Mechanism Of Membrane Binding Research Articles

Ligand-Receptor Interaction-Induced Intracellular Phase Separation: A Global Disruption Strategy for Resistance-Free Lethality of Pathogenic Bacteria.

Addressing the global challenge of bacterial resistance demands innovative approaches, among which multitargeting is a widely used strategy. Current strategies of multitargeting, typically achieved through drug combinations or single agents inherently aiming at multiple targets, face challenges such as stringent pharmacokinetic and pharmacodynamic requirements and cytotoxicity concerns. In this report, we propose a bacterial-specific global disruption approach as a vastly expanded multitargeting strategy that effectively disrupts bacterial subcellular organization. This effect is achieved through a pioneering chemical design of ligand-receptor interaction-induced aggregation of small molecules, i.e., DNA-induced aggregation of a diarginine peptidomimetic within bacterial cells. These intracellular aggregates display affinity toward various proteins and thus substantially interfere with essential bacterial functions and rupture bacterial cell membranes in an "inside-out" manner, leading to robust antibacterial activities and suppression of drug resistance. Additionally, biochemical analysis of macromolecule binding affinity, cytoplasmic localization patterns, and bacterial stress responses suggests that this bacterial-specific intracellular aggregation mechanism is fundamentally different from nonselective classic DNA or membrane binding mechanisms. These mechanistic distinctions, along with the peptidomimetic's selective permeation of bacterial membranes, contribute to its favorable biocompatibility and pharmacokinetic properties, enabling its in vivo antimicrobial efficacy in several animal models, including mice-based superficial wound models, subcutaneous abscess models, and septicemia infection models. These results highlight the great promise of ligand-receptor interaction-induced intracellular aggregation in achieving a globally disruptive multitargeting effect, thereby offering potential applications in the treatment of malignant cells, including pathogens, tumor cells, and infected tissues.

Leveraging Machine Learning-Guided Molecular Simulations Coupled with Experimental Data to Decipher Membrane Binding Mechanisms of Aminosterols.

Understanding the molecular mechanisms of the interactions between specific compounds and cellular membranes is essential for numerous biotechnological applications, including targeted drug delivery, elucidation of the drug mechanism of action, pathogen identification, and novel antibiotic development. However, estimation of the free energy landscape associated with solute binding to realistic biological systems is still a challenging task. In this work, we leverage the Time-lagged Independent Component Analysis (TICA) in combination with neural networks (NN) through the Deep-TICA approach for determining the free energy associated with the membrane insertion processes of two natural aminosterol compounds, trodusquemine (TRO), and squalamine (SQ). These compounds are particularly noteworthy because they interact with the outer layer of neuron membranes, protecting them from the toxic action of misfolded proteins involved in neurodegenerative disorders, in both their monomeric and oligomeric forms. We demonstrate how this strategy could be used to generate an effective collective variable for describing solute absorption in the membrane and for estimating free energy landscape of translocation via on-the-fly probability enhanced sampling (OPES) method. In this context, the computational protocol allowed an exhaustive characterization of the aminosterol entry pathway into a neuron-like lipid bilayer. Furthermore, it provided accurate prediction of membrane binding affinities, in close agreement with the experimental binding data obtained by using fluorescently labeled aminosterols and large unilamellar vesicles (LUVs). The findings contribute significantly to our understanding of aminosterol entry pathways and aminosterol-lipid membrane interactions. Finally, the computational methods deployed in this study further demonstrate considerable potential for investigating membrane binding processes.

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

Control of atypical PKCι membrane dissociation by tyrosine phosphorylation within a PB1-C1 interdomain interface

Atypical PKCs are cell polarity kinases that operate at the plasma membrane where they function within multiple molecular complexes to contribute to the establishment and maintenance of polarity. In contrast to the classical and novel PKCs, atypical PKCs do not respond to diacylglycerol cues to bind the membrane compartment. Until recently it was not clear how aPKCs are recruited; whether aPKCs can directly interact with membranes or whether they are dependent on other protein interactors to do so. Two recent studies identified the pseudo-substrate region and the C1 domain as direct membrane interaction modules, however their relative importance and coupling are unknown. We combined molecular modelling and functional assays to show that the regulatory module of aPKCι, comprising the PB1 pseudo-substrate and C1 domains, forms a cooperative and spatially continuous invariant membrane interaction platform. Furthermore, we show the coordinated orientation of membrane-binding elements within the regulatory module requires a key PB1-C1 interfacial β-strand (BSL). We show this element contains a highly conserved Tyr residue that can be phosphorylated and that negatively regulates the integrity of the regulatory module, leading to membrane release. We thus expose a hitherto unknown regulatory mechanism of aPKCι membrane binding and release during cell polarization.

