Biology subjects

Biswas, K. H.

Publications and source records attributed to Biswas, K. H..

8 recordsLinked to original sources

bioRxiv2024

Differential Kinetics of SARS-CoV-2 Proteases Revealed by a Dual-Color, BRET-based Protease Biosensor, DuProSense

While SARS-CoV-2 Mpro and PLpro proteases are known to cleave polyproteins pp1a and pp1ab at multiple sites, these have not been comprehensively characterized in living cells. Here we engineered a two-color Bioluminescence Resonance Energy Transfer (BRET)-based, dual protease (DuProSense) biosensor platform relying on a proximity-dependent energy transfer from a luciferase donor to two spectrally separated fluorescent protein acceptors enabling simultaneous monitoring of processing of two cleavage sites in a single assay with high specificity. DuProSense revealed a similar Mpro and PLpro cleavage kinetics for their N-terminal autocleavage sites. Importantly, systematic characterization of various Mpro and PLpro cleavage sites using DuProSense revealed significant differences in cleavage rates and nirmatrelvir potency of Mpro cleavage sites but no correlation between the cleavage rates and nirmatrelvir IC50 values. Overall, our results provide deeper insights into the proteolytic processing of SARS-CoV-2 polyproteins and the dual color BRET platform will find wider applications in the future. HighlightsO_LIEngineered a two-color BRET-based, dual protease biosensor (DuProSense) C_LIO_LIDuProSense biosensor enabled simultaneous and specific monitoring of Mpro and PLpro activities C_LIO_LIDuProSense platform revealed differential cleavage kinetics of Mpro cleavage sites in live cells C_LIO_LIDuProSense platform revealed Mpro cleavage site-dependent nirmatrelvir potency in live cells C_LI

biophysics↗

bioRxiv2024

Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

Ligand binding to GAF domains regulates the activity of associated catalytic domains in a wide variety of proteins. For instance, cGMP binding to the GAFa domain of phosphodiesterase 5 (PDE5) activates the cGMP-hydrolyzing catalytic domain in the protein. However, the residues involved and the mechanism of GAF domain function are not entirely clear. Here, combining computational and experimental analysis, we show that two highly coevolving residues distant from the ligand binding site play a critical role in GAF domain allostery. Specifically, Statistical Coupling Analysis (SCA) of GAF domain sequences revealed the highest coevolution score for residues L267 and F295. Molecular dynamics (MD) simulations of both apo and holo forms of the wild type and mutant (L267A and F295A) PDE5 GAFa domains revealed significant alterations in structural dynamics and interaction with cGMP. Incorporation of the mutations in a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor, which reports a ligand-induced conformational change, revealed a change in the conformation of the GAF domain and an increase in the EC50 of cGMP-induced conformational change. Similar results were obtained regarding cGMP-induced conformational change in the full-length PDE5 and in the fluorescence of the GAF domain fluorescent protein, miRFP670nano3. Finally, structural analysis of conformers observed in MD simulations revealed a possible mechanism underlying the impact of mutations of these two coevolving residues in the PDE5 GAFa domain. Our results provide insight into the role of distant, coevolving residues in GAF domain allostery, and may aid in understanding evolution of allostery in proteins.

molecular biology↗

bioRxiv2023

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity.

biophysics↗

bioRxiv2023

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

Recently, a miniaturized variant of an artificial luciferase (ALuc), named picALuc, with a molecular weight of 13 kDa and thus, the smallest luciferase, was reported. While picALuc was found to be as active as the ALuc, questions remained on the structural organization and residue-residue interactions in the protein. Here, combining structural modeling, molecular dynamics (MD) simulations and mutational analysis, we show that the loss of a salt bridge interaction formed by Glu50 (E50) residue results in an increased enzymatic activity of picALuc. Specifically, we generated a model of picALuc using the available structure of the Gaussia luciferase (GLuc) and performed a 1 s long Gaussian accelerated molecular dynamics (GaMD) simulation which revealed a general compaction of the protein structure as well as residue level interactions in the protein. Given that picALuc contains a number of charged residues, we focused our attention to salt bridge interactions and decided to mutate E10, E50 and D94 that were found to form a fluctuating, stable or a new salt bridge interaction, respectively. Live cell assays showed an enhanced bioluminescence in cells expressing the E50A mutant picALuc while in vitro assays revealed an increased Vmax of the E50A mutant without affecting its thermal stability. Dynamic cross-correlation and principal component analyses of the GaMD simulation trajectories revealed altered collective dynamics in the protein, in which residue E50 contributed substantially. Finally, we developed a protein fragment complementation assay using picALuc that allows monitoring protein-protein interaction in live cells. We envisage that the brighter variant of picALuc and the protein fragment complementation assay reported here will find a general applicability in developing bioluminescence-based assays and the strategy developed here will pave the way for further engineering of brighter variants of picALuc.

