Biology subjects

Mondal, A.

Publications and source records attributed to Mondal, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Enhancing NADPH to Restore Redox Homeostasis and Lysosomal Function in G6PD-Deficient Microglia

Microglia, the residential immune cells in the central nervous system (CNS) exhibited in multiple states from resting to activated, play a significant role in neurogenesis, myelination, synaptic transmission, immune surveillance, and neuroinflammation. The aggravated inflammatory response by microglia triggers the generation of superoxide, which often causes the degeneration of neurons, leading to the development of Parkinsons and Alzheimers. The oxidative stress is key to many neurological disorders, often regulated by many genes. The terminal neutralization of oxidative stress is mediated by NADPH and glutathione. The cytosolic NADPH level is majorly contributed by a key enzyme called glucose-6-phosphate dehydrogenase (G6PD). The deficiency of G6PD is associated with hemolytic anemia, diabetes, cardiovascular, autoimmune, and neurological disorders. Our recent study indicated that G6PD deficiency decreases cytosolic NADPH levels and alters redox homeostasis and lysosomal function in microglia. Therefore, replenishment of NADPH is crucial for targeting G6PD deficiency-mediated microglial dysfunctions. This research promotes alternate metabolic pathways by targeting the expression and activity of enzymes such as isocitrate dehydrogenase 1 (IDH1) and malic enzyme 1 (ME1), which are responsible for cytoplasmic NADPH production. Metabolites like citric and malic acid supplementation promote NADPH production and reduce microglial oxidative stress. Additionally, using another group of small-molecule metabolites, such as dieckol and resveratrol, enhances the expression of IDH1 and ME1 enzymes to resolve potential tissue heterogeneity. Finally, combining these metabolites supplementation increased NADPH production and restored redox homeostasis and lysosomal function in G6PD deficient microglia, indicating their further use as potential therapeutics against G6PD deficiency disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/607918v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@5e69f2org.highwire.dtl.DTLVardef@7d9015org.highwire.dtl.DTLVardef@17e5500org.highwire.dtl.DTLVardef@fe9bc5_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Early 2022 breakthrough infection sera from India target the conserved cryptic class 5 epitope to counteract immune escape by SARS-CoV-2 variants

During the COVID-19 pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here we report an analysis of 164 serum samples isolated from breakthrough infection patients in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wildtype, Kappa, Delta, and Omicron BA.1 viruses. Three sera with the highest neutralization breadth and potency were selected for epitope mapping using charged scanning mutagenesis coupled to yeast surface display and Next Generation Sequencing. All of these sera primarily targeted the recently identified class 5 cryptic epitope along with the class 1 and class 4 epitopes, targeted to a lesser extent. The class 5 epitope is completely conserved across all SARS-CoV-2 variants and for the majority of the Sarbecoviruses. In line with these observations, a major fraction of the serum samples, including the selected three, show broad neutralizing activity against recent Omicron subvariants including XBB.1.5. This is in contrast with the results obtained with the sera from individuals receiving multiple doses of original and updated mRNA vaccines, where impaired neutralization of XBB and later variant of concerns was observed. Our study demonstrates that two doses of the ChAdOx vaccine in a highly exposed population was sufficient to drive substantial neutralization breadth against emerging and upcoming variants of concern, thus paves the path towards the development of future vaccine candidates. ImportanceWorldwide implementation of COVID19 vaccine and parallel emergence of newer SARS-CoV-2 variants has shaped the humoral immune response in a population specific manner. While characterization of this immune response is important for monitoring the disease progression at population level, it is also imperative for the development of effective countermeasures in the form of novel vaccines and therapeutics. India has implemented the worlds second largest COVID19 vaccination drive and also encountered large number of post vaccination "breakthrough" infections. From a cohort of breakthrough infection patients, we identified individuals whose sera showed broadly cross-reactive immunity against different SARS-CoV-2 variants. Interestingly, these sera primarily target a novel cryptic epitope which was not identified in previous population level studies conducted in western countries. This rare cryptic epitope remains conserved across all SARS-CoV-2 variants including the recently emerged ones and also for the SARS-like coronaviruses that may cause future outbreaks, thus representing a potential target for future vaccines.