Investigating ps surface sensitivity and calcium dependency of hTIM-1 and hTIM-4.

Transmembrane immunoglobulin and mucin domain (TIM) protein plays an important role in the immune system. TIM protein utilizes the phosphatidylserine (PS) binding pocket of the IgV domain to recognize PS lipids on the surface of target cells, and the binding is calcium-mediated. Although all members of the TIM protein family possess PS-binding pockets, they are involved in different immune response and are expressed on the surface of different immune cells. To understand the variation of intercellular recognition mechanisms, it is important to investigate their surface sensitivities to PS and their dependency on calcium for binding. Previous work in our group showed that murine TIM1 (mTIM1) and TIM4 (mTIM4) have different PS surface sensitivities. Moreover, mTIM1 and mTIM4 can bind, to different extent, to the membrane without calcium ions, but calcium ions enhance binding. Since literature shows that genes of mTIM1 and mTIM4 are orthologs of human TIM1 (hTIM1) and TIM4 (hTIM4), respectively, we aim to understand the PS surface sensitivities and calcium dependency of hTIM1 and hTIM4, and elucidate if the difference of their PS surface sensitivities and calcium dependency preserve between murine and human TIM. hTIM1 and hTIM4 have three tryptophan amino acids, but only one of them buries into the membrane when they bind to the membrane. Using a tryptophan fluorescence assay, we quantify PS surface sensitivities and calcium dependency of hTIM1 and hTIM4. We have observed that hTIM1 binds to 7:3 POPC:POPS vesicles without calcium and the fluorescence spectrum blue shifts. By investigating factors of membrane-binding mechanism for hTIM1 and hTIM4, we help elucidate difference in TIM binding between human and murine, and may provide further insights on intercellular recognition mechanisms.

Abstract 2690: Mechanism of membrane binding in type I phosphatidylinositol phosphate kinases

Abstract 2690: Mechanism of membrane binding in type I phosphatidylinositol phosphate kinases

The structure of the human LACTB filament reveals the mechanisms of assembly and membrane binding.

Mitochondria are complex organelles that play a central role in metabolism. Dynamic membrane-associated processes regulate mitochondrial morphology and bioenergetics in response to cellular demand. In tumor cells, metabolic reprogramming requires active mitochondrial metabolism for providing key metabolites and building blocks for tumor growth and rapid proliferation. To counter this, the mitochondrial serine beta-lactamase-like protein (LACTB) alters mitochondrial lipid metabolism and potently inhibits the proliferation of a variety of tumor cells. Mammalian LACTB is localized in the mitochondrial intermembrane space (IMS), where it assembles into filaments to regulate the efficiency of essential metabolic processes. However, the structural basis of LACTB polymerization and regulation remains incompletely understood. Here, we describe how human LACTB self-assembles into micron-scale filaments that increase their catalytic activity. The electron cryo-microscopy (cryoEM) structure defines the mechanism of assembly and reveals how highly ordered filament bundles stabilize the active state of the enzyme. We identify and characterize residues that are located at the filament-forming interface and further show that mutations that disrupt filamentation reduce enzyme activity. Furthermore, our results provide evidence that LACTB filaments can bind lipid membranes. These data reveal the detailed molecular organization and polymerization-based regulation of human LACTB and provide new insights into the mechanism of mitochondrial membrane organization that modulates lipid metabolism.

Dissecting peripheral protein-membrane interfaces.

