Membrane Conformation Research Articles

Mechanistic study of SCOOPs recognition by MIK2–BAK1 complex reveals the role of N-glycans in plant ligand–receptor–coreceptor complex formation

Ligand-induced receptor and co-receptor heterodimerization is a common mechanism in receptor kinase (RK) signalling activation. SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) mediate the complex formation of Arabidopsis RK MIK2 and co-receptor BAK1, triggering immune responses. Through structural, biochemical and genetic analyses, we demonstrate that SCOOPs use their SxS motif and adjacent residues to bind MIK2 and the carboxy-terminal GGR residues to link MIK2 to BAK1. While N-glycosylation of plant RKs is typically associated with protein maturation, plasma membrane targeting and conformation maintenance, a surprising revelation emerges from our crystal structural analysis of MIK2–SCOOP–BAK1 complexes. Specific N-glycans on MIK2 directly interact with BAK1 upon SCOOP sensing. The absence of N-glycosylation at the specific site in MIK2 neither affects its subcellular localization and protein accumulation in plant cells nor alters its structural conformation, but markedly reduces its affinity for BAK1, abolishing SCOOP-triggered immune responses. This N-glycan-mediated receptor and co-receptor heterodimerization occurs in both Arabidopsis and Brassica napus. Our findings elucidate the molecular basis of SCOOP perception by the MIK2–BAK1 immune complex and underscore the crucial role of N-glycans in plant receptor–coreceptor interactions and signalling activation, shaping immune responses.

Exploring the allergenic potential of sesame oleosins: Isolation and bioinformatics analysis

This study examined two oleosins of 17 kDa and 15 kDa isolated from Yuzhi white sesame seeds through oil body extraction. The allergens were identified as oleosin H1 (Ses i 4) and oleosin L (Ses i 5) using SDS-PAGE, dot blot analysis, and LC-MS/MS. PCR analysis revealed high sequence homology for the oleosin proteins in the sesame seeds. Utilizing AlphaFold2, bioinformatics tools, and protein-protein docking, the structure and function of these oleosins were analyzed. Ten potential B cell epitope peptides were predicted and mapped onto the α-helix and random coil-dominated oleosome membrane conformation. IgE binding simulations identified key epitopes, B3 (FLTSGAFGL) and B4 (KRGVQEGTLY) for oleosin H1, and B8 (GGFGVAALSV) and B9 (DQLESAKTKL) for oleosin L. Mutational analysis highlighted Glu135, Phe102, Tyr128, Tyr139, Gly136, and Gly132 in oleosin H1, and Leu120, Lys119, and Leu113 in oleosin L as critical residues for binding stability, providing insights into the sensitization mechanism of these epitopes. The integration of bioinformatics and immunoinformatics in this study has contributed to a deeper understanding of the allergy properties of sesame oleosins.

Conformations of influenza A M2 protein in DOPC/DOPS and E. coli native lipids and proteins

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IM2) protein reconstituted in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC/DOPS) bilayers to those in isolated Escherichia coli (E. coli) membranes, having preserved its native proteins and lipids. IM2 is a single-pass transmembrane protein known to assemble into a homo-tetrameric proton channel. To represent this channel, we made a construct containing the IM2’s TMD region flanked by the juxtamembrane residues. The single cysteine substitution, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the electron spin resonance (ESR) study. For this particular residue, we probed the conformations of the spin-labeled IM2 reconstituted in DOPC/DOPS and isolated E. coli membranes using continuous-wave ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The continuous-wave ESR spectra corresponded to very slow spin-label motion in both environments. In all cases, the DEER data were reconstructed into distance distributions with well-resolved peaks at 1.68 and 2.37 nm in distance and amplitude ratios of 1.41 ± 0.2 and 2:1, respectively. This suggests four nitroxide spin labels located at the corners of a square, indicative of an axially symmetric tetramer. The distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IM2 TMD tetramer in agreement with the model. Thus, we can conclude that, under conditions of pH 7.4 used in this study, IM2 TMD has similar conformations in model lipid bilayers and membranes made of native E. coli lipids and proteins of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Differential Effects of Post-translational Modifications on the Membrane Interaction of Huntingtin Protein.

Huntington's disease is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple post-translational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the effect of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17-membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of the Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insights into the complex relationship between Nt17 structure, PTMs, and membrane binding.

Pixelated High-Q Metasurfaces for in Situ Biospectroscopy and Artificial Intelligence-Enabled Classification of Lipid Membrane Photoswitching Dynamics.