biophysics↗

bioRxiv2022

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein {beta}2-microglobulin ({beta}2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8+ T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of {beta}2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2. Significance StatementViruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with {beta}2m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of {beta}2m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.

immunology↗

bioRxiv2022

A Genetically Encoded BRET-based SARS-CoV-2 Mpro Protease Activity Sensor

The SARS-CoV-2 main protease, Mpro, is critical for its replication and is an appealing target for designing anti-SARS-CoV-2 agents. In this regard, a number of assays have been developed based on its cleavage sequence preferences to monitor its activity. These include the usage of Fluorescence Resonance Energy Transfer (FRET)-based substrates in vitro and a FlipGFP reporter, one which fluoresces after Mpro-mediated cleavage, in live cells. Here, we have engineered a pair of genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based sensors for detecting SARS-CoV-2 Mpro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the Mpro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in Mpro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based Mpro sensor. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensor developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

biophysics↗

bioRxiv2021

Probe the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly one micron and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances receptor phosphorylation. Single molecule imaging clearly resolves increased molecular binding dwell time at EphA2 clusters for both Grb2:SOS and NCK:NWASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

biophysics↗

bioRxiv2021

Stable Interaction Of The UK B.1.1.7 lineage SARS-CoV-2 S1 Spike N501Y Mutant With ACE2 Revealed By Molecular Dynamics Simulation

Corona Virus Disease of 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has caused a massive health crisis across the globe, with some genetic variants gaining enhanced infectivity and competitive fitness, and thus significantly aggravating the global health concern. In this regard, the recent SARS-CoV-2 alpha variant, B.1.1.7 lineage, reported from the United Kingdom (UK), is of great significance in that it contains several mutations that increase its infection and transmission rates as evident from clinical reports. Specifically, the N501Y mutation in the SARS-CoV-2 spike S1 receptor binding domain (S1-RBD) has been shown to possess an increased affinity for ACE2, although the basis for this is not entirely clear yet. Here, we dissect the mechanism underlying the increased affinity using molecular dynamics (MD) simulations of the available ACE2-S1-RBD complex structure (6M0J) and show a prolonged and stable interfacial interaction of the N501Y mutant S1-RBD with ACE2 compared to the wild type S1-RBD. Additionally, we find that the N501Y mutant S1-RBD displays altered dynamics that likely aids in its enhanced interaction with ACE2. By elucidating a mechanistic basis for the increased affinity of the N501Y mutant S1-RBD for ACE2, we believe that the results presented here will aid in developing therapeutic strategies against SARS-CoV-2 including designing drugs targeting the ACE2-S1-RBD interaction. SignificanceThe emergence of the new SARS-CoV-2 lineage in the UK in December 2020 has further aggravated the COVID-19 pandemic due to an increased ability of the variant to infect human hosts, likely due to mutations in the viral S1 spike protein including the N501Y S1-RBD mutation that is located at the interface of S1-RBD and ACE2, the host cell receptor for SARS-CoV-2. Given its location at the interface, N501Y S1-RBD mutation can therefore potentially alter the interfacial interaction. Multiple, all-atom, explicit solvent MD simulations of the ACE2-S1-RBD complex carried here indicated a more stable interaction between the N501Y mutant S1-RBD and ACE2 through stabilizing interfacial interactions of residues at one end of the interface that are either sequentially or physically near the mutation site. These mechanistic details will aid in better understanding the mechanism by which the alpha variant has increased infectivity as well as in designing better therapeutics including ACE2-S1 spike protein inhibitors that will, in turn, help thwarting the current and future pandemic. HighlightsO_LIN501 in the wild type SARS-CoV-2 S1-RBD forms unsustained hydrogen bonds with residues in the ACE2, namely Y41 and K353 C_LIO_LIY501 in the N501Y mutant SARS-CoV-2 S1-RBD is not capable of forming substantial hydrogen bonds with ACE2 within the time span of the current simulation C_LIO_LIEvidence from analyzing the simulation results suggests that Y501 of S1-RBD could form other types of non-covalent interactions with ACE2, such as van der Waals interactions C_LIO_LIN501Y S1-RBD mutation stabilizes the position of interfacial residues neighboring to the mutation site, as well as other non-interfacial residues that are distant from the mutation site C_LIO_LIThese altered dynamics results in more stable interaction of S1-RBD with ACE2 which could be the main reason underlying the reported enhanced affinity of S1-RBD in the SARS-CoV-2 alpha variant (UK B.1.1.7 lineage) to ACE2 C_LI

biophysics↗