Peripheral membrane proteins (PMPs) include a wide variety of proteins that have in common to bind transiently to the chemically complex interfacial region of membranes through their interfacial binding site (IBS). In contrast to protein-protein or protein-DNA/RNA interfaces, peripheral protein-membrane interfaces are poorly characterized. We collected a dataset of PMP domains representative of the variety of PMP functions: membrane-targeting domains (Annexin, C1, C2, discoidin C2, PH, PX), enzymes (PLA, PLC/D) and lipid-transfer proteins (START). The dataset contains 1328 experimental structures and 1194 AphaFold models. We mapped the amino acid composition and structural patterns of the IBS of each protein in this dataset, and evaluated which were more likely to be found at the IBS compared to the rest of the domains' accessible surface. In agreement with earlier work we find that about two thirds of the PMPs in the dataset have protruding hydrophobes (Leu, Ile, Phe, Tyr, Trp and Met) at their IBS. The three aromatic amino acids Trp, Tyr and Phe are a hallmark of PMPs IBS regardless of whether they protrude on loops or not. This is also the case for lysines but not arginines suggesting that, unlike for Arg-rich membrane-active peptides, the less membrane-disruptive lysine is preferred in PMPs. Another striking observation was the over-representation of glycines at the IBS of PMPs compared to the rest of their surface, possibly procuring IBS loops a much-needed flexibility to insert in-between membrane lipids. The analysis of the 9 superfamilies revealed amino acid distribution patterns in agreement with their known functions and membrane-binding mechanisms. Besides revealing novel amino acids patterns at protein-membrane interfaces, our work contributes a new PMP dataset and an analysis pipeline that can be further built upon for future studies of PMPs properties, or for developing PMPs prediction tools using for example, machine learning approaches.

Structure prediction-based insights into the patatin family of Mycobacterium tuberculosis.

Despite its genome sequencing more than two decades ago, the majority of the genes of Mycobacterium tuberculosis remain functionally uncharacterized. Patatins are one such class of proteins that, despite undergoing an expansion in this pathogenic species compared to their non-pathogenic cousins, remain largely unstudied. Recent advances in protein structure prediction using machine learning tools such as AlphaFold2 have provided high-confidence predicted structures for all M. tuberculosis proteins. Here we present detailed analyses of the patatin family of M. tuberculosis using AlphaFold-predicted structures, providing insights into likely modes of regulation, membrane interaction and substrate binding. Regulatory domains within this family of proteins include cyclic nucleotide binding, lid-like domains and other helical domains. Using structural homologues, we identified the likely membrane localization mechanisms and substrate-binding sites. These analyses reveal diversity in their regulatory capacity, mechanisms of membrane binding and likely length of fatty acid substrates. Together, this analysis suggests unique roles for the eight predicted patatins of M. tuberculosis.

An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65.

RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107-125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107-125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.

Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2-containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane-bound kinase.

Progressive membrane-binding mechanism of SARS-CoV-2 variant spike proteins

SummaryMembrane recognition by viral spike proteins is critical for infection. Here we show the host cell membrane-binding surfaces of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike variants Alpha, Beta, Gamma, Delta, Epsilon, Kappa, and Omicron as well as SARS-CoV-1 and pangolin and bat relatives. They show increases in membrane binding propensities over time, with all spike head mutations in variants, and particularly BA.1, impacting the protein’s affinity to cell membranes. Comparison of hundreds of structures yields a progressive model of membrane docking in which spike protein trimers shift from initial perpendicular stances to increasingly tilted positions that draw viral particles alongside host cell membranes before optionally engaging angiotensin-converting enzyme 2 (ACE2) receptors. This culminates in the assembly of the symmetric fusion apparatus, with enhanced membrane interactions of variants explaining their unique cell fusion capacities and COVID-19 disease transmission rates.

Atomic view of the HIV-1 matrix lattice; implications on virus assembly and envelope incorporation

During the late phase of HIV type 1 (HIV-1) infection cycle, the virally encoded Gag polyproteins are targeted to the inner leaflet of the plasma membrane (PM) for assembly, formation of immature particles, and virus release. Gag binding to the PM is mediated by interactions of the N-terminally myristoylated matrix (myrMA) domain with phosphatidylinositol 4,5-bisphosphate. Formation of a myrMA lattice on the PM is an obligatory step for the assembly of immature HIV-1 particles and envelope (Env) incorporation. Atomic details of the myrMA lattice and how it mediates Env incorporation are lacking. Herein, we present the X-ray structure of myrMA at 2.15 Å. The myrMA lattice is arranged as a hexamer of trimers with a central hole, thought to accommodate the C-terminal tail of Env to promote incorporation into virions. The trimer–trimer interactions in the lattice are mediated by the N-terminal loop of one myrMA molecule and α-helices I–II, as well as the 310 helix of a myrMA molecule from an adjacent trimer. We provide evidence that substitution of MA residues Leu13 and Leu31, previously shown to have adverse effects on Env incorporation, induced a conformational change in myrMA, which may destabilize the trimer–trimer interactions within the lattice. We also show that PI(4,5)P2 is capable of binding to alternating sites on MA, consistent with an MA–membrane binding mechanism during assembly of the immature particle and upon maturation. Altogether, these findings advance our understanding of a key mechanism in HIV-1 particle assembly.