Nanophotonic devices excel at confining light into intense hot spots of electromagnetic near fields, creating exceptional opportunities for light-matter coupling and surface-enhanced sensing. Recently, all-dielectric metasurfaces with ultrasharp resonances enabled by photonic bound states in the continuum (BICs) have unlocked additional functionalities for surface-enhanced biospectroscopy by precisely targeting and reading out the molecular absorption signatures of diverse molecular systems. However, BIC-driven molecular spectroscopy has so far focused on end point measurements in dry conditions, neglecting the crucial interaction dynamics of biological systems. Here, we combine the advantages of pixelated all-dielectric metasurfaces with deep learning-enabled feature extraction and prediction to realize an integrated optofluidic platform for time-resolved in situ biospectroscopy. Our approach harnesses high-Q metasurfaces specifically designed for operation in a lossy aqueous environment together with advanced spectral sampling techniques to temporally resolve the dynamic behavior of photoswitchable lipid membranes. Enabled by a software convolutional neural network, we further demonstrate the real-time classification of the characteristic cis and trans membrane conformations with 98% accuracy. Our synergistic sensing platform incorporating metasurfaces, optofluidics, and deep learning reveals exciting possibilities for studying multimolecular biological systems, ranging from the behavior of transmembrane proteins to the dynamic processes associated with cellular communication.

Molecular dynamics simulations of HIV-1 matrix-membrane interactions at different stages of viral maturation

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2-binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid residues such as ARG3 residues of both the MA monomers at the trimeric interface and highly basic region (HBR) residues (LYS29, LYS31) of one of the MA monomers. Our enhanced sampling simulations have explored the conformational space of phospholipids at different binding sites of the trimer-trimer interface of MA complexes that are not accessible by conventional unbiased molecular dynamics. Unlike the immature MA complex, the 2′ acyl tail of two PIP2 lipids at the trimeric interface of the mature MA complex is observed to sample stable binding pockets of MA consisting of helix-4 residues. Together, our results provide molecular-level insights into the interactions of MA trimeric complexes with membrane and different lipid conformations at the specific binding sites of MA protein before and after viral maturation.

Putrescine Improves the Resistance of Peach Fruit to Cold Stress via Elevating the Polyamines Conjugated to Plasma Membranes and Maintaining Membrane Conformation

Putrescine Improves the Resistance of Peach Fruit to Cold Stress via Elevating the Polyamines Conjugated to Plasma Membranes and Maintaining Membrane Conformation

Direct Modulators of K-Ras-Membrane Interactions.

Protein-membrane interactions (PMIs) are ubiquitous in cellular signaling. Initial steps of signal transduction cascades often rely on transient and dynamic interactions with the inner plasma membrane leaflet to populate and regulate signaling hotspots. Methods to target and modulate these interactions could yield attractive tool compounds and drug candidates. Here, we demonstrate that the conjugation of a medium-chain lipid tail to the covalent K-Ras(G12C) binder MRTX849 at a solvent-exposed site enables such direct modulation of PMIs. The conjugated lipid tail interacts with the tethered membrane and changes the relative membrane orientation and conformation of K-Ras(G12C), as shown by molecular dynamics (MD) simulation-supported NMR studies. In cells, this PMI modulation restricts the lateral mobility of K-Ras(G12C) and disrupts nanoclusters. The described strategy could be broadly applicable to selectively modulate transient PMIs.

Polyamines conjugated to the bio-membranes and membrane conformations are involved in the melatonin-mediated resistance of harvested plum fruit to cold stress

The mechanism underlying melatonin-mediated tolerance of postharvest fruit to cold is well explored. However, the relationship among cold tolerance, polyamines (PAs) conjugated to bio-membrane and membrane conformations remains to be elucidated in the melatonin-pretreated fruit. Therefore in this study, the polyamines conjugated to the bio-membranes (plasma membrane, mitochondrial membrane and tonoplast) and corresponding membrane conformations were detected in the melatonin-pretreated fruit under cold condition. The results demonstrated that cold stress caused damage to the normal conformations of the aforementioned bio-membranes and led to cold injury, which was indicated by the increased flesh browning area and plasma membrane permeability, and the decreased fruit firmness and sulphydryl content. However, melatonin pretreatment mitigated fruit cold injury and stabilized the conformations of the bio-membranes mentioned above, which was comitant with increased contents of non-covalently (NC)-conjugated spermidine (Spd) and spermine (Spm), rising by nearly 2.5 times, as well as covalently (C-) conjugated putrescine (Put) and Spd in the bio-membranes, which rose by over 3.5 and 4, respectively, compared to the cold-treated. From the results, it could be inferred that the polyamines conjugated to the bio-membranes and membrane conformation stabilization were involved in the melatonin-mediated resistance of harvested fruit to cold stress. This inference was confirmed by the complementary experiments with methylglyoxal-bis (guanylhydrazone) (MGBG) and phenanthroline, which inhibited S-adenosylmethionine decarboxylase and transglutaminase activities, respectively. MGBG and phenanthroline inhibited the melatonin-induced increases in the levels of NC-conjugated Spd and Spm, and C-conjugated PAs in the bio-membranes, respectively. Furthermore, both MGBG and phenanthroline reversed the mitigation effects of melatonin on fruit cold injury, in parallel with the decrease of the membrane conformation stabilization. Collectively, it could be concluded that melatonin-mediated resistance of harvested plum fruit to cold stress might involve in the increases in the PAs conjugated to the bio-membranes, as well as the maintenance of membrane conformation stabilization. The relationship between conjugated PAs in the bio-membranes and membrane conformation stabilization was discussed in the research.

