Alexander J. Sodt

Proteins and lipids of a biological cell work together to reshape the membranes in which they reside. Membrane reshaping is necessary for vesicular traffic and maintenance of organelle shape and structure. Curvature is the fundamental mathematical description of the local shape of a surface. Through the well-established Helfrich/Canham/Evans model of bilayer shape, curvature sensing and induction are both consequences of a protein’s spontaneous curvature , the local curvature at which the Helfrich model energy is a minimum. This paper describes a straightforward approach for estimating the strength and sign of a bilayer-associated molecule’s curvature induction from molecular simulation: Correlating the molecule’s lateral position with curvature on thermally-fluctuating bilayer modes. Example demonstrations range from a large multi-pass coarse-grained transmembrane protein to an all-atom amphipathic helical segment. Quantification of these systems is placed in the context of experiments seeking the same strength and sign of curvature induction.

Viral entry requires remodeling the host membrane with a short fusion peptide (FP) that anchors and deforms the target lipid bilayer. However, the contribution of the FP throughout the full fusion pathway remains unknown. Here, all-atom molecular dynamics simulations define the membrane-deformation mechanisms associated with two distinct insertion configurations of membrane-bound human parainfluenza virus type 3 (HPIV3) FP. Molecular simulations quantifying the linear response of curvature to the peptide are complemented with analyses of lipid organization and FP-lipid interactions to define the underlying membrane-deformation mechanisms. The surface-parallel FP configuration generates positive membrane curvature through deformation of the proximal leaflet, consistent with the classical hydrophobic insertion (wedging) mechanism. In contrast, the transmembrane FP configuration generates curvature through localized deformation of the distal leaflet, a distinct mechanism that would strongly influence later stages of fusion. Cryo-electron tomography shows that target membrane curvature at virus-host contact sites is consistent with the molecular dynamics prediction. In the absence of F-protein activation and FP insertion, the cryo-ET-determined curvature distribution shifts toward negative values, thereby isolating the role of the fusion protein in inducing early-stage curvature that enables the host and virus membranes to approach one another. Modeling key mutational changes isolates how aromatic and electrostatic interactions act together to promote curvature generation. Taken together, these findings establish FP insertion configuration as a determinant of productive membrane curvature across the full fusion pathway.

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.

The structure of phospholipid headgroups and chains are well-established drivers of membrane elastic properties, but functions for different chemistries that join these moieties together are poorly understood. While canonical phospholipids feature ester linkages, alkyl ether- and plasmenyl-linked species emerged in prokaryotes, are highly abundant in metazoans, and have been implicated in neurodegeneration and aging. Ether phospholipid chemistry, and plasmenyl linkages in particular, arose independently several times in evolution, suggesting conserved functions in the structure of cell membranes. Here we combine experiments and molecular simulations to determine how backbone linkage chemistry modulates membrane mechanics. We find that ether linkages additively promote negative intrinsic curvature, destabilizing bilayers and enhancing membrane fusion. They also decouple membrane stiffness from viscosity, softening membranes while maintaining packing in the hydrophobic core. The plasmenyl linkage uniquely stabilizes the inverted hexagonal phase by lowering the energetic cost of chain stretching, providing a rationale for the evolution of its biosynthesis. These results explain the fusogenicity of ether lipids and show how they regulate membrane topology through multiple physical mechanisms. We propose that phospholipid backbone linkage chemistry constitutes a modular control element for membrane mechanics and topology. Significance statement: The structure and dynamics of cell membranes can be sensitive to small chemical changes in their phospholipid building blocks. Phospholipids with ether bonds connecting their glycerol backbone and hydrocarbon chains have long been proposed to impart chemical stability to thermo- and acidophile microbial membranes, but have more recently been identified as major components of mammalian tissues. We show that ether linkages promote membrane dynamics through imposition of a canonical molecular geometry and by decoupling of bending stiffness from chain ordering. Plasmalogen lipids, in which the ether linkage is modified with a vicinal double bond, further promote non-lamellar topologies through a chain-stretching mechanism. These biophysical features suggest a basis for the repeated emergence of ether phospholipids in evolution and their observed functions in membrane trafficking. The thermodynamic and structural bases of plasmalogen function are especially notable as these lipids have been increasingly implicated in neurodegenerative and cardiovascular disease.

