Anionic Lipid Bilayers Research Articles

Against Expectations: Unassisted RNA Adsorption onto Negatively Charged Lipid Bilayers.

The composition and physicochemical properties of biological membranes can be altered by diverse membrane integral and peripheral proteins as well as by small molecules, natural and synthetic. Diverse oligonucleotides have been shown to electrostatically interact with cationic and bivalent ion loaded zwitterionic liposomes, leading to the formation of oligonucleotide-liposome aggregates. However, interaction of RNAs with other membrane surfaces remains ill understood. We used the nonnatural RNA10 to investigate RNA binding to anionic and net-uncharged membrane surfaces. RNA10 had initially been selected in a screen for nonnatural RNA motives that bind to phosphatidylcholine liposomes in the presence of Mg2+. Here we show that interaction of defined RNA molecules with membrane surfaces crucially depends on electrostatic surface properties. Furthermore, RNA10 electrostatically binds to anionic lipid bilayers in the absence of Mg2+ or other bivalent cations, and this interaction leads to measurably changed physicochemical properties of the bilayer and the oligonucleotide. Thus, the structure of polyanionic RNA can be modulated via contact with negatively charged membrane surfaces and vice versa.

Probing the Conformational and Energy Landscapes of KRAS Membrane Orientation.

Membrane reorientation of oncogenic RAS proteins is emerging as an important modulator of their functions. Previous studies have shown that the most common orientations include those with either the three C-terminal α-helices (OS1) or N-terminal β-strands (OS2) of the catalytic domain facing the membrane. OS1 and OS2 differ by the degree to which the effector-interacting surface is occluded by the membrane. However, the relative stability of these states and the rates of transition between them remained undetermined. How mutations might modulate preferences for specific orientation states is also far from clear. The current work attempted to address these questions through a comprehensive analysis of two 20 μs-long atomistic molecular dynamics simulations. The simulations were conducted on the oncogenic G12D and Q61H KRAS mutants bound to an anionic lipid bilayer. G12D and Q61H are among the most prevalent cancer-causing mutations at the P-loop and switch 2 regions of KRAS, respectively. We found that both mutants fluctuate in a similar manner between OS1 and OS2 via an intermediate orientation OS0, and both favor the signaling competent OS1 and OS0 over the occluded OS2. However, they differ in the details, such as in the extent to which they sample OS1. Analysis of the orientation free-energy landscapes estimated from the simulations indicate that OS1 and OS2 are the most stable states. However, the overall free energy surface is rugged, indicating a large diversity of conformations including at least two substates in each orientation state that differ in stability only by about 0.5-1.0 kcal/mol. Reversible transitions between OS1 and OS2 occur via two well-defined pathways that traverse OS0. In the minimum energy path, helix 4 remains close to the membrane as the angle of the catalytic domain from the membrane plane changes, resulting in a barrier of ∼1 kcal/mol for OS1/OS2 interconversions. Estimation of the rates of the various transitions based on survival probabilities yielded two rate constants in the order of 107 and 106 s-1, which we attribute to intrinsic protein conformational dynamics and transient protein-lipid interactions, respectively. The faster process dominates every transition, confirming a previous suggestion that RAS membrane reorientation is driven by conformational fluctuations rather than protein-lipid interactions.