Facile metal-ion infiltration into polyimide membranes with coordination crosslinking for efficient gas separation

We report a facile metal-ion infiltration method for engineering the properties of 6FDA-DAM:DABA (6FDA: 4,4′-(hexafluoroisopropylidene)diphthalic anhydride, DAM: 2,4,6-trimethyl-1,3-diaminobenzene, DABA: 3,5-diaminobenzoic acid; referred to as 6FDD) polyimide membranes. The 6FDD polyimide membrane samples were simply immersed in metal ion (copper, zinc, and cobalt) solutions of various concentrations (1 and 10 wt%) to obtain metal-ion-infiltrated 6FDD membranes. The metal ions and the carboxylate group of the 6FDD polyimide form a coordination bond, which can increase the d-spacing between the inter-polymer chain distances and reduce polymer chain mobility. The coordination between the metal ion and the carboxylated polymer chain enhances the mechanical properties of the polyimide membranes. The metal ion coordination provides not only increased stiffness but also high resistance to the breaking point under elongation. The metal ions were distributed uniformly across the entire 6FDD membrane without any noticeable defects, which indicated the effectiveness and simplicity of the proposed method, which can be applied to versatile membrane module types and conformations. The metal-ion infiltrated 6FDD membrane exhibited improved gas permeability compared to that of the pure 6FDD membrane because of the enhanced diffusivity coefficient resulting from the increased d-spacing. Particularly, the metal-ion-infiltrated 6FDD membranes exhibited significantly higher plasticization resistance because of a decrease in the normalized CO2 permeability at 750 psi compared to that of the pure 6FDD membrane. For example, the normalized CO2 permeability of the 10 wt% copper-ion-infiltrated 6FDD membrane increased by 120% but that of pure 6FDD membrane increased by 315%. This was because the metal–carboxylate group coordination strongly prevented relaxation of the polymer chain when it was exposed to plasticizing gases at high gas feed pressures.

Phenotypes controlled by the Brucella abortus two component system BvrR/BvrS are differentially impacted by BvrR phosphorylation.

Brucella abortus is a zoonotic pathogen whose virulence depends on its ability to survive intracellularly at the endoplasmic reticulum derived compartment. The two-component system BvrR/BvrS (BvrRS) is essential for intracellular survival due to the transcriptional control of the type IV secretion system VirB and its transcriptional regulator VjbR. It is a master regulator of several traits including membrane homeostasis by controlling gene expression of membrane components, such as Omp25. BvrR phosphorylation is related to DNA binding at target regions, thereby repressing or activating gene transcription. To understand the role of BvrR phosphorylation we generated dominant positive and negative versions of this response regulator, mimicking phosphorylated and non-phosphorylated BvrR states and, in addition to the wild-type version, these variants were introduced in a BvrR negative background. We then characterized BvrRS-controlled phenotypes and assessed the expression of proteins regulated by the system. We found two regulatory patterns exerted by BvrR. The first pattern was represented by resistance to polymyxin and expression of Omp25 (membrane conformation) which were restored to normal levels by the dominant positive and the wild-type version, but not the dominant negative BvrR. The second pattern was represented by intracellular survival and expression of VjbR and VirB (virulence) which were, again, complemented by the wild-type and the dominant positive variants of BvrR but were also significantly restored by complementation with the dominant negative BvrR. These results indicate a differential transcriptional response of the genes controlled to the phosphorylation status of BvrR and suggest that unphosphorylated BvrR binds and impacts the expression of a subset of genes. We confirmed this hypothesis by showing that the dominant negative BvrR did not interact with the omp25 promoter whereas it could interact with vjbR promoter. Furthermore, a global transcriptional analysis revealed that a subset of genes responds to the presence of the dominant negative BvrR. Thus, BvrR possesses diverse strategies to exert transcriptional control on the genes it regulates and, consequently, impacting on the phenotypes controlled by this response regulator.