Rod and cone photoreceptors are sensory neurons required for the first steps of image-forming vision. Mutations in PRPH2 damage photoreceptor viability and produce a broad range of inherited retinal degenerations (IRDs) for which no treatments are available. The gene product, PRPH2 (peripherin-2), is an integral membrane tetraspanin that is required for the biogenesis and normal structure of photoreceptor outer segment (OS) disks. Peripherin-2 and its homologous partner Rom1 (rom1) self-assemble into non-covalent dimers that organize into three parallel belts that encircle each disk, forming a supramolecular scaffold that imposes extreme curvature on the disk rim. The mechanism by which curvature is generated and maintained is not yet understood. Here, we report the development of blue native (BN)-PAGE and molecular dynamics (MD) simulation approaches that suggest these proteins form an interaction web characterized by multiple interaction modes. BN-PAGE reveals that disk rim belts consist of a mixture of free peripherin-2 dimers and variously-sized chains of disulfide-linked dimers. MD simulations find that although free dimers can generate spontaneous curvature in a model membrane, dimers that are disulfide-linked into linear chains generate highly anisotropic curvature - a key hallmark of normal disk rims. The MD findings further suggest that although individual belts likely provide sufficient thermodynamic driving force to generate extreme curvature, non-covalent inter -belt interactions may also constrain rim diameter. The new findings advance understanding of peripherin-2 and rom1 structure and function in health and disease, and simultaneously provide promising new strategies for molecular phenotyping these clinically important proteins.

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 fingolimod’s preference for pore-favoring curvature with its strong interactions with lipids and trans-leaflet translocation. These findings establish a molecular mechanism for fingolimod’s off-target activity and provide a starting point for understanding how some CAD structures can drive membrane-specific effects that compromise bacterial physiology. Importance: Many 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.

GTPase-activating proteins are important regulators of small GTPases; among these, ASAP1 stimulates GTP hydrolysis on Arf1 and is implicated in cancer progression. ASAP1 contains a Pleckstrin Homology (PH) domain essential for maximum Arf·GTP hydrolysis. The prevailing view of PH domains is that they regulate proteins through passive mechanisms like membrane recruitment. In sharp contrast, we show that the PH domain of ASAP1 actively contributes to Arf1 GTP hydrolysis. By combining NMR, molecular dynamics simulations, kinetic assays, and mutational analysis, we find that the PH domain binds Arf·GTP at the membrane, to establish an active state primed for GTP hydrolysis. We identify key residues on the PH domain and Arf that drive this allosteric mechanism, which mathematical modeling shows contributes as much to GTPase activation as membrane recruitment. The finding that PH domains directly modulate small GTPases has broad implications for the Ras and Rho oncoprotein families.

Magnesium (Mg²⁺) uptake systems are present in all domains of life, consistent with the vital role of this ion. P-type ATPase Mg²⁺ importers are required for bacterial growth when Mg²⁺ is limiting or during pathogenesis. However, insights into their mechanisms of action are missing. Here we solved the cryo-EM structure of the Mg²⁺ transporter MgtA from Escherichia coli. We obtained high-resolution structures of both homodimeric (2.9 Å) and monomeric (3.6 Å) forms. The dimer structure is formed by multiple contacts between residues in adjacent soluble N and P subdomains. Our structures revealed an ion, assigned as Mg²⁺, in the transmembrane segment. Moreover, we detected two cytoplasmic ion-binding sites and determined the structure of the N-terminal tail. Sequence conservation, mutagenesis and ATPase assays indicate dimerization, the ion-binding sites and the N-terminal tail facilitate cation transport or serve regulatory roles.

GTPase-activating proteins (GAPs) are important regulators of small GTPases with a wide range of cellular functions; among these, ASAP1 stimulates GTP hydrolysis on Arf1 and is implicated in cancer progression. ASAP1 contains a Pleckstrin Homology (PH) domain critical for maximum hydrolysis of GTP bound to the small GTPase Arf. The prevailing view of PH domains is that they regulate proteins by passive mechanisms such as recruitment to the membrane surface. In sharp contrast to this model of regulation, our research reveals that the PH domain of ASAP1 actively contributes to Arf1 GTP hydrolysis. By combining NMR, molecular dynamics simulations, kinetic assays, and mutational analysis, we found that the PH domain directly interacts with Arf·GTP at the membrane, to drive conformational rearrangements of the GTP binding site. These structural changes establish an active state primed for GTP hydrolysis, facilitating charge stabilization which in turn, significantly enhances the catalytic rate of the GTPase reaction. Specifically, we identified key residues on both the PH domain and Arf responsible for this allosteric mechanism. Further, through mathematical modeling, we quantified the contribution of this newly discovered allosteric mechanism to ASAP1 GTPase-activating protein activity and found that it contributes equally to GTPase activation as membrane recruitment. The discovery that PH domains can directly affect nucleotide hydrolysis by a small GTPase has ramifications for the larger group of small GTPases, that include Ras and Rho proteins, that are regulated by proteins with PH domains, control diverse cellular functions and are oncoproteins.

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 vacuole’s capacity to phase separate into ordered membrane domains. 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 sterol’s 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.