Abstract 2763: Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface

Abstract Ras proteins are classical members of small GTPases, controlling signal transduction pathways and promoting cell proliferation. Ras consists of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and activation is terminated by GTPase-activating proteins that induce the GTP hydrolysis. Ras has multiple partners, signals through several key pathways and fulfills critical functions in the cell life. Mutations in Ras are common in a variety of cancers; yet it is still undruggable. KRas4B is frequently mutated in cancer. Elucidation of Ras conformational ensembles at the signaling platforms in the plasma membrane including the ligand-bound conformations, membrane orientations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, and transition states are essential for deciphering Ras functions from its conformational landscapes. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. Raf kinases contain Ras binding domain (RBD) and cysteine-rich domain (CRD) at conserved region 1 (CR1), and kinase domain at CR3. The high-affinity interaction of Raf RBD with Ras was solved in the absence of CRD. It is known that Raf CRD play a key role in the membrane anchoring mechanism. Recently, pivotal roles of CRD in the membrane interactions of KRas4B-Raf-1 RBD-CRD complex were reported. Here, we employ all-atom molecular dynamics (MD) simulations to investigate dimeric KRas4B-Raf-1 complex at the anionic lipid bilayer composed of DOPC and DOPS (4:1). Active KRas4B dimer modulates Raf-1 activation, promoting dimerization of Raf-1’s kinase domain. In the dimeric complex of KRas4B-Raf-1, Raf-1 CRD stably binds anionic lipid bilayers inserting its positively-charged loop into the amphipathic interface. The key basic residues at the loop are responsible for the membrane association, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. Our simulations suggest that Raf-1 CRD not only offers an additional membrane anchor for the KRas4B-Raf-1 complex, but also reduces the fluctuations of Ras-RBD, enhancing the high affinity interaction between Ras and Raf. Raf-1 CRD supports the active-state Ras orientation, promoting Ras dimerization through the allosteric lobe helical interface at the membrane. The enhanced stability of the complex at the membrane rendered by CRD promotes Raf dimerization in the MAPK signaling, which is the key Ras proliferative pathway. Here we suggest these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Ruth Nussinov. Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2763.

Membrane Binding of Synaptotagmin-Like Protein 4: Insight from Molecular Dynamics Simulations

Synaptotagmin-like proteins (Slps) are a family of Rab GTPase effectors involved in secretory vesicle trafficking and membrane docking. Slps possess an N-terminal domain that binds Rab27 on large dense-core secretory vesicles and one or two C-terminal C2 domains that dock to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). In particular, Slp-4, also known as granuphilin, docks secretory vesicles to the plasma membrane primarily via its C2A domain, which has a high affinity for anionic lipid bilayers. Although the structure of the Slp-4 C2A domain is known and some of its key PIP2-binding residues have been identified, the full structural mechanism of its strong membrane binding remains unclear. Here, we employ molecular dynamics simulations of the Slp-4 C2A domain to determine contributions of particular residues in binding to a lipid bilayer containing physiologically relevant levels of the anionic target lipids PS and PIP2. Simulations include mutations of two important residues: Lys398, which is known to participate in PIP2 coordination, and Phe452, which is suspected to insert into target membranes hydrophobically. Preliminary results support the experimental finding that both the K398A and F452A mutants are capable of binding membranes but suggest a different balance of lipid interactions in the two mutants. Overall, experimental and computational results indicate that high-affinity membrane binding of the Slp-4 C2A domain is sustained via many lipid-interacting residues distributed over a large electropositive protein surface.

Lipid bilayer disruption induced by amphiphilic Janus nanoparticles: the non-monotonic effect of charged lipids.

In this study, we report the complex effects of charged lipids on the interaction between amphiphilic Janus nanoparticles and lipid bilayers. Janus nanoparticles are cationic on one hemisphere and hydrophobic on the other. We show that the nanoparticles, beyond threshold concentrations, induce holes in both cationic and anionic lipid bilayers mainly driven by hydrophobic interactions. However, the formation of these defects is non-monotonically dependent on ionic lipid composition. The electrostatic attraction between the particles and anionic lipid bilayers enhances particle adsorption and lowers the particle concentration threshold for defect initiation, but leads to more localized membrane disruption. Electrostatic repulsion leads to reduced particle adsorption on cationic bilayers and extensive defect formation that peaks at intermediate contents of cationic lipids. This study elucidates the significant role lipid composition plays in influencing how amphiphilic Janus nanoparticles interact with and perturb lipid membranes.

Could Cardiolipin Protect Membranes against the Action of Certain Antimicrobial Peptides? Aurein 1.2, a Case Study.

The activity of a host of antimicrobial peptides has been examined against a range of lipid bilayers mimicking bacterial and eukaryotic membranes. Despite this, the molecular mechanisms and the nature of the physicochemical properties underlying the peptide–lipid interactions that lead to membrane disruption are yet to be fully elucidated. In this study, the interaction of the short antimicrobial peptide aurein 1.2 was examined in the presence of an anionic cardiolipin-containing lipid bilayer using molecular dynamics simulations. Aurein 1.2 is known to interact strongly with anionic lipid membranes. In the simulations, the binding of aurein 1.2 was associated with buckling of the lipid bilayer, the degree of which varied with the peptide concentration. The simulations suggest that the intrinsic properties of cardiolipin, especially the fact that it promotes negative membrane curvature, may help protect membranes against the action of peptides such as aurein 1.2 by counteracting the tendency of the peptide to induce positive curvature in target membranes.

Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein Nanoparticles.

Modulations of synaptic membranes play an essential role in the physiological and pathological functions of the presynaptic protein α-synuclein (αSyn). Here we used solution atomic force microscopy (AFM) and electron paramagnetic resonance (EPR) spectroscopy to investigate membrane modulations caused by αSyn. We used several lipid bilayers to explore how different lipid species may regulate αSyn–membrane interactions. We found that at a protein-to-lipid ratio of ∼1/9, αSyn perturbed lipid bilayers by generating semi-transmembrane defects that only span one leaflet. In addition, αSyn coaggregates with lipid molecules to produce ∼10 nm-sized lipoprotein nanoparticles. The obtained AFM data are consistent with the apolipoprotein characteristic of αSyn. The role of anionic lipids was elucidated by comparing results from zwitterionic and anionic lipid bilayers. Specifically, our AFM measurements showed that anionic bilayers had a larger tendency of forming bilayer defects; similarly, our EPR measurements revealed that anionic bilayers exhibited more substantial changes in lipid chain mobility and bilayer polarity. We also studied the effect of cholesterol. We found that cholesterol increased the capability of αSyn in inducing bilayer defects and altering lipid chain mobility and bilayer polarity. These data can be explained by an increase in the lipid headgroup–headgroup spacing and/or specific cholesterol−αSyn interactions. Interestingly, we found an inhibitory effect of the cone-shaped phosphatidylethanolamine lipids on αSyn-induced bilayer remodeling. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization and suppress lipid extraction. Our results of lipid-dependent membrane modulations are likely relevant to αSyn functioning.

Interfacial charges drive the organization of supported lipid membranes and their interaction with nanoparticles.

In this work, we investigated the interaction of cationic gold nanoparticles (AuNPs) with an anionic solid-supported lipid bilayer (SSLB) prepared via the spontaneous fusion of vesicles of phosphatidylserine (DPPS) and phosphatidylcholine (DPPC) on SiO2. We combined sum frequency generation (SFG) spectroscopy at the SSLBs interfaces with electrophoretic light scattering at the vesicles/liquid interfaces, and we provided further insight into the formation of organized DPPS-DPPC films on SiO2 and their interaction with NPs. We found that there is a critical threshold of the relative vesicles/substrate interfacial zeta potentials, beyond which the conformational organization of SSLBs failed. Moreover, we also demonstrated that the presence of anionic DPPS lipids in model mixed DPPS-DPPC membranes accelerated the interaction rate with cationic AuNPs.

Abstract 678: Raf-1 cysteine-rich domain (CRD) supports active orientations of KRas4B/Raf-1 at the membrane

Abstract Ras is a small GTPase, controlling signal transduction pathways and promoting cell proliferation and survival. In cancer, there are two independent pathways in tumor proliferation: one includes Raf/MEK/ERK (MAPK) and PI3K/Akt/mTOR. KRas4B is the most abundant oncogenic isoform. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. The high-affinity interaction of Ras binding domain (RBD) of Raf with Ras was solved, but the relative position of Raf's cysteine-rich domain (CRD) in the Ras/Raf complex at the membrane and the key question of exactly how it affects Raf signaling are daunting. Here, we employ all-atom MD simulations to investigate membrane-anchored Raf-1 CRD and suggest a model of KRas4B/Raf-1 complex at the anionic lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) in mole ratio 4:1. Active Ras modulates Raf activation, with KRas4B preferentially activating Raf-1. We show that the Raf-1 CRD stably binds anionic lipid bilayers inserting a positively-charged loop into the amphipathic interface. The loop contains key basic residues, responsible for the membrane association. Importantly, when in complex with Ras/RBD, covalently-connected CRD presents the same membrane interaction mechanism, with CRD locating at the space between the RBD and membrane, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. To date, CRD's role was viewed in terms of stabilizing Raf-membrane interaction. Our observations argue for a key role of CRD that Raf-1 CRD not only offers an additional anchor for the KRas4B/Raf-1 complex, but by reducing the fluctuations of Ras/RBD it also increases the population of Ras/RBD at the membrane and enhances the already high affinity between Ras and RBD. In the KRas4B/RBD-CRD complex, Raf-1 CRD supports the active-state Ras orientation at the membrane, releasing the catalytic domain from the transiently confined orientation. This promotes MAPK signaling, the key Ras proliferative pathway, above other pathways. Even though in the absence of CRD the Ras/Raf-1 RBD interaction can elicit MAPK signaling, the enhanced stability of the complex at the membrane rendered by CRD can considerably intensify it, by helping in Raf's dimerization. Our data reveal these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Ruth Nussinov. Raf-1 cysteine-rich domain (CRD) supports active orientations of KRas4B/Raf-1 at the membrane [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 678.

Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs.

Amylin (hIAPP) Aggregates on the Membrane

Amylin is a 37 residue intrinsically disordered peptide whose aggregation is associated with Type II diabetes. Small oligomers (n-mers, with n<20) bind strongly to small unilamellar vesicles and are likely to be the key to its toxicity. However, we do not know which n-mer is the key. Here we use Single Molecule Photo-bleaching (smPB) techniques to study the size (‘n’) distribution of the oligomers in physiological solutions (at a sub-nM concentration) as well as on supported anionic lipid bilayers (POPC:POPG:Cholesterol = 1:1:1) exposed to such solutions. Single molecule imaging results indicate that solution state and membrane-bound oligomers mostly consist of monomers to trimers (neglecting any pre-measurement photobleaching). Dimers and trimers have higher membrane affinity than the monomers. This higher membrane affinity is possibly linked to the conformational changes which occur during aggregation. We explore aggregation-associated conformational transitions using fluorescence, vibrational and Solid State NMR (ssNMR) based methods that we have established earlier for amyloid beta. We find that the high membrane affinity species contains a mixture of both alpha-helices and beta-sheets. Our study, therefore, quantifies the structure and membrane interaction of small n-mers of amylin as a function of n, paving the way for size-specific drug-targeting of amylin aggregates.

Development of coated liposomes loaded with ghrelin for nose-to-brain delivery for the treatment of cachexia.

The aim of the present study was to develop a ghrelin-containing formulation based on liposomes coated with chitosan intended for nose–brain delivery for the treatment of cachexia. Among the three types of liposomes developed, anionic liposomes provided the best results in terms of encapsulation efficiency (56%) and enzymatic protection against trypsin (20.6% vs 0% for ghrelin alone) and carboxylesterase (81.6% vs 17.2% for ghrelin alone). Ghrelin presented both electrostatic and hydrophobic interactions with the anionic lipid bilayer, as demonstrated by isothermal titration calorimetry. Then, anionic liposomes were coated with N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride. The coating involved a size increment from 146.9±2.7 to 194±6.1 nm, for uncoated and coated liposomes, respectively. The ζ-potential was similarly increased from -0.3±1.2 mV to 6±0.4 mV before and after coating, respectively. Chitosan provided mucoadhesion, with an increase in mucin adsorption of 22.9%. Enhancement of permeation through the Calu3 epithelial monolayer was also observed with 10.8% of ghrelin recovered in the basal compartment in comparison to 0% for ghrelin alone. Finally, aerosols generated from two nasal devices (VP3 and SP270) intended for aqueous dispersion were characterized with either coated or uncoated liposomes. Contrarily to the SP270 device, VP3 device showed minor changes between coated and uncoated liposome aerosols, as shown by their median volume diameters of 38.4±5.76 and 37.6±5.74 µm, respectively. Overall, the results obtained in this study show that the developed formulation delivered by the VP3 device can be considered as a potential candidate for nose–brain delivery of ghrelin.