molecular biology↗

bioRxiv2024

MELD-Adapt: On-the-Fly Belief Updating in Integrative Molecular Dynamics

Integrative structural biology synergizes experimental data with computational methods to elucidate the structures and interactions within biomolecules, a task that becomes critical in the absence of high-resolution structural data. A critical step in integrating the data, is knowing the expected accuracy or belief in the dataset. We previously showed that the Modeling Employing Limited Data (MELD) approach succeeds at predicting structures and finding the best interpretation of the data when the initial belief is equal or slightly lower than the real value. But, this initial belief is not known for many data sources. Here we introduce MELD-Adapt, designed to dynamically evaluate and infer the reliability of input data, thereby enhancing the accuracy of structure predictions. We demonstrate the utility of this method across different systems, particularly emphasizing its capability to correct initial assumptions and identify the correct fraction of data to produce reliable structural models. The approach is tested with two benchmark sets: the folding of twelve proteins with coarse physical insights and the binding of peptides with varying affinities to the Extrateterminal (ET) domain. Our findings also show the importance of how data is distributed in the system (locally clustered or globally distributed) and the subtle balance between data, beliefs, and force fields.

biophysics↗

bioRxiv2024

Protein Retrieval via Integrative Molecular Ensembles (PRIME) through extended similarity indices

Molecular dynamics (MD) simulations are ideally suited to describe conformational ensembles of biomolecules such as proteins and nucleic acids. Microsecond-long simulations are now routine, facilitated by the emergence of graphical processing units. Processing such ensembles on the basis of statistical mechanics can bring insights about different biologically relevant states, their representative structures, states, and even dynamics between states. Clustering, which groups objects based on structural similarity, is typically used to process ensembles, leading to different states, their populations, and the identification of representative structures. For some purposes, such as in protein structure prediction, we are interested in identifying the representative structure that is more similar to the native state of the protein. The traditional pipeline combines hierarchical clustering for clustering and selecting the cluster centroid as representative of the cluster. However, even when the first cluster represents the native basin, the centroid can be several angstroms away in RMSD from the native state - and many other structures inside this cluster could be better choices of representative structures, reducing the need for protein structure refinement. In this study, we developed a module--Protein Retrieval via Integrative Molecular Ensemble (PRIME), that consists of tools to determine the most prevalent states in an ensemble using extended continuous similarity. PRIME is integrated with our Molecular Dynamics Analysis with N -ary Clustering Ensembles (MDANCE) package and can be used as a post-processing tool for arbitrary clustering algorithms, compatible with several MD suites. PRIME was validated with ensembles of different protein and protein complex systems for their ability to reliably identify the most native-like state, which we compare to their experimental structure, and to the traditional approach. Systems were chosen to represent different degrees of difficulty such as folding processes and binding which require large conformational changes. PRIME predictions produced structures that when aligned to the experimental structure were better superposed (lower RMSD). A further benefit of PRIME is its linear scaling - rather than the traditional O(N 2) traditionally associated to comparisons of elements in a set.

biophysics↗

bioRxiv2024

Sifting Through the Noise: A Computational Pipeline for Accurate Prioritization of Protein- Protein Binding Candidates in High-Throughput Protein Libraries

Identifying the interactome for a protein of interest is challenging due to the large number of possible binders. High-throughput experimental approaches narrow down possible binding partners, but often include false positives. Furthermore, they provide no information about what the binding region is (e.g. the binding epitope). We introduce a novel computational pipeline based on an AlphaFold2 (AF) Competition Assay (AF-CBA) to identify proteins that bind a target of interest from a pull-down experiment, along with the binding epitope. Our focus is on proteins that bind the Extraterminal (ET) domain of Bromo and Extraterminal domain (BET) proteins, but we also introduce nine additional systems to show transferability to other peptide-protein systems. We describe a series of limitations to the methodology based on intrinsic deficiencies to AF and AF-CBA, to help users identify scenarios where the approach will be most useful. Given the speed and accuracy of the methodology, we expect it to be generally applicable to facilitate target selection for experimental verification starting from high-throughput protein libraries. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/576374v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b90071org.highwire.dtl.DTLVardef@1cc2f66org.highwire.dtl.DTLVardef@3c1109org.highwire.dtl.DTLVardef@1827353_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

