Biology subjects

Sodt, A.

Publications and source records attributed to Sodt, A..

4 recordsLinked to original sources

bioRxiv2026

The Influenza Hemagglutinin Cytoplasmic Tail Domain Interacts with Phosphatidylinositol 4,5-bisphosphate

During the influenza viral life cycle, the viral glycoprotein hemagglutinin (HA) mediates binding, entry, and fusion. Densely packed clusters of HA trimers at the plasma membrane are required to produce infectious virions; however, the mechanism of HA clustering is still unknown. We have shown previously that HA co-clusters with and modulates phosphatidylinositol 4,5-bisphosphate (PIP2) in host cell plasma membranes (PM). Here, we further characterize the relationship between HA and PIP2 using molecular dynamics simulations (MD) and fluorescence photoactivation localization microscopy (FPALM) to elucidate a mechanism of HA-PIP2 interaction. We found that the interaction occurs largely between the PIP2 head group and the cytoplasmic tail domain (CTD) of HA. Mutations of the CTD were made to alter charge (HARE, HARREQ), palmitoylation sites (HAMAY), or a combination thereof (HAREMAY, RREQMAY). MD showed that HARREQ and RREQMAY had the strongest effect on HA-PIP2 interactions through a depletion in the radial distribution function of PIP2 around HA at distances [≤]2.5 nm. FPALM revealed that HA cluster density at the PM was significantly reduced by CTD mutations, with the largest reduction occurring in mutants where the CTD charge and acylation were both altered (HAREMAY). HAREMAY clusters were also found to have larger circularities and perimeters, implying a structural change to the clusters. Mutations in the HA transmembrane domain also caused modest changes to the cluster properties of HA and its co-clustering with PIP2. FPALM showed PIP2 co-clustering with HA was also affected by HA mutations with more free PIP2 localized under HAREMAY clusters. A chemical model of simultaneous HA-PIP2 and PH-PIP2 binding enables interpretation of HA-PIP2 interactions and reveals quantitative differences between PIP2 binding by HA CTD mutants. We conclude that the mechanism of HA-PIP2 interaction consists of at least electrostatic and hydrophobic components. Our insights into the mechanism of HA-PIP2 interaction, and the prevalence of putative PIP2-interacting domains in a number of viral spike proteins suggest it may be fruitful to identify methods of disrupting interactions between phosphoinositides and viral proteins.

biophysics↗

bioRxiv2026

Multiple Sclerosis Drug Fingolimod Exhibits Antibacterial Activity through Bacterial Membrane Permeabilization

Although receptor-mediated mechanisms account for the therapeutic action of numerous FDA-approved drugs, emerging evidence suggests that many of these therapeutics have off-target antimicrobial activities. One example is fingolimod, an immunomodulator used to treat multiple sclerosis, that has been reported to have antimicrobial effects associated with membrane permeabilization. Yet the molecular mechanism by which fingolimod alters bacterial membranes remains unknown. As a cationic amphiphilic drug (CAD), fingolimod is comprised of both hydrophobic and positively charged regions that can enable membrane interactions. We show that fingolimod compromises membrane integrity in E. coli and P. aeruginosa, contributing to its antimicrobial activity. To determine how fingolimod disrupts membrane integrity, we used planar lipid bilayer electrophysiology with phospholipid compositions mimicking E. coli membranes. Using gramicidin A channels as molecular biosensors, we show that fingolimod alters both mechanical properties and surface charge of lipid bilayers at concentrations that have antimicrobial effects. At higher concentrations, fingolimod directly permeabilizes lipid bilayers, as revealed by conductance measurements and Bilayer Overtone Analysis. Molecular dynamics simulations correlate fingolimods preference for pore-favoring curvature with its strong interactions with lipids and trans-leaflet translocation. These findings establish a molecular mechanism for fingolimods off-target activity and provide a starting point for understanding how some CAD structures can drive membrane-specific effects that compromise bacterial physiology. ImportanceMany commonly prescribed drugs, beyond their primary action via receptor targets, modify cell membranes. A mechanistic understanding of how these drugs interact with bacterial membranes will have a significant impact on drug design and on the evaluation of potential side effects. Furthermore, the emerging need for new antimicrobial drugs has led to increased interest in drug repurposing. Elucidating the molecular mechanisms of these compounds interactions with bacterial membranes can ultimately provide critical insights into redesigning existing drugs as antimicrobials and into identifying unintended membrane-related effects that may contribute to their therapeutic or off-target effects.

microbiology↗

bioRxiv2025

Dissection of ergosterol metabolism reveals a pathway optimized for membrane phase separation

Sterols are among the most abundant lipids in eukaryotic cells, yet are synthesized through notoriously long metabolic pathways. It has been proposed that the molecular evolution of such pathways must have required each step to increase the capacity of its product to condense and order phospholipids. Here we carry out a systematic analysis of the ergosterol pathway that leverages the yeast vacuoles capacity to phase-separate as a predictive biophysical readout for each intermediate. In the post-synthetic steps specific to ergosterol biosynthesis, we find that successive modifications act to oscillate ordering capacity, settling on a level that supports phase separation while retaining fluidity of the resulting domains. Simulations carried out with each intermediate showed how conformers in the sterols alkyl tail are capable of modulating long-range ordering of phospholipids, which could underlie changes in phase behavior. Our results indicate that the complexity of sterol metabolism could have resulted from the need to balance lipid interactions required for membrane organization.

biophysics↗

bioRxiv2023

Point mutations in Arf1 reveal cooperative effects of the N-terminal region and myristate for GTPase-activating protein catalytic activity

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1*GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs). Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. We hypothesized that the myristate moiety associates with the N-terminal extension to alter its structure, thereby affecting its function. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

biochemistry↗