Conformation of influenza AM2 membrane protein in nanodiscs and liposomes

The influenza A M2 protein (AM2) is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle including viral assembly and budding. An atomic-level conformational understanding of this key player in the influenza life cycle could inform new antiviral strategies. For conformational studies of complex systems like the AM2 membrane protein, a multipronged approach using different biophysical methods and different model membranes is a powerful way to incorporate complementary data and achieve a fuller, more robust understanding of the system. However, one must be aware of how the sample composition required for a particular method impacts the data collected and how conclusions are drawn. In that spirit, we systematically compared the properties of AM2 in two different model membranes: nanodiscs and liposomes. Electron paramagnetic spectroscopy of spin-labeled AM2 showed that the conformation and dynamics were strikingly similar in both AM2-nanodiscs and AM2-liposomes consistent with similar conformations in both model membranes. Analysis of spin labeled lipids embedded in both model membranes revealed that the bilayer in AM2-liposomes was more fluid and permeable to oxygen than AM2-nanodiscs with the same lipid composition. Once the difference in the partitioning of the paramagnetic oxygen relaxation agent was taken into account, the membrane topology of AM2 appeared to be the same in both liposomes and nanodiscs. Finally, functionally relevant AM2 conformational shifts previously seen in liposomes due to the addition of cholesterol were also observed in nanodiscs.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases.In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution ofinflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Influence of Cholesterol on the Membrane Binding and Conformation of α-Synuclein.

The α-Synuclein (α-Syn) plays an important role in the pathology of Parkinson's disease (PD), and its oligomers and fibrils are toxic to the nervous system. As organisms age, the cholesterol content in biological membranes increases, which is a potential cause of PD. Cholesterol may affect the membrane binding of α-Syn and its abnormal aggregation, but the mechanism remains unclear. Here, we present our molecular dynamics simulation studies on the interaction between α-Syn and lipid membranes, with or without cholesterol. It is demonstrated that cholesterol provides additional hydrogen bond interaction with α-Syn; however, the coulomb interaction and hydrophobic interaction between α-Syn and lipid membranes could be weakened by cholesterol. In addition, cholesterol leads to the shrinking of lipid packing defects and the decrease of lipid fluidity, thereby shortening the membrane binding region of α-Syn. Under these multifaceted effects of cholesterol, membrane-bound α-Syn shows signs of forming a β-sheet structure, which may further induce the formation of abnormal α-Syn fibrils. These results provide important information for the understanding of membrane binding of α-Syn, and they are expected to promote the bridging between cholesterol and the pathological aggregation of α-Syn.

Direct observation of Piezo1 conformational states.

PIEZOs are mechanosensitive ion channels that convert force into chemoelectrical signals. Mechanotransduction is thought to be initiated through the deformation of extensive arms of transmembrane domains emanating from a central ion-conducting pore. Structural studies show these arms adopt a symmetric bowl-shaped conformation, forming a dome of membrane that can be flatted and expanded by force. However, direct observation of channel conformation in a cell and an understanding of how the arms are engaged during mechanotransduction is lacking. Here, we use site-specific labeling and intramolecular fluorescent imaging to examine PIEZO1 conformation in a cell membrane. We show the arms of PIEZO1 are bent by the plasma membrane at rest and exhibit pronounced variation in flexibility along their length. Using stimuli which are known to modulate channel activity, we also show that the degree of arm extension from the central pore correlates with channel activation and inhibition. Our findings highlight how the cellular environment can shape PIEZO1 conformation and demonstrate a structure-function relationship between channel conformation and activation. More broadly, this study provides a basic understanding of how PIEZO1 senses mechanical force and presents a technical framework for the study of single-molecule protein conformation using direct nanoscopic imaging.

Insight into Bcl-2 proteins' functioning at mitochondrial membrane level.

Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes. 1. A. Ul Mushtaq, J. Åden, L.A. Clifton, H. Wacklin-Knecht, M. Campana, A.P.G. Dingeldein, C. Persson, T. Sparrman, G. Gröbner. Neutron reflectometry and NMR spectroscopy of full length Bcl-2 protein reveal its membrane localization and conformation. Communications Biology 4 (2021) 507.

How ostreolysin A detects SM/cholesterol in membranes.