G6PD deficiency mediated impairment of iNOS and lysosomal acidification affecting phagocytotic clearance in microglia in response to SARS-CoV-2

The glucose-6-phosphate dehydrogenase (G6PD) deficiency is X-linked and is the most common enzymatic deficiency disorder globally. It is a crucial enzyme for the pentose phosphate pathway and produces NADPH, which plays a vital role in the regulation of oxidative stress of many cell types. The deficiency of G6PD causes hemolytic anemia, diabetes, cardiovascular and neurological disorders. Notably, the patient with G6PD deficiency was severely affected by SARS-CoV-2 and showed prolonged COVID-19 symptoms, neurological impacts, and high mortality. However, the mechanism of COVID-19 severity in G6PD deficient patients is still ambiguous. Here, using a CRISPR-edited G6PD deficient human microglia cell culture model, we observed a significant reduction in NADPH and an increase in basal reactive oxygen species (ROS) in microglia. Interestingly, the deficiency of the G6PD-NAPDH axis impairs induced nitric oxide synthase (iNOS) mediated nitric oxide (NO) production which plays a fundamental role in inhibiting viral replication. Surprisingly, we also observed that the deficiency of the G6PD-NADPH axis reduced lysosomal acidification, which further abrogates the lysosomal clearance of viral particles. Thus, impairment of NO production and lysosomal acidification as well as redox dysregulation in G6PD deficient microglia altered innate immune response, promoting the severity of SARS-CoV-2 pathogenesis.

cell biology↗

bioRxiv2023

Long-term cold adaptation leads to increased thermal plasticity of male body size and thermal canalization of fecundity in populations of Drosophila melanogaster: an experimental evolution study

From laboratory populations of Drosophila melanogaster maintained at 25{degrees}C for many decades, new populations were derived and maintained at 17{degrees}C for over 60 generations. Fitness traits such as body size and fecundity of the cold and warm selected populations were quantified at both 17{degrees}C and 25{degrees}C. Flies from both selection regimes had similar body weight when grown at 25{degrees}C, but 17{degrees}C selected males had greater dry weight and less relative water content than 25{degrees}C selected males, when grown at 17{degrees}C. Cold selected males thus evolved greater thermal plasticity of body weight and density compared to controls. Fecundity data showed, 25{degrees}C selected females laid less eggs at 17{degrees}C compared to 25{degrees}C treatment, but 17{degrees}C selected females did not show significant difference in fecundity between 25{degrees}C and 17{degrees}C. Cold evolved flies thus showed thermal canalization of fecundity, possibly buffering the repressing effect of cold. Whether or not (i) greater body mass and density of cold evolved males, and (b) unrepressed fecundity of cold evolved females, both observed when reared at cold, are causally related, remain to be investigated. The study thus shows thermal selection can lead to evolution of greater thermal plasticity in a trait, while result in thermal canalization of another.

evolutionary biology↗

bioRxiv2023

Development of covalent chemogenetic K2P channel activators

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (Covalent Activation of TREK family K+ channels to cLAmp Membrane Potential) that leverages the discovery of a site in the K2P modulator pocket that reacts with electrophile-bearing derivatives of a TREK subfamily small molecule activator, ML335, to activate the channel irreversibly. We show that the CATKLAMP strategy can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a new means to alter K2P channel activity that should facilitate studies both molecular and systems level studies of K2P function and enable the search for new K2P modulators.

biophysics↗

bioRxiv2023

MGDrivE 3: A decoupled vector-human framework for epidemiological simulation of mosquito genetic control tools and their surveillance

