Biology subjects

Uday, A.

Publications and source records attributed to Uday, A..

3 recordsLinked to original sources

bioRxiv2025

Mechanism of substrate binding by the SARS-CoV-2 NiRAN domain and modulation of its activities during replication

The SARS-CoV-2 Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain initiates viral genome capping by RNAylating nsp9 with the 5'-pppA-end of the genome followed by GDP-dependent deRNAylation to form the core capped GpppA-genome. Additionally, it cycles nsp9 through NMPylation-deNMPylation to generate GpppN. It is unclear how the distinct substrates, 5'-pppA-RNA and NTP, are bound, and how NiRAN balances RNAylation versus NMPylation. Earlier models proposed a common base-up pose for both the substrates. Here, structure-guided mutagenesis and reconstitution assays show that 5'-pppA indeed binds base-up during RNAylation, revealing that nsp12-Asp711 confers adenine selectivity, whereas, NTP adopts a perpendicular base-out pose during NMPylation. NiRAN intrinsically favors NMPylation over RNAylation, but nsp13 NTPase activity flips this preference. RNAylation weakens when RdRp is RNA-bound or replicating it, suggesting that a trans-acting NiRAN associated with an RNA-free RdRp performs capping. These findings provide insights into the orchestration of NiRAN activities and potential druggable sites for anti-viral therapeutics.

molecular biology↗

bioRxiv2023

Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?

Calmodulin (CaM) is a ubiquitous calcium-sensitive messenger in eukaryotic cells. It was previously shown that CaM possesses an affinity for diverse lipid moieties, including those found on CaM-binding proteins. These facts together with our observation that CaM accumulates in membrane-rich protrusions of HeLa cells upon increased cytosolic calcium, motivated us to perform a systematic search for unmediated CaM interactions with model lipid membranes mimicking the cytosolic leaflet of plasma membranes. A range of experimental techniques and Molecular Dynamics simulations proves unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. Lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM-membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but its binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role - it forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer to increase exposure of hydrophobic acyl chains to the interacting proteins. Our findings suggest that upon increased intracellular calcium concentration, CaM and the cytosolic leaflet of cellular membranes can be functionally connected.

biophysics↗

bioRxiv2023

FRET-GP - A Local Measure of the Impact of Transmembrane Peptide on Lipids

Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of the protein with lipids. In this work we describe the FRET-GP method, which utilizes Forster resonance energy transfer (FRET) to specifically probe nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe, is quantified using generalized polarization (GP) to reports on lipid fluidity in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contribute to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GPFRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters, but the local effects were considerably stronger (GPFRET >> GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby increasing comprehensibility of the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/554931v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1d8fd57org.highwire.dtl.DTLVardef@e0713eorg.highwire.dtl.DTLVardef@12ab78aorg.highwire.dtl.DTLVardef@177dc30_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