Post-translational modification sites are present in hydrophilic cavities of alpha-synuclein, tau, FUS, and TDP-43 fibrils: A molecular dynamics study.

Hydration plays a crucial role in the refolding of intrinsically disordered proteins into amyloid fibrils; however, the specific interactions between water and protein that may contribute to this process are still unknown. In our previous studies of alpha-synuclein (aSyn), we have shown that waters confined in fibril cavities are stabilizing features of this pathological fold; and that amino acids that hydrogen bond with these confined waters modulate primary and seeded aggregation. Here, we extend our aSyn molecular dynamics (MD) simulations with three new polymorphs and correlate MD trajectory information with known post-translational modifications (PTMs) and experimental data. We show that cavity residues are more evolutionarily conserved than non-cavity residues and are enriched with PTM sites. As expected, the confinement within hydrophilic cavities results in more stably hydrated amino acids. Interestingly, cavity PTM sites display the longest protein-water hydrogen bond lifetimes, three-fold greater than non-PTM cavity sites. Utilizing the deep mutational screen dataset by Newberry et al. and the Thioflavin T aggregation review by Pancoe et al. parsed using a fibril cavity/non-cavity definition, we show that hydrophobic changes to amino acids in cavities have a larger effect on fitness and aggregation rate than residues outside cavities, supporting our hypothesis that these sites are involved in the inhibition of aSyn toxic fibrillization. Finally, we expand our study to include analysis of fibril structures of tau, FUS, TDP-43, prion, and hnRNPA1; all of which contained hydrated cavities, with tau, FUS, and TDP-43 recapitulating our PTM results in aSyn fibril cavities.

Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency That Does Not Affect TNFR2 Function.

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.

Risk Stratification in Primary Care: Value-Based Contributions of Provider Adjudication.

BACKGROUND: In primary care risk stratification, automated algorithms do not consider the same factors as providers. The process of adjudication, in which providers review and adjust algorithm-derived risk scores, may improve the prediction of adverse outcomes. OBJECTIVE: We assessed the patient factors that influenced provider adjudication behavior and evaluated the performance of an adjudicated risk model against a commercial algorithm. DESIGN: (1) Structured interviews with primary care providers (PCP) and multivariable regression analysis and (2) receiver operating characteristic curves (ROC) with sensitivity analyses. PARTICIPANTS: Primary care patients aged 18 years and older with an adjudicated risk score. APPROACH AND MAIN MEASURES: (1) Themes from structured interviews and discrete variables associated with provider adjudication behavior; (2) comparison of concordance statistics and sensitivities between risk models. KEY RESULTS: 47,940 patients were adjudicated by PCPs in 2018. Interviews revealed that, in adjudication, providers consider disease severity, presence of self-management skills, behavioral health, and whether a risk score is actionable. Provider up-scoring from the algorithmic risk score was significantly associated with patient male sex (OR 1.24, CI 1.15-1.34), age > 65 (OR 2.55, CI 2.24-2.91), Black race (1.26, CI 1.02-1.55), polypharmacy >10 medications (OR 4.87, CI 4.27-5.56), a positive depression screen (OR 1.57, CI 1.43-1.72), and hemoglobin A1c >9 (OR 1.89, CI 1.52-2.33). Overall, the adjudicated risk model performed better than the commercial algorithm for all outcomes: ED visits (c-statistic 0.689 vs. 0.684, p < 0.01), hospital admissions (c-statistic 0.663 vs. 0.649, p < 0.01), and death (c-statistic 0.753 vs. 0.721, p < 0.01). When limited to males or seniors, the adjudicated models displayed either improved or non-inferior performance compared to the commercial model. CONCLUSIONS: Provider adjudication of risk stratification improves model performance because providers have a personal understanding of their patients and are able to apply their training to clinical decision-making.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2ß2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.

Fatty acid-induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor-receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.

Multiscale Computational Modeling of Tubulin-Tubulin Lateral Interaction.