Some pore-forming toxins target cells by binding specific lipids on the plasma membrane. Structural investigation of these protein-lipid interactions can provide insight into how membrane lipids are presented to proteins on the extracellular surface of cells. We use toxin proteins that recognize distinct regulatory pools of cholesterol in mammalian plasma membranes to better understand cholesterols membrane organization. The fungal toxin Ostreolysin A (OlyA) specifically binds sphingomyelin (SM) only when SM is complexed with cholesterol in the SM-sequestered pool. We’ve rigorously tested the lipid specificity of OlyA in reconstituted and cellular systems and discovered a single mutation in OlyA that abolishes SM/cholesterol specificity. This mutant, OlyA (E69A), binds SM containing membranes regardless of cholesterols presence. High-resolution X-ray structures of WT and mutant OlyA have led to a working model proposing these proteins recognize different membrane conformations of SM. Unfortunately, our solution structures lack direct information about sphingomyelins conformation in a membrane environment. Using single-particle Cryo-EM, we’ve now solved structures of OlyA bound to SM containing membranes with and without cholesterol. These structures provide the first direct insights into how WT and mutant OlyA recognize SM in these two forms. They also provide high-resolution information about the protein-protein interactions that drive pore assembly and shed light on how protein-lipid interactions initiate this process.

Ab initio prediction and characterization of membrane binding of HIV-1 broadly neutralizing antibodies by multi-scale simulations.

Rare but potent broadly neutralizing antibodies (bnAbs) engage the HIV virion membrane and the membrane proximal exposed region (MPER) peptide during neutralization. A preliminary lipid mediated binding step is suggested to increase antigen avidity but it remains unclear how initial membrane association occurs and thus what features are essential to mature bnAbs against a lipid-antigen epitope. In this study, we derived computational methods to investigate biophysical features of MPER bnAbs driving membrane association. We performed microsecond atomistic simulations of Fabs docked in a lipid bilayer. Phospholipid binding in CDR loops is predicted in all 4 replicates of PGZL1, 4E10 and 10E8 where a phospholipid headgroup position closely matches the phosphate position in lipid-soaked x-ray crystallography structures (mean RMSD 0.6, 0.7, 2.7 Å respectively). Interaction profiles also highlight how framework regions FRH3 (PGZL1 and 4E10) or FRL3 (10E8) significantly associate with lipids. We then conducted fixed velocity pulling simulations for 4E10 and mutants with reduced affinity for lipid vesicles in vitro. Max force calculates differences in energy required to dissociate Fabs, establishing a bona fide tool to approximate membrane affinities. We expanded this platform to track evolution of binding affinity with inferred germline, intermediate, and mature PGZL1 variants that show increasing binding strengths. To reduce bias from pre-docked membrane conformations, we generated coarse grained models with a Fab initiated above a bilayer. Spontaneous membrane binding events (>1 µs) are detected in over half of the 18 independent 14 µs bnAb trajectories but only in 2 of 18 replicates for negative control BSA. Altogether we present a computational toolkit to biophysically evaluate residue-specific and surface level features of membrane binding antibodies and differentiate membrane affinities, facilitating future studies of complete MPER or other auto-reactive bnAb maturation pathways.

Stabilization of membrane topologies by proteinaceous remorin scaffolds

In plants, the topological organization of membranes has mainly been attributed to the cell wall and the cytoskeleton. Additionally, few proteins, such as plant-specific remorins have been shown to function as protein and lipid organizers. Root nodule symbiosis requires continuous membrane re-arrangements, with bacteria being finally released from infection threads into membrane-confined symbiosomes. We found that mutations in the symbiosis-specific SYMREM1 gene result in highly disorganized perimicrobial membranes. AlphaFold modelling and biochemical analyses reveal that SYMREM1 oligomerizes into antiparallel dimers and may form a higher-order membrane scaffolding structure. This was experimentally confirmed when expressing this and other remorins in wall-less protoplasts is sufficient where they significantly alter and stabilize de novo membrane topologies ranging from membrane blebs to long membrane tubes with a central actin filament. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. Taken together we describe a plant-specific mechanism that allows the stabilization of large-scale membrane conformations independent of the cell wall.

Potential of Small‐Angle Neutron Scattering for Evaluating Protein Locus within a Polymersome

Inside Cover: In article 2200300 by Albena Lederer and co-workers, a specific amphiphilic copolymer structure defines the formation of polymersomes, which mimic diffusion processes through biological membranes. The locus of the cargo and the membrane conformation after diffusion are still open questions. Small-angle neutron scattering in combination with multidetecton fractionation opens new opportunities to evaluate membrane alteration after post-loading.