Specific Lipid-protein Interactions Research Articles

Elucidating the role of lipid interactions in stabilizing the membrane protein KcsA

The significant effects of lipid binding on the functionality of potassium channel KcsA have been validated by brilliant studies. However, the specific interactions between lipids and KcsA, such as binding parameters for each binding event, have not been fully elucidated. In this study, we employed native mass spectrometry to investigate the binding of lipids to KcsA and their effects on the channel. The tetrameric structure of KcsA remains intact even in the absence of lipid binding. However, the subunit architecture of the E71A mutant, which is constantly open at low pH, relies on tightly associated copurified lipids. Furthermore, we observed that lipids exhibit weak binding to KcsA at high pH when the channel is at a closed/inactivation state in the absence of permeant cation K+. This feeble interaction potentially facilitates the association of K+ ions, leading to the transition of the channel to a resting closed/open state. Interestingly, both anionic and zwitterionic lipids strongly bind to KcsA at low pH when the channel is in an open/inactivation state. We also investigated the binding patterns of KcsA with natural lipids derived from E. coli and Streptomyces lividans. Interestingly, lipids from E. coli exhibited much stronger binding affinity compared to the lipids from S. lividans. Among the natural lipids from S. lividans, free fatty acids and triacylglycerols demonstrated the tightest binding to KcsA, whereas no detectable binding events were observed with natural phosphatidic acid lipids. These findings suggest that the lipid association pattern in S. lividans, the natural host for KcsA, warrants further investigation. In conclusion, our study sheds light on the role of lipids in stabilizing KcsA and highlights the importance of specific lipid-protein interactions in modulating its conformational states.

Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

Protein-lipid interactions and protein anchoring modulate the modes of association of the globular domain of the Prion protein and Doppel protein to model membrane patches.

The Prion protein is the molecular hallmark of the incurable prion diseases affecting mammals, including humans. The protein-only hypothesis states that the misfolding, accumulation, and deposition of the Prion protein play a critical role in toxicity. The cellular Prion protein (PrPC) anchors to the extracellular leaflet of the plasma membrane and prefers cholesterol- and sphingomyelin-rich membrane domains. Conformational Prion protein conversion into the pathological isoform happens on the cell surface. In vitro and in vivo experiments indicate that Prion protein misfolding, aggregation, and toxicity are sensitive to the lipid composition of plasma membranes and vesicles. A picture of the underlying biophysical driving forces that explain the effect of Prion protein - lipid interactions in physiological conditions is needed to develop a structural model of Prion protein conformational conversion. To this end, we use molecular dynamics simulations that mimic the interactions between the globular domain of PrPC anchored to model membrane patches. In addition, we also simulate the Doppel protein anchored to such membrane patches. The Doppel protein is the closest in the phylogenetic tree to PrPC, localizes in an extracellular milieu similar to that of PrPC, and exhibits a similar topology to PrPC even if the amino acid sequence is only 25% identical. Our simulations show that specific protein-lipid interactions and conformational constraints imposed by GPI anchoring together favor specific binding sites in globular PrPC but not in Doppel. Interestingly, the binding sites we found in PrPC correspond to prion protein loops, which are critical in aggregation and prion disease transmission barrier (β2-α2 loop) and in initial spontaneous misfolding (α2-α3 loop). We also found that the membrane re-arranges locally to accommodate protein residues inserted in the membrane surface as a response to protein binding.

Membrane binding and lipid-protein interaction of the C2 domain from coagulation factor V

Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV–C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2–25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1–3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed “open” and “closed” states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both “open” and “closed” states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1–3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.

The molecular basis for the increased stability of the FUS-LC fibril at the anionic membrane- and air-water interfaces.

Self-organization of biomolecules can lead to the formation of liquid droplets, hydrogels, and irreversible aggregates that bear immense significance in biology and diseases. Despite the considerable number of studies conducted on biomolecular condensation in bulk solution, there is still a lack of understanding of how different surfaces regulate the condensation process. In this context, recent studies showed that, in contrast to zwitterionic lipid membranes, anionic membranes promoted the production of liquid droplets of FUsed in Sarcoma Low Complexity domain (FUS-LC) despite exhibiting no specific protein-lipid interactions. Moreover, the air-water interface led to a solid fibril-like aggregate of FUS-LC. The molecular mechanism of condensation/aggregation of proteins in response to surfaces of various charged states or levels of hydrophobicity remains to be better elucidated. Here, we provide initial insights into this question by investigating the stability of a small β fibril state of FUS-LC in bulk solution vs. membrane- and air-water interfaces. We perform multiple independent molecular dynamics simulations with distinct starting conformations for each system to demonstrate the statistical significance of our findings. Our study demonstrates the stability of the FUS-LC fibril in the presence of anionic membranes on the μs timescale while the fibril falls apart in bulk solution. We observe that a zwitterionic membrane does not enhance the stability of the fibril and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) has a higher propensity to stabilize the fibril than dioleoylphosphatidylglycerol (DOPG), in qualitative agreement with experiments. We further show that the fibril becomes more stable at the air-water interface. We pinpoint interfacial solvation at the membrane- and air-water interfaces as a key factor that contributes to the stabilization of the peptide assembly.

Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed nonequilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multicomponent lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

Cardiomyocyte sarcolemma modelling and lipid-protein interactions.

The cardiomyocyte sarcolemma is a crucial cellular organelle in heart function. The vital components of the sarcolemma are membrane proteins that generate and control the cardiac action potential, responsible for cardiomyocyte contractibility and heart rhythm. Their malfunction can be caused by congenital mutations and side effects of antiarrhythmic therapies and can lead to life-threatening conditions related to heart failure. Other main components are represented by several lipid types, which not only provide the matrix for membrane proteins but also play active roles in regulating protein function. Shifts in membrane physiology through changes in the lipidomic profile or lipid-protein interactions can also cause cardiac disorders. Our goal is to characterize specific lipid-protein interactions in the sarcolemma by studying lipid redistribution around proteins and identifying lipid binding sites that can potentially regulate protein function. We combined lipidomic, cryo-electron microscopy and X-ray crystallography studies to build computational models of the cardiomyocyte sarcolemma. The membrane models consist of 16 lipid species, which differ in headgroup type, levels of acyl tail unsaturation and length. We embedded a selected membrane protein (cardiac ion channel, transporter or receptor) in a lipid bilayer and performed molecular dynamics simulations with the Martini coarse-grained models. We simulated each system with a corresponding membrane protein for 120 μs and analyzed lipid-protein interactions in terms of lipid redistribution around the proteins via computing lipid depletion-enrichment index, lipid density, and predicting lipid binding sites.Our results reproduce experimental findings of the interactions between lipids and the Kir2.2 potassium channel. We propose potential lipid binding sites for other proteins, highlighting similarities and differences in lipid redistribution and interaction with the cardiomyocyte proteins. Such findings unveil new possible lipid-protein interactions in the cardiomyocyte sarcolemma and provide an understanding of their relevance in heart function.

Protein-protein interactions of the phenylpropanoid pathway in sorghum bicolor.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of the phenylpropanoid pathway followed by cinnamate 4-hydroxylase (C4H), a cytochrome P450 monooxygenase associated with the endoplasmic reticulum of plant cells. PAL and C4H are important for the plant cells due to their involvement in kickstarting the production of primary and secondary metabolites such as phenylpropanoids and lignin. C4H also interacts with cytochrome P450 reductase (CPR; Sorghum bicolor CPR in this study) to hydrolyze trans-cinnamic acid into p-coumaric acid. This study uses fluorescence correlation spectroscopy and single-protein fluorescence tracking of labeled proteins to directly measure the interactions of all three proteins in the absence and presence of their respective substrates. This study also explores specific protein-lipid interactions as a function of state (i.e. oxidation state, heterodimeric state, substrate occupancy, etc.)

Combining native mass spectrometry and lipidomics to uncover specific membrane protein-lipid interactions from natural lipid sources.

While it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative approach that combines native mass spectrometry (MS) and lipidomics to identify lipids retained by membrane proteins from natural lipid extracts. Our results reveal that the bacterial ammonia channel (AmtB) enriches specific cardiolipin (CDL) and phosphatidylethanolamine (PE) from natural headgroup extracts. When the two extracts are mixed, AmtB retains more species, wherein selectivity is tuned to bias headgroup selection. Using a series of natural headgroup extracts, we show TRAAK, a two-pore domain K+ channel (K2P), retains specific acyl chains that is independent of the headgroup. A brain polar lipid extract was then combined with the K2Ps, TRAAK and TREK2, to understand lipid specificity. More than a hundred lipids demonstrated affinity for each protein, and both channels were found to retain specific fatty acids and lysophospholipids known to stimulate channel activity, even after several column washes. Natural lipid extracts provide the unique opportunity to not only present natural lipid diversity to purified membrane proteins but also identify lipids that may be important for membrane protein structure and function.

A BAK subdomain that binds mitochondrial lipids selectively and releases cytochrome C.

How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.

TMEM16 scramblases thin the membrane to enable lipid scrambling

TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. Scrambling was proposed to occur with lipid headgroups moving between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryogenic electron microscopy structure of the nanodisc-reconstituted Ca2+-bound afTMEM16 scramblase showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove’s intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not require specific protein-lipid interactions with the extracellular vestibule. We find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes, despite an open pathway. Our results show afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove.