Microtubules are multistranded polymers in eukaryotic cells that support key cellular functions such as chromosome segregation, motor-based cargo transport, and maintenance of cell polarity. Microtubules self-assemble via "dynamic instability," in which the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e., what is different between the GTP- and GDP-tubulin states that enables microtubule growth and shortening, respectively? More generally, a major challenge in biology is how to connect theoretical frameworks across length- and timescales, from atoms to cellular behavior. In this study, we describe a multiscale model by linking atomistic molecular dynamics (MD), molecular Brownian dynamics (BD), and cellular-level thermokinetic modeling of microtubules. Here, we investigated the underlying interaction energy when tubulin dimers associate laterally by performing all-atom MD simulations. We found that the lateral potential energy is not significantly different among three nucleotide states of tubulin, GTP, GDP, and GMPCPP and is estimated to be ≅ -11 kBT. Furthermore, using MD potential energy in our BD simulations of tubulin dimers confirms that the lateral bond is weak on its own, with a mean lifetime of ∼0.1 µs, implying that the longitudinal bond is required for microtubule assembly. We conclude that nucleotide-dependent lateral-bond strength is not the key mediator microtubule dynamic instability, implying that GTP acts elsewhere to exert its stabilizing influence on microtubule polymer. Furthermore, the estimated lateral-bond strength (ΔGlat0≅ -5 kBT) is well-aligned with earlier estimates based on thermokinetic modeling and light microscopy measurements. Thus, we have computationally connected atomistic-level structural information, obtained by cryo-electron microscopy, to cellular-scale microtubule assembly dynamics using a combination of MD, BD, and thermokinetic models to bridge from Ångstroms to micrometers and from femtoseconds to minutes.

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies.

OBJECTIVE: Understanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. BACKGROUND: Tauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. NEW/UPDATED HYPOTHESIS: We hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. MAJOR CHALLENGES FOR THE HYPOTHESIS: Our approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. LINKAGE TO OTHER MAJOR THEORIES: Our biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease-associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.

Antibiotic Utilization and the Role of Suspected and Diagnosed Mosquito-borne Illness Among Adults and Children With Acute Febrile Illness in Pune, India.

Background: Antibiotic resistance mechanisms originating in low- and middle- income countries are among the most common worldwide. Reducing unnecessary antibiotic use in India, the world's largest antibiotic consumer, is crucial to control antimicrobial resistance globally. Limited data describing factors influencing Indian clinicians to start or stop antibiotics are available. Methods: Febrile adults and children admitted to a public tertiary care hospital in Pune, India, were enrolled. Antibiotic usage and clinical history were recorded. Immunoassays for mosquito-borne disease and bacterial cultures were performed by protocol and clinician-directed testing. Clinical factors were assessed for association with empiric antibiotic initiation and discontinuation by day 5 using multivariable logistic regression and propensity score-matched Cox proportional hazard models. Results: Among 1486 participants, 683 (82%) adults and 614 (94%) children received empiric antibiotics. Participants suspected of having mosquito-borne disease were less likely to receive empiric antibiotics (adjusted odds ratio [AOR], 0.5; 95% confidence interval [CI], .4-.8). Empiric antibiotics were discontinued in 450 (35%) participants by day 5. Dengue or malaria testing performed before day 4 was positive in 162 (12%) participants, and was associated with antibiotic discontinuation (AOR, 1.7; 95% CI, 1.2-2.4). In a propensity score-matched model accounting for admission suspicion of mosquito-borne disease, positive dengue or malaria tests increased hazard of antibiotic discontinuation (hazard ratio, 1.6; 95% CI, 1.2-2.0). Conclusions: Most patients with acute febrile illness in an Indian public hospital setting receive empiric antibiotics. Mosquito-borne disease identification is associated with reduced empiric antibiotic use and faster antibiotic discontinuation.

Correction to: α-Synuclein's Uniquely Long Amphipathic Helix Enhances its Membrane Binding and Remodeling Capacity.

The original version of the article unfortunately contained error in author group; two authors were not submitted and published in the original version. Also the funding information is erroneously omitted.

Oxidation increases the strength of the methionine-aromatic interaction.

Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we found that oxidation increased the strength of the methionine-aromatic interaction motif, a driving force for protein folding and protein-protein interaction, by 0.5-1.4 kcal/mol. We found that non-hydrogen-bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups were enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR spectroscopy experiments on model peptides demonstrated that oxidation of methionine stabilized the interaction by 0.5-0.6 kcal/mol. We confirmed the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on (i) calmodulin structure and dynamics, and (ii) lymphotoxin-α binding toTNFR1. Thus, the methionine-aromatic motif was a determinant of protein structural and functional sensitivity to oxidative stress.