Polyethersulfone ultrafiltration membranes modified with MAA-EGDMA imprinted polymers for selective atrazine separation

The molecular imprinting polymer (MIP) technology is widely known for producing polymers with molecular selectivity in separation and adsorption. The combination of this technology with membrane technology has been extensively explored to maximize the usage of both technologies. To selectively segregate the target molecules, membranes were produced by copolymerizing commercial scaffold monomers with template-containing monomers. The MIP, in combination with the membrane structure, may be utilised as filtering materials for selective separation, which is a fundamental feature of this technology. To produce the imprinted polymer embedded in polyethersulfone (PES) membranes, we employed phase inversion in water as a non-solvent for membrane synthesis. The porosity, mean pore radius, morphology, and contact angle of membranes were studied to investigate the possibility of enhancing the membrane-binding mechanism and the membrane flux. All tests were carried out with a 50 mg·L−1 solution of atrazine (Atr) with a pH of 7.7. The optimal membrane concentration is shown by the 17% PES membrane. It shows the highest value of binding capacity that can be retained with high permeate flux. Higher surface hydrophilicity of MIP blended membranes increases the Atr rejection.

An Integrated Pharmacological, Structural, and Genetic Analysis of Extracellular Versus Intracellular ROS Production in Neutrophils

The neutrophil NADPH oxidase produces both intracellular and extracellular reactive oxygen species (ROS). Although oxidase activity is essential for microbial killing, and ROS can act as signaling molecules in the inflammatory process, excessive extracellular ROS directly contributes to inflammatory tissue damage, as well as to cancer progression and immune dysregulation in the tumor microenvironment. How specific signaling pathways contribute to ROS localization is unclear. Here we used a systems pharmacology approach to identify the specific Class I PI3-K isoform p110β, and PLD1, but not PLD2, as critical regulators of extracellular, but not intracellular ROS production in primary neutrophils. Combined crystallographic and molecular dynamics analysis of the PX domain of the oxidase component p47phox, which binds the lipid products of PI 3-K and PLD, was used to clarify the membrane-binding mechanism and guide the design of mutant mice whose p47phox is unable to bind 3-phosphorylated inositol phospholipids. Neutrophils from these K43A mutant animals were specifically deficient in extracellular, but not intracellular, ROS production, and showed increased dependency on signaling through the remaining PLD1 arm. These findings identify the PX domain of p47phox as a critical integrator of PLD1 and p110β signaling for extracellular ROS production, and as a potential therapeutic target for modulating tissue damage and extracellular signaling during inflammation.

Modeling the membrane binding mechanism of a lipid transport protein Osh4 to single membranes

All-atom (AA) molecular dynamics simulations are used to unravel the binding mechanism of yeast oxysterol binding protein (Osh4) to model membranes with varying anionic lipid concentration using AA and the highly mobile membrane mimetic (HMMM) representations. For certain protein-lipid interactions, an improved forcefield description is used (CUFIX) to accurately describe lipid-protein electrostatic interactions. Our detailed computational studies have identified a single, β-crease oriented, membrane-bound conformation of Osh4 for all anionic membranes. The penetration of the PHE-239 residue below the membrane phosphate plane is the characteristic signature of the membrane-bound state of Osh4. As the phenylalanine loop anchors itself deeply in the membrane; the other regions of the Osh4, namely, ALPS motif, β6- β7 loop, β14- β15 loop, and β16- β17 loop, maximize their contact with the membrane. Furthermore, loose lipid packing and higher mobility of HMMM enable stronger association of the ALPS motif with the membrane lipids through its hydrophobic surface. After the HMMM is converted to AA and equilibrated, the binding is two to three times stronger compared with simulations started with the AA representation, yielding the major importance of the ALPS motif to binding. Quantitative estimation of binding energy revealed that the phenylalanine loop plays a crucial role in stable membrane attachment of Osh4 and contributes significantly toward overall binding process. The CUFIX parameters provide a more balanced picture of hydrophobic and electrostatic interactions between the protein and the membrane, which differs from our past work that showed salt bridges alone stabilized Osh4-membrane contact. Our study provides a comprehensive picture of the binding mechanism of Osh4 with model single membranes and, thus, understanding of the initial interactions is important for elucidating the biological function of this protein to shuttle lipids between organelles.

