Various enhanced sampling methods have been proposed to overcome these challenges. Despite their effectiveness, many suffer from artifacts from energetic biases guiding the simulations, or lack of effective progress coordinates. Proteins normal modes uniquely defined by their 3D fold capture their intrinsic dynamics and could provide unbiased guidance, but how to combine these modes with molecular dynamics to generate continuous, energetically unbiased pathways has been challenging. In this study, we demonstrate that conformations generated along from normal modes using adaptive anisotropic network model provide a physical, intuitive, and generalizable progress coordinate for weighted ensemble simulations, providing a boost in efficiency and a means to generate pathways for any protein system without prior knowledge.

biophysics↗

bioRxiv2025

Small molecule activator of phosphatase PP2A remodels scaffold PR65 structural dynamics to promote holoenzyme assembly

Small molecule activators of protein phosphatase 2A (PP2A), hereafter SMAPs, have attracted substantial interest, for their potential to inhibit cancer cell proliferation by targeting PR65, the scaffold subunit of the PP2A heterotrimer. PR65 is a uniquely flexible and stable molecule composed of 15 tandem HEAT (Huntingtin, Elongation factor 3-PP2A-TOR1) repeats. We characterized the binding sites and interactions of two SMAPs ATUX-8385 and DT-061 with PR65 and evaluated effects on PR65 structural dynamics using docking and molecular dynamics simulations. We initiated SMAP-bound PR65 simulations starting from two binding sites: S1, determined by cryo-electron microscopy for DT-061 bound to PP2A, on the inner helices of the HEAT repeats 2 and 3 (2i and 3i); and S2, predicted by docking of ATUX-8385 onto PR65, on 4i and 5i and outer helices 5o and 6o consistent with footprinting experiments. S2 proved to be a stable site for both SMAPs when initiating the simulations at S2. However, neither DT-061 nor ATUX-8385 demonstrated stable binding to S1. DT-061 rapidly dissociated from S1 to settle instead at a neighboring site S4 overlapping with our previously identified S3 for PR65 in extended form, suggesting that binding to S1 may be a 2-step process: an initial binding to PR65 alone, either to S3/S4 or S2, followed by movement to S3/S4, and then an induced relocation to S1 upon complexation with the regulatory and catalytic subunits. Targeted in silico mutagenesis showed that mutations at S2 and S4 destabilized SMAP binding to the PR65 (subunit). Heterotrimeric PP2A simulations showed that S3 and S4 binding were not persistent upon complexation. Together, these results corroborate our findings. Furthermore, preferentially stabilized a relatively extended PR65 conformation that would accommodate, if not promote, the assembly of the catalytic and regulatory subunits to prompt the activation of the trimeric phosphatase.

biophysics↗

bioRxiv2025

Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.

biophysics↗

bioRxiv2025

Cryo-electron microscopy structures of a high-affinity zinc ABC transporter

Zn2+ is an essential micronutrient to all living organisms and plays a key role in various physiological functions. Microorganisms employ high-affinity Zn2+ ABC transporters to uptake zinc from the environment when it is scarce. However, the mechanism of zinc uptake and its regulation remain poorly understood. Here, we report the cryo-electron microscopy structure of the Zn2+ ABC transporter complex ZnuB-ZnuC from Escherichia Coli. The complex has two ZnuB subunits for transport and two ZnuC subunits for regulation. The ZnuB homodimer is in an outward-facing, closed conformation with a large hydrophilic cavity at the dimer interface. The ZnuC subunits contain an N-terminal ATP-binding cassette (ABC) domain (NBD) and a C-terminal zinc-sensing domain (ZSD). Zn2+ binding to the ZSD locks the transporter in a closed state regardless of nucleotide, whereas under low-Zn2+ conditions, ZSD C-terminal disorder permits ATP-driven zinc uptake. High-affinity zinc ABC transporters are ubiquitously utilized by pathogenic bacteria to evade host immune systems in competing for essential Zn2+. These findings highlight new pharmaceutical targets for disrupting Zn2+ homeostasis in antibiotic-resistant pathogens. One-sentence summaryHigh-affinity zinc ABC transporter structures show a self-regulating mechanism via a built-in zinc sensor.

biochemistry↗

bioRxiv2025

Substrate binding and channeling allosterically modulate the interactions within the AlkB-AlkG electron transfer complex