Novel mosquito genetic control tools, such as CRISPR-based gene drives, hold great promise in reducing the global burden of vector-borne diseases. As these technologies advance through the research and development pipeline, there is a growing need for modeling frameworks incorporating increasing levels of entomological and epidemiological detail in order to address questions regarding logistics and biosafety. Epidemiological predictions are becoming increasingly relevant to the development of target product profiles and the design of field trials and interventions, while entomological surveillance is becoming increasingly important to regulation and biosafety. We present MGDrivE 3 (Mosquito Gene Drive Explorer 3), a new version of a previously-developed framework, MGDrivE 2, that investigates the spatial population dynamics of mosquito genetic control systems and their epidemiological implications. The new framework incorporates three major developments: i) a decoupled sampling algorithm allowing the vector portion of the MGDrivE framework to be paired with a more detailed epidemiological framework, ii) a version of the Imperial College London malaria transmission model, which incorporates age structure, various forms of immunity, and human and vector interventions, and iii) a surveillance module that tracks mosquitoes captured by traps throughout the simulation. Example MGDrivE 3 simulations are presented demonstrating the application of the framework to a CRISPR-based homing gene drive linked to dual disease-refractory genes and their potential to interrupt local malaria transmission. Simulations are also presented demonstrating surveillance of such a system by a network of mosquito traps. MGDrivE 3 is freely available as an open-source R package on CRAN (https://cran.r-project.org/package=MGDrivE2) (version 2.1.0), and extensive examples and vignettes are provided. We intend the software to aid in understanding of human health impacts and biosafety of mosquito genetic control tools, and continue to iterate per feedback from the genetic control community. Author summaryVector-borne diseases such as malaria cause massive morbidity and mortality throughout much of the world. Currently-available control measures, such as insecticide-based tools and antimalarial drugs, have limited impact and are waning in effectiveness, hence there is a need for novel tools to complement existing ones. Mosquito genetic control tools, such as gene drive systems and genetic versions of the sterile insect technique, offer a range of promising options, the development of which has greatly expanded since the advent of CRISPR-based gene-editing. Recently, we proposed MGDrivE 2 (Mosquito Gene Drive Explorer 2), which incorporates epidemiology into simulations of the dynamics of these systems in spatially-structured mosquito populations; however, that framework relied on simple model representations of vector-borne diseases. Here, we present MGDrivE 3, which decouples the vector portion of the model from the human portion, allowing the mosquito genetic control framework to be paired with more-detailed epidemiological frameworks. As an example, we implement the human transmission dynamics of the Imperial College London malaria model. We also incorporate a network of mosquito traps for surveillance. As genetic control technology edges closer towards field implementation, more detailed predictions of its epidemiological and biosafety implications are needed. We propose MGDrivE 3 to fulfill this role.

ecology↗

bioRxiv2023

Cost-effectiveness of Precision Guided SIT for Control of Anopheles gambiae in the Upper River Region, The Gambia.

