Biology subjects

Narayanan, K.

Publications and source records attributed to Narayanan, K..

4 recordsLinked to original sources

bioRxiv2024

Deep mutational scanning reveals sequence to function constraints for SWEET family transporters

Protein science is entering a transformative phase enabled by deep mutational scans that provide an unbiased view of the residue level interactions that mediate function. However, it has yet to be extensively used to characterize the mutational and evolutionary landscapes of plant proteins. Here, we apply the method to explore sequence-function relationships within the sugar transporter AtSWEET13. DMS results describe how mutational interrogation throughout different regions of the protein affects AtSWEET13 abundance and transport function. Our results identify novel transport-enhancing mutations that are validated using the FRET sensor assays. Extending DMS results to phylogenetic analyses reveal the role of transmembrane helix 4 (TM4) which makes the SWEET family transporters distinct from prokaryotic SemiSWEETs. We show that transmembrane helix 4 is intolerant to motif swapping with other clade-specific SWEET TM4 compositions, despite accommodating single point-mutations towards aromatic and charged polar amino acids. We further show that the transfer learning approaches based on physics and ML based In silico variant prediction tools have limited utility for engineering plant proteins as they were unable to reproduce our experimental results. We conclude that DMS can produce datasets which, when combined with the right predictive computational frameworks, can direct plant engineering efforts through derivative phenotype selection and evolutionary insights.

plant biology↗

bioRxiv2023

The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins

Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and functions as a cell entry receptor for several henipaviruses including Nipah virus (NiV), a pathogenic zoonotic virus with pandemic potential. To understand the sequence basis of promiscuity for EFNB2 binding to the attachment glycoprotein of NiV (NiV-G) and Eph receptors, we performed deep mutagenesis on EFNB2 to identify mutations that enhance binding to NiV-G over EphB2, one of the highest affinity Eph receptors. The mutations highlight how different EFNB2 conformations are selected by NiV-G versus EphB2. Specificity mutations are enriched at the base of the G-H binding loop of EFNB2, especially surrounding a phenylalanine hinge upon which the G-H loop pivots, and at a phenylalanine hook that rotates away from the EFNB2 core to engage Eph receptors. One EFNB2 mutant, D62Q, possesses pan-specificity to the attachment glycoproteins of closely related henipaviruses and has markedly diminished binding to the six Eph receptors. However, EFNB2-D62Q has high residual binding to EphB3 and EphB4. A second deep mutational scan of EFNB2 identified combinatorial mutations to further enhance specificity to NiV-G. A triple mutant of soluble EFNB2, D62Q-Q130L-V167L, has minimal binding to Eph receptors but maintains binding, albeit reduced, to NiV-G. Soluble EFNB2 decoy receptors carrying the specificity mutations were potent neutralizers of chimeric henipaviruses. These findings demonstrate how specific residue changes at the shared binding interface of a promiscuous ligand (EFNB2) can influence selectivity for multiple receptors, and may also offer insight towards the development of henipavirus therapeutics and diagnostics. IMPORTANCEEphrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.

microbiology↗

bioRxiv2022

Parsing the role of NSP1 in SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo, in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.

microbiology↗

bioRxiv2020

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures. Article SummaryScientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.

microbiology↗