The transmembrane alkane monooxygenase AlkB and rubredoxin AlkG form an electron transfer complex that hydroxylates terminal alkanes to produce alcohols. The recent cryoEM study of Fontimonas thermophila AlkB-AlkG complex (FtAlkBG) revealed its architecture, including a dodecane (D12) substrate in the diiron active site. However, the molecular mechanism of action of FtAlkBG remains unknown. Here, we examined the FtAlkBG dynamics and interactions by multiscale computations, including molecular dynamics (MD) simulations, elastic network model (ENM), and QM/MM study of the oxidative mechanism at the catalytic site of AlkB. D12 remained stably bound to the catalytic site during MD runs, coordinated by hydrophobic residues I267, L263, L264, and I133, critical to substrate stabilization. MD simulations further showed that D12 could exit the catalytic site via a well-defined translocation pathway gated by I54, S49, and F46, through two intermediate states before nearly leaving the protein, and diffuse back to the active site. Several salt bridges and conserved AlkB-AlkG contacts were enhanced upon substrate binding. Substrate binding also mediated the association/dissociation events required for efficient electron transfer. It promoted tighter coupling between the diiron center in AlkB and the iron in AlkG. The allosteric effects relevant to enzymatic activity regulated by the substrate binding and channeling were further delineated by ENM analysis which confirmed a strong coupling spanning the site of entry from the membrane, the catalytic site and the AlkB-AlkG interface. Our study provides new insights into key sites that could be targeted for developing AlkB-variants with desirable alkane conversion functions.

molecular biology↗

bioRxiv2025

Accurate Identification and Mechanistic Evaluation of Pathogenic Missense Variants with Rhapsody-2

Understanding the effects of missense mutations or single amino acid variants (SAVs) on protein function is crucial for elucidating the molecular basis of diseases/disorders and designing rational therapies. We introduce here Rhapsody-2, a machine learning tool for discriminating pathogenic and neutral SAVs, significantly expanding on a precursor limited by the availability of structural data. With the advent of AlphaFold2 as a powerful tool for structure prediction, Rhapsody-2 is trained on a significantly expanded dataset of 117,525 SAVs corresponding to 12,094 human proteins reported in the ClinVar database. Adopting a broad set of descriptors composed of sequence evolutionary, structural, dynamic, and energetics features in the training algorithm, Rhapsody-2 achieved an AUROC of 0.94 in 10-fold cross-validation when all SAVs of a particular test protein (mutant) were excluded from the training set. Benchmarking against a variety of testing datasets demonstrated the high performance of Rhapsody-2. While sequence evolutionary descriptors play a dominant role in pathogenicity prediction, those based on structural dynamics provide a mechanistic interpretation. Notably, residues involved in allosteric communication, and those distinguished by pronounced fluctuations in the high frequency modes of motion or subject to spatial constraints in soft modes usually give rise to pathogenicity when mutated. Overall, Rhapsody-2 provides an efficient and transparent tool for accurately predicting the pathogenicity of SAVs and unraveling the mechanistic basis of the observed behavior, thus advancing our understanding of genotype-to-phenotype relations. Significance StatementUnderstanding the impact of single amino acid variants (SAVs) on protein function is pivotal for detecting and treating genetic disorders. We present Rhapsody-2, a machine learning tool that leverages AlphaFold2-predicted protein structures to accurately predict the pathogenicity of SAVs. By integrating sequence, structure, and dynamics properties, Rhapsody-2 achieves a high performance in distinguishing pathogenic variants across multiple benchmark datasets. Our tool not only provides a robust framework for predicting the impact of SAVs but also allows for mechanistic interpretability of pathogenicity, offering a valuable resource for understanding genotype-to-phenotype relationships toward assisting in precision medicine.

bioinformatics↗

bioRxiv2024

GraphVelo allows inference of multi-modal single cell velocities and molecular mechanisms

RNA velocities and generalizations emerge as powerful approaches for extracting time-resolved information from high-throughput snapshot single-cell data. Yet, several inherent limitations restrict applying the approaches to genes not suitable for RNA velocity inference due to complex transcriptional dynamics, low expression, or lacking splicing dynamics, or data of non-transcriptomic modality. Here, we present GraphVelo, a graph-based machine learning procedure that uses as input the RNA velocities inferred from existing methods and infers velocity vectors lying in the tangent space of the low-dimensional manifold formed by the single cell data. GraphVelo preserves vector magnitude and direction information during transformations across different data representations. Tests on synthetic and experimental single-cell data including viral-host interactome, multi-omics, and spatial genomics datasets demonstrate that GraphVelo, together with downstream generalized dynamo analyses, extends RNA velocities to multi-modal data and reveals quantitative nonlinear regulation relations between genes, virus and host cells, and different layers of gene regulation.

bioinformatics↗

bioRxiv2024

Modeling brain signaling as Markovian helps explain its structure-function relationship