Precision-guided sterile insect technique (pgSIT) is an extremely promising vector control intervention that can reduce and potentially eliminate the unacceptable malaria burden, particularly in sub-Saharan Africa. The deployment of pgSIT shows the greatest promise and does not have a near peer competitor in the fight to eradicate malaria. Here we explore the cost effectiveness of using this approach in Africa using mathematical modeling and economical analysis. Overall, we find that pgSIT represents a cost-effective and promising approach to A. gambiae control in The Gambia, with the potential to deliver significant economic and social benefits. SummaryPrecision-guided sterile insect technique (pgSIT) is an extremely promising vector control intervention that can reduce and potentially eliminate the unacceptable malaria burden, particularly in sub-Saharan Africa. pgSIT is a safe, innovative, and highly targeted approach to mosquito control that combines the principles of the sterile insect technique (SIT) with advanced state-of-the-art technologies of genetic engineering (Akbari et al. 2023; M. Li et al. 2021; Kandul, Liu, and Akbari 2021; Kandul et al. 2019, 2022). The technique involves inundative releases of genetically sterile male mosquitoes into the environment to mate with their wild counterparts, sterilizing them in the process. After multiple releases, this method can suppress, and even eradicate pest populations without the use of chemical pesticides or other less specific agents. The use of pgSIT has the potential to be a lasting, safe, cost-effective, sustainable, and environmentally friendly method to suppress a target species, acting as a chemical-free, species-specific insecticide. Before this work is considered for field application, however, we need to have a robust, data-driven modeling framework that will accurately predict the outcome of pgSIT release scenarios for malaria control, and we need an assessment of the costs and health benefits of implementing this technology in a region in Africa. This cost assessment evaluates the economic feasibility of both capacity building and establishing the infrastructure for a pgSIT facility in The Gambia to control the deadly malaria mosquito vector Anopheles gambiae. We focused on the Upper River region (URR) for three key reasons: (1) this area is known to have the highest per capita malaria rates in The Gambia (2) this region has more comprehensive historical and current data on malaria incidence and prevalence, malaria-associated healthcare and prevention costs, and human demographic data and (3) its location could feasibly demonstrate that a single pgSIT production facility can support phased suppression on a regional scale and then upon local extinction be repurposed to support vector suppression on a country or continent-wide scale. The pgSIT treatment of the URR ([~]2069 km2) is predicted to prevent approximately 230 deaths and about 48,000 sick days per year. This estimate is based on a model for localized extinction of A. gambiae, reaching full epidemiological impact by the third year. There are multiple ways to calculate the value of this intervention monetarily, from the value of statistical life (VSL) to quality adjusted life years (QALY), which ranges from 367 million to 880 million USD saved in the first ten years of the facility being active. Other metrics such as willingness-to-pay (WTP), estimates the willingness of locals to contribute to malaria prevention financially, and estimates based on gross domestic product (GDP) growth predict this model to save either 53 million or 551 million USD, respectively, in the first ten years of intervention. This model assumes localized extinction of A. gambiae by the second year of intervention with repeated releases to maintain extinction despite seasonal mosquito migration from beyond the treatment area. Localized extinction, however, is expected to have a year-to-year suppression effect making it easier to suppress mosquito populations with reduced sterile male releases in subsequent years. It is, therefore, likely an underestimate of the costs and benefits of pgSIT sterile male releases. In later years, the release of sterile males from this facility could be redirected to new areas to expand the suppression region. Additionally, this facility would have a significant off-season where the facility is not producing sterile male A. gambiae. This off-season could, therefore, produce sterile males to suppress A. gambiae populations in other regions with seasonal malaria during the off-season in The Gambiae. It could even be used to mass produce pgSIT sterile males for other mosquito species, such as the dengue vector, Aedes aegypti, which have eggs that can be stored for many months and consequently can be stockpiled to aid in the suppression of dengue outbreaks, which are also common in the area. Initially, however, the off-season can be used to build local capacity for genetically engineered (GE) mosquitos, which would consist of training staff, optimizing procedures, and troubleshooting any issues that arose during the higher production phases. The costs for this approach will vary as there are some unknowns and variability in the expected efficiencies of the facility, equipment, and procedures. In particular, mosquito survival rates and fecundity may vary more widely at scale and selection of the rearing equipment and protocols, and the mosquito sorting technology will dictate production levels and procedures. This variability is factored into the cost, and, therefore, a range of technologies and costs are considered. The expected start-up costs range from 6 to 11.5 million USD, which includes all necessary development, field trials, equipment, facility construction, staffing, and other establishment costs. The major upfront costs vary by facility size and capital equipment, much of which is dependent on mosquito rearing efficiencies. The annual estimated operational costs range from approximately 315,000 USD, depending on equipment selection and the size of the facility. Annually, this intervention costs less than 1 USD per person to suppress mosquitoes in the URR and prevent malaria transmission, which is about 6% of a malaria prevention WTP based on current interventions or 0.3-11% when using the VSL or QALY metrics, with most variation from the VSL calculation method. This intervention costs 15 to 124 USD (2022) to save one life-year and prevents malaria infections at 13 to 113 USD per case prevented, making this method competitive with many current interventions. WTP demonstrates that this facility could be made locally sustainable long term by local funding. Overall, the estimates of the capital and operational costs associated with the production of pgSIT sterile males, and the construction and management of the production facility indicate the cost savings associated with the annual decrease in morbidity and mortality (value of life) resulting from the use of pgSIT are significantly higher than the implementation costs. These estimates suggest that pgSIT represents a cost-effective and promising approach to A. gambiae control in The Gambia, with the potential to deliver significant economic and social benefits.