Regulation of human A2A adenosine receptor conformational dynamics by endogenous phospholipids

Endogenous phospholipids are widely recognized to regulate membrane protein structure and activity through specific protein-lipid interactions and by altering membrane bulk chemical and physical properties. Protein-lipid interactions are important regulators of G protein-coupled receptors (GPCRs), sensory membrane proteins that control many physiological processes and targets of over one-third of FDA-approved drugs. However, molecular mechanisms by which phospholipids regulate GPCRs are not well understood.

Lipid-Protein Interactions in Plasma Membrane Organization and Function.

Lipid-protein interactions in cells are involved in various biological processes, including metabolism, trafficking, signaling, host-pathogen interactions, and transmembrane transport. At the plasma membrane, lipid-protein interactions play major roles in membrane organization and function. Several membrane proteins have motifs for specific lipid binding, which modulate protein conformation and consequent function. In addition to such specific lipid-protein interactions, protein function can be regulated by the dynamic, collective behavior of lipids in membranes. Emerging analytical, biochemical, and computational technologies allow us to study the influence of specific lipid-protein interactions, as well as the collective behavior of membranes on protein function. In this article, we review the recent literature on lipid-protein interactions with a specific focus on the current state-of-the-art technologies that enable novel insights into these interactions.

Lipid coated protein condensates as stable protocells with selective uptake abilities for biomolecules.

To create cell-like synthetic systems, spatial confinement that is stable against environmental changes and selective uptake of diverse biomolecules into these compartments are key initial conditions. However, fabrication of protocells with these two features has been extremely difficult. Here, we used fully protein-based liquid condensates and a lipid coating on these condensates to construct highly stable protocells with an uptake ability for outside biomolecules. Condensates with an extremely high density of 6His-tagged proteins were coated with Ni(ii)-NTA(nitrilotriacetic acid)-modified lipids. High condensate rigidity and specific 6His-Ni-NTA interactions enabled the formation of lipid-protein protocells, which are stable even after centrifugations. In addition, immobile lipid coatings on condensates were permeable to outside biomolecules. When binding modules were fused into condensate-forming proteins, the resulting functionalized condensate-protocells could strongly and selectively uptake various outside proteins through specific protein interactions.

The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters.

Solute carrier (SLC) transporters represent the second-largest fraction of the membrane proteome after G-protein-coupled receptors, but have been underutilized as drug targets and the function of many members of this family is still unknown. They are technically challenging to work with as they are difficult to express and highly dynamic, making them unstable in detergent solution. Many SLCs lack known inhibitors that could be utilized for stabilization. Furthermore, as they bind their physiological substrates with high micromolar to low millimolar affinities, binding and transport assays have proven to be particularly challenging to implement. Previously, we reported a GFP-based method for the overexpression and purification of membrane proteins in Saccharomyces cerevisiae. Here, we extend this expression platform with the GFP thermal shift (GFP-TS) assay, which is a simplified version of fluorescence-detection size-exclusion chromatography that combines the sample versatility of fluorescence-detection size-exclusion chromatography with the high-throughput capability of dye-based thermal shift assays. We demonstrate how GFP-TS can be used for detecting specific ligand interactions of SLC transporter fusions and measuring their affinities in crude detergent-solubilized membranes. We further show how GFP-TS can be employed on purified SLC transporter fusions to screen for specific lipid-protein interactions, which is an important complement to native mass spectrometry approaches that cannot cope easily with crude lipid-mixture preparations. This protocol is simple to perform and can be followed by researchers with a basic background in protein chemistry. Starting with an SLC transporter construct that can be expressed and purified from S. cerevisiae in a well-folded state, this protocol extension can be completed in ~4-5 d.

Abstract 83: Structural and biochemical properties of DIRAS3 N-terminal extension that permit its function as a tumor suppressor of oncogenic K-RAS