Drug-resistant Enterobacteriaceae colonization is associated with healthcare utilization and antimicrobial use among inpatients in Pune, India.

BACKGROUND: Healthcare exposure may increase drug-resistant Enterobacteriaceae colonization risk. Nascent antimicrobial stewardship efforts in low- and middle-income countries require setting-specific data. We aimed to evaluate risk factors for inpatient drug resistant Enterobacteriaceae colonization in a resource-limited setting in India. METHODS: Patients age ≥ 6 months admitted with ≥24 h of fever to a tertiary hospital in Pune, India were enrolled in a prospective cohort. Perirectal swabs, collected on admission and hospitalization day 3 or 4, were cultured in vancomycin- and ceftriaxone-impregnated media to assess for ceftriaxone-resistant Enterobacteriaceae (CTRE) and carbapenem-resistant Enterobacteriaceae (CPRE). Multivariable analyses assessed risk factors for drug-resistant Enterobacteriaceae colonization among participants without admission colonization. RESULTS: Admission perirectal swabs were collected on 897 participants; 87 (10%) had CTRE and 14 (1.6%) had CPRE colonization. Admission CTRE colonization was associated with recent healthcare contact (p < 0.01). Follow-up samples were collected from 620 participants, 67 (11%) had CTRE and 21 (3.4%) had CPRE colonization. Among 561 participants without enrollment CTRE colonization, 49 (9%) participants were colonized with CTRE at follow-up. Detection of CTRE colonization among participants not colonized with CTRE at admission was independently associated with empiric third generation cephalosporin treatment (adjusted odds ratio [OR] 2.9, 95% CI 1.5-5.8). Follow-up transition to CPRE colonization detection was associated with ICU admission (OR 3.0, 95% CI 1.0-8.5). CONCLUSIONS: Patients who receive empiric third generation cephalosporins and are admitted to the ICU rapidly develop detectable CTRE and CPRE colonization. Improved antimicrobial stewardship and infection control measures are urgently needed upon hospital admission.

High Burden of Antimicrobial Resistance and Mortality Among Adults and Children With Community-Onset Bacterial Infections in India.

Background: In India, antimicrobial consumption is high, yet systematically collected data on the epidemiology, risk factors, and outcomes of antimicrobial-resistant infections are limited. Methods: A prospective study of adults and children hospitalized for acute febrile illness was conducted between August 2013 and December 2015. In-hospital outcomes were recorded, and logistic regression was performed to identify independent predictors of community-onset antimicrobial-resistant infections. Results: Among 1524 patients hospitalized with acute febrile illness, 133 isolates were found among 115 patients with community-onset infections; 66 isolates (50.0%) were multidrug resistant and, of 33 isolates tested for carbapenem susceptibility, 12 (36%) were resistant. Multidrug-resistant infections were associated with recent antecedent antibiotic use (adjusted odds ratio [aOR], 4.17; 95% confidence interval [CI], 1.19-19.7) and were independently associated with mortality (aOR, 6.06; 95% CI, 1.2-55.7). Conclusion: We found a high burden of community-onset antimicrobial-resistant infection among patients with acute febrile illness in India. Multidrug-resistant infection was associated with prior antibiotic use and an increased risk of mortality.

Death Receptor 5 Activation Is Energetically Coupled to Opening of the Transmembrane Domain Dimer.

The precise mechanism by which binding of tumor necrosis factor ligands to the extracellular domain of their corresponding receptors transmits signals across the plasma membrane has remained elusive. Recent studies have proposed that activation of several tumor necrosis factor receptors, including Death Receptor 5, involves a scissorlike opening of the disulfide-linked transmembrane (TM) dimer. Using time-resolved fluorescence resonance energy transfer, we provide, to our knowledge, the first direct biophysical evidence that Death Receptor 5 TM-dimers open in response to ligand binding. Then, to probe the importance of the closed-to-open TM domain transition in the overall energetics of receptor activation, we designed point-mutants (alanine to phenylalanine) in the predicted, tightly packed TM domain dimer interface. We hypothesized that the bulky residues should destabilize the closed conformation and eliminate the ∼3 kcal/mol energy barrier to TM domain opening and the âˆ¼2 kcal/mol energy difference between the closed and open states, thus oversensitizing the receptor. To test this, we used all-atom molecular dynamics simulations of the isolated TM domain in explicit lipid bilayers coupled to thermodynamic potential of mean force calculations. We showed that single point mutants at the interface altered the energy landscape as predicted, but were not enough to completely eliminate the barrier to opening. However, the computational model did predict that a double mutation at i, i+4 positions at the center of the TM domain dimer eliminates the barrier and stabilizes the open conformation relative to the closed. We tested these mutants in cells with time-resolved fluorescence resonance energy transfer and death assays, and show remarkable agreement with the calculations. The single mutants had a small effect on TM domain separation and cell death, whereas the double mutant significantly increased the TM domain separation and more than doubled the sensitivity of cells to ligand stimulation.