genetics↗

bioRxiv2023

Eliminating Malaria Vectors with Precision Guided Sterile Males

Controlling the principal African malaria vector, the mosquito Anopheles gambiae, is considered essential to curtail malaria transmission. However existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass-releases of non-biting, non-driving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here we develop precision guided Sterile Insect Technique (pgSIT) in the mosquito A. gambiae for inducible, programmed male-sterilization and female-elimination for wide scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male-sterility and >99.9% female-lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, for the first time enabling scalable SIT-like confinable suppression in the species.

bioengineering↗

bioRxiv2023

Connexin 43 mediated collective cell migration is independent of Golgi orientation

Cell migration is vital for multiple physiological functions and is involved in the metastatic dissemination of tumour cells in various cancers. For effective directional migration, cells often reorient their secretory traffic towards the leading edge by reorienting the Golgi apparatus. However, conflicting results on the positioning of the Golgi relative to the migrating cell raise questions about its regulation. Herein, we address the role of gap junction protein Connexin 43, which connects cells allowing the direct exchange of molecules and also controls the distribution of microtubules. We utilized HeLa WT (wild-type cells lacking Connexin 43) and HeLa 43 cells (stably expressing Connexin 43). We found that functional Connexin 43 channels affected Golgi morphology and reduced the ability of reorientation of Golgi (towards the leading edge) during cell migration. Although Connexin 43 also reduced migration velocity, the front displayed enhanced coherence in movement, implying an augmented collective nature of migration compared to HeLa WT cells lacking the cell-cell connectivity evidenced in HeLa 43. The increase in vimentin and basal actin in HeLa 43 shows that Connexin 43 expression alters the cytoskeleton. Non-invasive measurement of basal membrane height fluctuations revealed that HeLa 43 had a lower membrane tension and a more uniform intra- and inter-cellular distribution than HeLa WT, similar to their coherence in migration. We, therefore, propose that the reduced Golgi reorientation in HeLa 43 might be linked to lesser dependence on directed secretory trafficking, which is believed to be required to buffer the increasing tension caused by migration.

biophysics↗

bioRxiv2022

GENERALIST: An efficient generative model for protein sequence families

Generative models of protein sequence families are an important tool in the repertoire of protein scientists and engineers alike. However, state-of-the-art generative approaches face inference, accuracy, and overfitting-related obstacles when modeling moderately sized to large proteins and/or protein families with low sequence coverage. To that end, we present a simple to learn, tunable, and accurate generative model, GENERALIST: GENERAtive nonLInear tenSor-factorizaTion for protein sequences. Compared to state-of-the-art methods, GENERALIST accurately captures several high order summary statistics of amino acid covariation. GENERALIST also predicts conservative local optimal sequences which are likely to fold in stable 3D structure. Importantly, unlike other methods, the density of sequences in GENERALIST-modeled sequence ensembles closely resembles the corresponding natural ensembles. GENERALIST will be an important tool to study protein sequence variability.

biophysics↗

bioRxiv2022

Structural basis for CaVα2δ:gabapentin binding

Gabapentinoid drugs for pain and anxiety act on the CaV2{delta}-1 and CaV2{delta}-2 subunits of high-voltage activated calcium channels (CaV1s and CaV2s). Here, we present the cryo-EM structure of the gabapentin-bound brain and cardiac CaV1.2/CaV{beta}3/CaV2{delta}-1 channel. The data reveal a binding pocket in the CaV2{delta}-1 dCache1 domain that completely encapsulates gabapentin and define CaV2{delta} isoform sequence variations that explain gabapentin binding selectivity of CaV2{delta}-1 and CaV2{delta}-2.