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations.

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50-60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm2 in the search for a second endosome with which to interact.

Membrane-Facilitated Receptor Access and Binding Mechanisms of Long-Acting β2-Adrenergic Receptor Agonists

The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment of asthma and other chronic pulmonary diseases. These drugs activate the β2-adrenergic receptors (β2-AR), a class A G protein-coupled receptor (GPCR), and differ significantly in their clinical onset and duration of actions. According to the microkinetic model, the long duration of action of salmeterol and formoterol compared with salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classic and enhanced molecular dynamics simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known to be critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other β2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process. SIGNIFICANCE STATEMENT: The cell membrane's functional role behind the duration of action of long-acting β2-adrenergic receptor (β2-AR) agonists such as salmeterol has been a subject of debate for a long time. This study investigated the binding and unbinding mechanisms of the three commonly used β2-AR agonists, salmeterol, formoterol, and salbutamol, using advanced simulation techniques. The obtained results offer unprecedented insights into the active role of membrane lipids in facilitating access and binding of the ligands, affecting the molecular recognition process and thus their pharmacology.

Membrane binding properties of the C-terminal segment of retinol dehydrogenase 8

Light absorption by rhodopsin leads to the release of all-trans retinal (ATRal) in the lipid phase of photoreceptor disc membranes. Retinol dehydrogenase 8 (RDH8) then reduces ATRal into all-trans retinol, which is the first step of the visual cycle. The membrane binding of RDH8 has been postulated to be mediated by one or more palmitoylated cysteines located in its C-terminus. Different peptide variants of the C-terminus of RDH8 were thus used to obtain information on the mechanism of membrane binding of this enzyme. Steady-state and time-resolved fluorescence measurements were performed using short and long C-terminal segments of bovine RDH8, comprising one or two tryptophan residues. The data demonstrate that the amphipathic alpha helical structure of the first portion of the C-terminus of RDH8 strongly contributes to its membrane binding, which is also favored by palmitoylation of at least one of the cysteines located in the last portion of the C-terminus.

Characterization of Beta-2-Glycoprotein I Domain V interaction with Plasma Membrane

Beta-2-Glycoprotein I (β2GPI), also known as apolipoprotein H, is a 48kDa protein which circulates in blood at a high concentration [0.2 mg/ml]. β2GPI is the main target of autoantibodies in antiphospholipid syndrome (APS) which results in thrombosis and/or recurrent miscarriage. Therefore, investigating membrane binding mechanism of β2GPI is important to elucidate how this protein become immunogenic upon binding to plasma membrane and to help target specific drug development for APS. It is known that β2GPI binds to anionic phospholipids in the plasma membrane via its positively charged domain V (DV) but the mechanism of binding is still elusive. in this study, we performed multiple 500-ns molecular dynamics simulations of β2GPI-DV in the presence of membranes containing PC and PS in varying compositions employing the HMMM (highly mobile membrane mimetic) model. We observed stable membrane binding in eight out of ten replicas. Our results show that in seven out of eight replicas, β2GPI-DV interacts with the membrane via its flexible loop (311-317) which insert into the membrane below the PO4 level. interestingly, in one replica we observe insertion of the loop carrying three lysine residues, a mode which closely corresponds to an alternative binding mode reported previously. The orientation of membrane-bound states was characterized by measuring the tilt angle between the protein and the membrane. After insertion of the flexible loop, β2GPI-DV held an average tilt angle of 92 with respect to the membrane normal. This study establishes the significance of the flexible loop for membrane binding of β2GPI which could lead to a more rational approach in drug design for treatment of APS.