Abstract DIRAS3 is an imprinted tumor suppressor gene that encodes a small GTPase of the RAS superfamily. In contrast to oncogenic RAS proteins, DIRAS3 inhibits tumor growth and migration, while inducing autophagy and tumor dormancy. DIRAS3 inhibits PI3K/AKT/mTOR and RAS/MAP signaling. DIRAS3 inhibits oncogenic, mutant K-RAS signaling by binding directly to mutant K-RAS and disrupting K-RAS dimers and clusters on the inner leaflet of the plasma membrane. DIRAS3 bears 60% amino acid homology to K-RAS, differing by the addition of a distinctive 34 amino acid N-terminal extension (NTE). Both the NTE and an intact C-terminal CAAX box are required for the suppressor function of DIRAS3 in breast and ovarian cancers. While prenylation of the CAAX box is thought to facilitate binding of DIRAS3 to the inner leaflet of the cell surface membrane through a lipid tail, how the NTE influences membrane association is not well understood. In this study, we apply biochemical and biophysical methods to investigate NTE structure and ligand binding activities using synthetic wild type and mutated NTE peptides. Using a lipid overlay assay and surface plasmon resonance (SPR) analysis, we found that the DIRAS3 NTE has affinity for phosphatidylinositol phosphate lipids, especially PI(3,4,5)P3 and PI(4,5)P2. SPR demonstrated a strong interaction between NTE and PIP3 (dissociation constant of ~36 nM) with a cooperative self-association of NTE on the PIP3 sensor surface. Isothermal titration calorimetry revealed that NTE-PIP3 binding is complex, involving multiple sites and cooperativity, that appeared dominated by entropic interaction. Structural analysis of NTE mutants with circular dichroism spectroscopy indicated that the NTE is mainly unstructured in solution, however PIP3-binding induces a conformational rearrangement of the NTE that involves a hydrophobic surface of an amphipathic helix region. Furthermore, mass spectrometry identified N-myrisoylation of glycine in the NTE region of DIRAS3, which is unique among RAS family proteins. In addition, SPR screening of a K-RAS peptide library and kinetic analysis of the interaction between DIRAS3 NTE and K-RAS revealed engagement of the NTE with the effector lobe of K-RAS and a conformational change in the NTE•K-RAS complex. Together, our study suggests that DIRAS3 NTE may play an important role in inhibiting K-RAS and AKT signaling by facilitating specific protein-lipid interactions at the plasma membrane. The affinity of the NTE for PIP3 could contribute to the known regulation of PI3K by DIRAS3. Moreover, functional DIRAS3 may be tethered to the inner leaflet of the cell membrane at two points, by prenylation of the CAAX box at the C-terminal and by myristoylation of the NTE. Citation Format: Xiaowen Liang, Kwangkook Lee, Sung Y. Jung, Gamze Bildik, Weiqun Mao, Joshua P. Gray, Steven W. Millward, Zhen Lu, Robert C. Bast. Structural and biochemical properties of DIRAS3 N-terminal extension that permit its function as a tumor suppressor of oncogenic K-RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 83.

Nano-scale resolution of native retinal rod disk membranes reveals differences in lipid composition.

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.

Probing the Membrane Environment of Plasma Membrane Proteins: A Micropatterning Approach

It is well established that lipids and proteins are not just independent components of the plasma membrane of eukaryotic cells but that their arrangement, dynamics and function are interdependent. Besides specific lipid-protein interaction, transmembrane proteins are thought to bind a shell of annular lipids, which are more or less tightly associated with the proteins. Furthermore, highly ordered nanoscopic membrane domains have been proposed to act to compartmentalize proteins and their interactions, but have thus far not been directly observed. Here, we use a combination of single molecule tracking and protein micropatterning to examine these interactions directly in the plasma membrane of living cells. In our experimental approach, different proteins of interest are immobilized within defined patterns in the plasma membrane, where they act as steric obstacles to the diffusion of lipid tracers and thus locally decrease their mobility. In the presence of lipid-protein interactions of any type, lipid mobility will be even further decreased within protein patterns. We used different types of proteins for patterning - single- and multi-spanning transmembrane proteins, a multi-subunit protein and a GPI-anchored protein. For all proteins we found that lipid tracer diffusion within protein patterns was slowed down only by steric hindrance since the size of the protein “sensed” by the diffusing lipid corresponded well with the size estimated from the protein crystal structure. These findings indicate that none of the examined proteins influence their membrane environment beyond their physical size, neither via tightly associated annular lipids nor via more ordered membrane domains.

Modeling Protein-Lipid Interactions during Viral Assembly of SARS-CoV-2

Specific protein-lipid interactions are key for cellular processes as well as for pathogens replication. Over the past months, SARS-CoV-2, the causative agent for COVID-19, infected more than thirty million people around the world, causing over a million deaths. The infectivity and transmission rate of the virus is unprecedented, and scientists have made important advances in record time to understand the virus mechanisms and develop therapies to combat it. A molecular level understanding of the interactions of key viral proteins with the cellular host is needed to continue the development of therapies that block or control the infection.

Inositol Phospholipid Interactions with Inward Rectifier Potassium Channels and Their Role in Disease

Ion channels are frequently modulated by specific protein-lipid interactions in the phospholipid bilayer. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised in biophysical studies. Here, we provide an extensive application of a coarse-grained molecular dynamics free-energy perturbation (CG-MD-FEP) methodology to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step protocol, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid and in the presence of anionic phospholipid such as phosphatidylserine (PS). By perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K has increased PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. This is in good agreement with electrophysiological studies showing the E179K mutant exhibits a reduction in neomycin sensitivity, which implies that PIP2 binds more tightly to E179K mutant channels. We conclude that our application provides a valuable method to compare affinities between lipid species, and for annotating amino acid side chains within lipid-binding pockets.