biophysics↗

bioRxiv2022

EMC holdase:CaV1.2/CaVβ3 complex and CaV1.2 channel structures reveal CaV assembly and drug binding mechanisms

Voltage-gated ion channels (VGICs) comprise multiple structural units whose assembly is required for function1,2. There is scant structural understanding of how VGIC subunits assemble and whether chaperone proteins are required. High-voltage activated calcium channels (CaVs)3,4 are paradigmatic multi-subunit VGICs from electrically excitable tissues whose function and trafficking is powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaV13 and auxiliary CaV{beta}5, and CaV2{delta} subunits6,7. Here, we present cryo-EM structures of human brain and cardiac CaV1.2 bound with CaV{beta}3 to a chaperone, the endoplasmic reticulum membrane protein complex (EMC)8,9, and of the isolated CaV1.2/CaV{beta}3/CaV2{delta}-1 channel. These provide an unprecedented view of an EMC holdase:client complex and define EMC sites, the TM and Cyto docks, whose interaction with the client channel cause partial extraction of a pore subunit and splay open the CaV2{delta} interaction site. The structures further identify the CaV2{delta} binding site for gabapentinoid anti-pain and anti-anxiety drugs6, show that EMC and CaV2{delta} channel interactions are mutually exclusive, and indicate that EMC to CaV2{delta} handoff involves a Ca2+-dependent step and ordering of multiple CaV1.2 elements. Together, the structures unveil a CaV assembly intermediate and previously unknown EMC client binding sites that have broad implications for biogenesis of VGICs and other membrane proteins.

biophysics↗

bioRxiv2022

Targeting an evolutionarily conserved "E-L-L" motif in the spike protein to develop a small molecule fusion inhibitor against SARS-CoV-2

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved "E-L-L" motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing the post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this "E-L-L" motif resulting in effective inhibition of SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy towards blocking S protein-mediated viral entry, mutations within the "E-L-L" motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential "E-L-L" motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

biochemistry↗

bioRxiv2022

Structure determination of protein-peptide complexes from NMR chemical shift data using MELD.

Intrinsically disordered regions of proteins often mediate important protein-protein interactions. However, the folding upon binding nature of many polypeptide-protein interactions limits the ability of modeling tools to predict structures of such complexes. To address this problem, we have taken a tandem approach combining NMR chemical shift data and molecular simulations to determine structures of peptide-protein complexes. Here, we demonstrate this approach for polypeptide complexes formed with the extraterminal (ET) domain of bromo and extraterminal domain (BET) proteins, which exhibit a high degree of binding plasticity. This system is particularly challenging as the binding process includes allosteric changes across the ET receptor upon binding, and the polypeptide binding partners can form different conformations (e.g., helices and hairpins) in the complex. In a blind study, the new approach successfully modeled bound-state conformations and binding poses, using only backbone chemical shift data, in excellent agreement with experimentally-determined structures. The approach also predicts relative binding affinities of different peptides. This hybrid MELD-NMR approach provides a powerful new tool for structural analysis of protein-polypeptide complexes in the low NMR information content regime, which can be used successfully for flexible systems where one polypeptide binding partner folds upon complex formation.

biophysics↗

bioRxiv2021

A Comprehensive Roadmap Towards Generation of Influenza B Reporter Assay Using a Single DNA Polymerase Based Cloning of Reporter RNA Construct

Mini-genome reporter assay is a key tool for conducting RNA virus research. But, procedural complications and lack of adequate literature pose a major challenge in developing these assay systems. Here, we present a novel yet generic and simple cloning strategy for the construction of influenza B virus reporter RNA template and describe an extensive standardization of the reporter RNP/ polymerase activity assay for monitoring viral RNA synthesis in an infection free setting. Using this assay system, we, for the first time showed the effect of viral protein NS1 and host protein PKC-Delta upon influenza B virus RNA synthesis. Additionally, the assay system showed promising results in evaluating the efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for standardization of influenza virus mini-genome assay and an excellent tool for screening of host factors and antivirals in a fast, user-friendly and high throughput manner.

molecular biology↗