Structure determines function. However, this universal theme in biology has been surprisingly difficult to observe in human brain neuroimaging data. Here, we link structure to function by hypothesizing that brain signals propagate as a Markovian process on an underlying structure. We focus on a metric called commute time: the average number of steps for a random walker to go from region A to B and then back to A. Commute times based on white matter tracts from diffusion MRI exhibit an average {+/-} standard deviation Spearman correlation of -0.26 {+/-} 0.08 with functional MRI connectivity data across 434 UK Biobank individuals and -0.24 {+/-} 0.06 across 400 HCP Young Adult brain scans. The correlation increases to -0.36 {+/-} 0.14 and to -0.32 {+/-} 0.12 when the principal contributions of both commute time and functional connectivity are compared for both datasets. The correlations are stronger by 33% compared to broadly used communication measures such as search information and communicability. The difference further widens to a factor of 5 when commute times are correlated to the principal mode of functional connectivity from its eigenvalue decomposition. Overall, the study points to the utility of commute time to account for the role of polysynaptic (indirect) connectivity underlying brain function by assuming that signals randomly traverse along the underlying brain structure.

neuroscience↗

bioRxiv2024

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease. One Sentence SummaryPulmonary arterial hypertension pathophenotypes arise from allele-specific NCOA7 regulation of lysosome function and inflammatory oxysterol generation, as demonstrated by genomic and metabolomic association studies coupled with genetic and pharmacologic mechanistic evidence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/582142v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@896f6forg.highwire.dtl.DTLVardef@f31c2eorg.highwire.dtl.DTLVardef@bf46dcorg.highwire.dtl.DTLVardef@1c55686_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. C_FIG

physiology↗

bioRxiv2023

Graph-Dynamo: Learning stochastic cellular state transition dynamics from single cell data

Modeling cellular processes in the framework of dynamical systems theories is a focused area in systems and mathematical biology, but a bottleneck to extend the efforts to genome-wide modeling is lack of quantitative data to constrain model parameters. With advances of single cell techniques, learning dynamical information from high throughput snapshot single cell data emerges as an exciting direction in single cell studies. Our previously developed dynamo framework reconstructs generally nonlinear genome-wide gene regulation relations from single cell expression state and either splicing- or metabolic labeling-based RNA velocity data. In this work, we first developed a graph-based machine learning procedure that imposes a mathematical constraint that the RNA velocity vectors lie in the tangent space of the low-dimensional manifold formed by the single cell expression data. Unlike a popular cosine correlation kernel used in literature, this tangent space projection (TSP) preserves the magnitude information of a vector when one transforms between different representations of the data manifold. Next, we formulated a data-driven graph Fokker-Planck (FPE) equation formalism that models the full cellular state transition dynamics as a convection-diffusion process on a data-formed graph network. The formalism is invariant under representation transformation and preserves the topological and dynamical properties of the system dynamics. Numerical tests on synthetic data and experimental scRNA-seq data demonstrate that the graph TSP/FPE formalism built from snapshot single cell data can recapitulate system dynamics. Significance StatementA cell is a dynamical system, which responds to extracellular and intracellular cues and changes its internal state. In practice the internal state of a cell is often characterized by its gene expression profile such as its transcriptome measured through destructive single cell techniques. Just like one can use Newtons equations to describe motions of the celestial bodies in the solar system, the state change of a cell in principle can also be described by a set of mathematical equations. Determining the form and associated parameters of such equations, however, is challenging. This work presented a general framework of reconstructing dynamical equations from snapshot single cell data.

biophysics↗

bioRxiv2023

Structural Mechanisms for VMAT2 inhibition by tetrabenazine

The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinsons disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2s importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here we report a 3.1 [A] resolution cryo-EM structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntingtons chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.

biochemistry↗

bioRxiv2023

Structural basis of CMKLR1 signaling induced by chemerin9

Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) or chemerin receptor 1, is a chemoattractant G protein-coupled receptor (GPCR) that responds to the adipokine chemerin and is highly expressed in innate immune cells, including macrophages and neutrophils. The signaling pathways of CMKLR1 can lead to both pro- and anti-inflammatory effects depending on the ligands and physiological contexts. To understand the molecular mechanisms of CMKLR1 signaling, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of the CMKLR1-Gi signaling complex with chemerin9, a nanopeptide agonist derived from chemerin, which induced complex phenotypic changes of macrophages in our assays. The cryo-EM structure, together with molecular dynamics simulations and mutagenesis studies, revealed the molecular basis of CMKLR1 signaling by elucidating the interactions at the ligand-binding pocket and the agonist-induced conformational changes. Our results are expected to facilitate the development of small molecule CMKLR1 agonists that mimic the action of chemerin9 to promote the resolution of inflammation.