The IM30/Vipp1 C-terminus associates with the lipid bilayer and modulates membrane fusion.

IM30/Vipp1 proteins are crucial for thylakoid membrane biogenesis in chloroplasts and cyanobacteria. A characteristic C-terminal extension distinguishes these proteins from the homologous bacterial PspA proteins, and this extension has been discussed to be key for the IM30/Vipp1 activity. Here we report that the extension of the Synechocystis IM30 protein is indispensable, and argue that both, the N-terminal PspA-domain as well as the C-terminal extension are needed in order for the IM30 protein to conduct its in vivo function. In vitro, we show that the PspA-domain of IM30 is vital for stability/folding and oligomer formation of IM30 as well as for IM30-triggered membrane fusion. In contrast, the IM30 C-terminal domain is involved in and necessary to stabilize defined contacts to negatively charged membrane surfaces, and to modulate the IM30-induced membrane fusion activity. Although the two IM30 protein domains have distinct functional roles, only together they enable IM30 to work properly.

Membrane remodeling and mechanics: Experiments and simulations of α-Synuclein.

We review experimental and simulation approaches that have been used to determine curvature generation and remodeling of lipid bilayers by membrane-bending proteins. Particular emphasis is placed on the complementary approaches used to study α-Synuclein (αSyn), a major protein involved in Parkinson's disease (PD). Recent cellular and biophysical experiments have shown that the protein 1) deforms the native structure of mitochondrial and model membranes; and 2) inhibits vesicular fusion. Today's advanced experimental and computational technology has made it possible to quantify these protein-induced changes in membrane shape and material properties. Collectively, experiments, theory and multi-scale simulation techniques have established the key physical determinants of membrane remodeling and rigidity: protein binding energy, protein partition depth, protein density, and membrane tension. Despite the exciting and significant progress made in recent years in these areas, challenges remain in connecting biophysical insights to the cellular processes that lead to disease. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Polyunsaturated chains in asymmetric lipids disorder raft mixtures and preferentially associate with α-Synuclein.

Using molecular dynamics simulations, we have explored the effect of asymmetric lipids-specifically those that contain one polyunsaturated (PUFA) and one saturated fatty acid chain-on phase separation in heterogeneous membranes. These lipids are prevalent in neuronal membranes, particularly in synaptic membranes, where the Parkinson's Disease protein α-Synuclein (αS) is found. We have therefore explored the relationship between asymmetric, PUFA-containing lipids, and αS. The simulations show that asymmetric lipids partition to the liquid disordered (Ld) phase of canonical raft mixtures because of the highly disordered PUFA chain. In the case of a membrane built to mimic the lipid composition of a synaptic vesicle, the PUFA-containing asymmetric lipids completely disrupt phase separation. Because αS is positively charged, we show that it partitions with negatively charged lipids, regardless of the saturation state of the chains. Additionally, αS preferentially associates with the polyunsaturated fatty acid tails of both charged and neutral lipids. This is a consequence of those chains' ability to accommodate the void beneath the amphipathic helix. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Piecing it together: Unraveling the elusive structure-function relationship in single-pass membrane receptors.