biophysics↗

bioRxiv2022

Syntaxin1 Ser14 Phosphorylation is Required for Non-Vesicular Dopamine Release

Amphetamine (AMPH), a psychostimulant commonly prescribed for the treatment of neuropsychiatric and neurological disorders, has a high liability for abuse. The abuse and psychomotor stimulant properties of AMPH are primarily associated with its ability to increase dopamine (DA) neurotransmission. This increase is mediated, in large part, by non-vesicular DA release (DA efflux). DA efflux is the result of reversal of the DA transporter (DAT) promoted by AMPH. Syntaxin 1 (Stx1) is a SNARE protein that plays a pivotal role in vesicular release. Previously, we have shown that Stx1 also interacts with the distal DAT N-terminus, an event promoted by AMPH. Stx1 is phosphorylated at Ser14 by casein kinase II (CK2). Using Drosophila Melanogaster as an animal model, we show that this phosphorylation event is critical for non-vesicular DA release and regulates the expression of AMPH preference as well as the ability of AMPH to promote mating drive. We also show that reverse transport of DA mediated by DAT underlies these complex behaviors promoted by AMPH. Our molecular dynamics (MD) simulations of the phosphorylated DAT/Stx1 complex demonstrate that the phosphorylation state of these proteins plays a key role in allowing DAT to dwell in an efflux-willing state. This state also supports constitutive DA efflux (CDE), an event that occurs in the absence of AMPH. The DAT-Stx1 phosphorylated complex is characterized by the breakdown of two key salt bridges in DAT, K66-D345 and E428-R445, which are critical for the formation of the intracellular (IC) gate and for transport function. The breaking of these salt bridges leads to an opening and hydration of the DAT intracellular vestibule, allowing DA to bind from the cytosol, a mechanism that we hypothesize leads to CDE. We further determine the importance of Stx1 phosphorylation in CDE by pharmacologically inhibiting CK2 with CX-4945, a molecule currently in phase II clinical trials for cancer treatment. CX-4945 treatment prevented the expression of CDE in isolated Drosophila Melanogaster brains as well as behaviors associated with CDE. Thus, our results suggest that Stx1 phosphorylation is a possible pharmacological target for the treatment of AMPH abuse.

neuroscience↗

bioRxiv2022

Structural and Dynamic Effects of PTEN C-terminal Tail Phosphorylation

The phosphatase and tensin homolog deleted on chromosome ten (PTEN) tumor suppressor gene encodes a tightly regulated dual-specificity phosphatase that serves as the master regulator of PI3K/AKT/mTOR signaling. The carboxy-terminal tail (CTT) is key to regulation and harbors multiple phosphorylation sites (Ser/Thr residues 380-385). CTT phosphorylation suppresses the phosphatase activity by inducing a stable, closed conformation. However, little is known about the mechanisms of phosphorylation-induced CTT-deactivation dynamics. Using explicit solvent microsecond molecular dynamics simulations, we show that CTT phosphorylation leads to a partially collapsed conformation, which alters the secondary structure of PTEN and induces long-range conformational rearrangements that encompass the active site. The active site rearrangements prevent localization of PTEN to the membrane, precluding lipid phosphatase activity. Notably, we have identified phosphorylation-induced allosteric coupling between the interdomain region and a hydrophobic site neighboring the active site in the phosphatase domain. Collectively, the results provide a mechanistic understanding of CTT phosphorylation dynamics and reveal potential druggable allosteric sites in a previously believed clinically undruggable protein.

biophysics↗

bioRxiv2022

Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

immunology↗

bioRxiv2021

Bile acids gate dopamine transporter mediated currents

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Interestingly, sterols such as cholesterol interact with monoamine transporters (MAT), including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward circuitries in the brain. The present study explores interactions of the BA, obeticholic acid (OCA), with DAT and mechanistically defines the regulation of DAT activity via both electrophysiology and molecular modeling. We express murine DAT (mDAT) in Xenopus laevis oocytes and confirm that DA induces an inward current that reaches a steady-state at a negative membrane voltage. Next, we show that OCA triggers an inward current through DAT that is Na+ dependent and not regulated by intracellular calcium. OCA also inhibits the DAT-mediated Li+ leak current, a feature that parallels DA action and indicates direct binding to the transporter. Interestingly, OCA does not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, in regard to DA. The current induced by OCA is transient in nature, returning to baseline in the continued presence of the BA. To understand the molecular mechanism of how OCA affects DAT electrical activity, we performed docking simulations. These simulations revealed two potential binding sites that provide important insights into the potential functional relevance of the OCA-DAT interaction. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., 2014;Cheng and Bahar, 2015). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is facilitated through the residues DAT R445 and D436. This binding may stabilize the inward-facing open (IFo) state by preventing the re-formation of the IF gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

neuroscience↗