The challenge of crystallizing single-pass plasma membrane receptors has remained an obstacle to understanding the structural mechanisms that connect extracellular ligand binding to cytosolic activation. For example, the complex interplay between receptor oligomerization and conformational dynamics has been, historically, only inferred from static structures of isolated receptor domains. A fundamental challenge in the field of membrane receptor biology, then, has been to integrate experimentally observable dynamics of full-length receptors (e.g. diffusion and conformational flexibility) into static structural models of the disparate domains. In certain receptor families, e.g. the ErbB receptors, structures have led somewhat linearly to a putative model of activation. In other families, e.g. the tumor necrosis factor (TNF) receptors, structures have produced divergent hypothetical mechanisms of activation and transduction. Here, we discuss in detail these and other related receptors, with the goal of illuminating the current challenges and opportunities in building comprehensive models of single-pass receptor activation. The deepening understanding of these receptors has recently been accelerated by new experimental and computational tools that offer orthogonal perspectives on both structure and dynamics. As such, this review aims to contextualize those technological developments as we highlight the elegant and complex conformational communication between receptor domains. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

α-Synuclein's Uniquely Long Amphipathic Helix Enhances its Membrane Binding and Remodeling Capacity.

α-Synuclein is the primary protein found in Lewy bodies, the protein and lipid aggregates associated with Parkinson's disease and Lewy body dementia. The protein folds into a uniquely long amphipathic α-helix (AH) when bound to a membrane, and at high enough concentrations, it induces large-scale remodeling of membranes (tubulation and vesiculation). By engineering a less hydrophobic variant of α-Synuclein, we previously showed that the energy associated with binding of α-Synuclein's AH correlates with the extent of membrane remodeling (Braun et al. in J Am Chem Soc 136:9962-9972, 2014). In this study, we combine fluorescence correlation spectroscopy, electron microscopy, and vesicle clearance assays with coarse-grained molecular dynamics simulations to test the impact of decreasing the length of the amphipathic helix on membrane binding energy and tubulation. We show that truncation of α-Synuclein's AH length by approximately 15% reduces both its membrane binding affinity (by fivefold) and membrane remodeling capacity (by nearly 50% on per mole of bound protein basis). Results from simulations correlate well with the experiments and lend support to the idea that at high protein density there is a stabilization of individual, protein-induced membrane curvature fields. The extent to which these curvature fields are stabilized, a function of binding energy, dictates the extent of tubulation. Somewhat surprisingly, we find that this stabilization does not correlate directly with the geometric distribution of the proteins on the membrane surface.

α-Synuclein Reduces Tension and Increases Undulations in Simulations of Small Unilamellar Vesicles.

Using coarse-grained molecular dynamics simulations we have explored the effect of α-Synuclein (αSyn) on the structural and mechanical properties of small unilamellar vesicles in the fluid-phase. The study is motivated by observations that a high density of membrane-bound αSyn inhibits the fusion of synthetic small unilamellar vesicles. By combining three-dimensional pressure tensor calculations with our recently developed spherical harmonics fluctuation analysis approach, we show a reduction in membrane surface tension and increased membrane undulations when αSyn is bound to the vesicle's outer leaflet at a 200:1 L/P. The protein effects these changes by decreasing the negative pressure in the headgroup region of the outer leaflet and increasing the positive pressure throughout the hydrocarbon core.

Biophysics of α-synuclein induced membrane remodelling.

α-Synuclein is an intrinsically disordered protein whose aggregation is a hallmark of Parkinson's disease. In neurons, α-synuclein is thought to play important roles in mediating both endo- and exocytosis of synaptic vesicles through interactions with either the lipid bilayer or other proteins. Upon membrane binding, the N-terminus of α-synuclein forms a helical structure and inserts into the hydrophobic region of the outer membrane leaflet. However, membrane structural changes induced by α-synuclein are still largely unclear. Here we report a substantial membrane area expansion induced by the binding of α-synuclein monomers. This measurement is accomplished by observing the increase of membrane area during the binding of α-synuclein to pipette-aspirated giant vesicles. The extent of membrane area expansion correlates linearly with the density of α-synuclein on the membrane, revealing a constant area increase induced by the binding per α-synuclein molecule. The area expansion per synuclein is found to exhibit a strong dependence on lipid composition, but is independent of membrane tension and vesicle size. Fragmentation or tubulation of the membrane follows the membrane expansion process. However, contrary to BAR domain proteins, no distinct tubulation-transition density can apparently be identified for α-synuclein, suggesting a more complex membrane curvature generation mechanism. Consideration of α-synuclein's membrane binding free energy and biophysical properties of the lipid bilayer leads us to conclude that membrane expansion by α-synuclein results in thinning of the bilayer. These membrane thinning and tubulation effects may underlie α-synuclein's role in mediating cell trafficking processes such as endo- and exocytosis.