Binding of cationic model peptides (KX)4K to anionic lipid bilayers: Lipid headgroup size influences secondary structure of bound peptides

Differential Scanning Calorimetry (DSC) and Fourier transformed Infrared (FT-IR) spectroscopy were used to test the influence of acyl chain length, acyl chain saturation, and chemical structure of anionic phospholipids on the interaction with cationic model peptides (KX)4K, with amino acid X=A, Abu, and L. The lipids used were phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and cardiolipin (CL). DSC was used to monitor the phase transition of lipid vesicles before and after peptide binding. The electrostatic attraction is the main driving force for binding. The hydrophobicity of the amino acid X influences the binding strength as well as the secondary structure of the bound peptide. Binding of peptides leads to an upshift of the lipid phase transition. Lipids with smaller headgroups show a larger upshift of the main phase transition temperature.Data from FT-IR spectroscopy show in addition that the stability of the bound β-sheets of (KX)4K depends on the hydrophobicity of the uncharged amino acid X and on the size of the lipid headgroup. For lipids with large anionic headgroups, such as PS, the antiparallel β-sheet of (KAbu)4K bound to gel phase bilayers is converted to an unordered structure upon heating through the lipid phase transition. Reducing the size of the headgroup, as in PG, increases the stability of the bound peptide β-sheets. For the smallest headgroups, present in PA and CL, stably bound β-sheets are observed even above the lipid phase transition.

Interfacial activation of M37 lipase: A multi-scale simulation study

Lipases are enzymes of biotechnological importance that function at the interface formed between hydrophobic and aqueous environments. Hydrophobic interfaces can induce structural transitions in lipases that result in an increase in enzyme activity, although the detailed mechanism of this process is currently not well understood for many lipases. Here, we present a multi-scale molecular dynamics simulation study of how different interfaces affect the conformational dynamics of the psychrophilic lipase M37. Our simulations show that M37 lipase is able to interact both with anionic lipid bilayers and with triglyceride surfaces. Interfacial interactions with triglyceride surfaces promote large-scale motions of the lid region of M37, spanning residues 235–283, revealing an entry pathway to the catalytic site for substrates. Importantly, these results suggest a potential activation mechanism for M37 that deviates from other related enzymes, such as Thermomyces lanuginosus lipase. We also investigated substrate binding in M37 by using steered MD simulations, confirming the open state of this lipase. The exposure of hydrophobic residues within lid and active site flap regions (residues 94–110) during the activation process provides insights into the functional effect of hydrophobic surfaces on lipase activation.

Modulation of Elasticity and Interactions in Charged Lipid Multibilayers: Monovalent Salt Solutions.

We have studied the electrostatic screening effect of NaCl solutions on the interactions between anionic lipid bilayers in the fluid lamellar phase using a Poisson–Boltzmann-based mean-field approach with constant charge and constant potential limiting charge regulation boundary conditions. The full DLVO potential, including the electrostatic, hydration and van der Waals interactions, was coupled to thermal bending fluctuations of the membranes via a variational Gaussian Ansatz. This allowed us to analyze the coupling between the osmotic pressure and the fluctuation amplitudes and compare them both simultaneously with their measured dependence on the bilayer separation, determined by the small-angle X-ray scattering experiments. High-structural resolution analysis of the scattering data revealed no significant changes of membrane structure as a function of salt concentration. Parsimonious description of our results is consistent with the constant charge limit of the general charge regulation phenomenology, with fully dissociated lipid charge groups, together with a 6-fold reduction of the membranes’ bending rigidity upon increasing NaCl concentration.

Phosphatidylinositol 4,5-Bisphosphate Modulates the Affinity of Talin-1 for Phospholipid Bilayers and Activates Its Autoinhibited Form.

Integrins are vital transmembrane receptors that mediate cell-cell and cell-extracellular matrix interactions and signaling. Talin is a 270 kDa protein and is considered a key regulator of integrin activity. The interaction between talin and integrin is commonly regarded as the final step of inside-out activation. In the cytosol, talin adopts an autoinhibited conformation, in which the C-terminal rod domain binds the N-terminal head domain, preventing the interactions of the head domain with the membrane surface and the integrin cytoplasmic domain. It has long been suggested that the presence of phosphatidylinositol 4,5-bisphosphate (PIP2) at focal adhesions plays a role in activating talin. However, a detailed picture and mechanism of PIP2 activation of autoinhibited talin remains elusive. Here, we use a fluorescence resonance energy transfer-based binding assay to measure the affinity of talin and lipid bilayers harboring anionic lipids. Results show that the R9 and R12R13 segments of the talin rod domain inhibit the binding of the talin head domain (THD) to anionic lipid bilayers. In contrast, we show that the binding of the THD to bilayers containing PIP2 is insensitive to the presence of the inhibitor domains, thereby directly implicating PIP2 as an effective activator of talin. Furthermore, we have mapped the activation to the interaction of PIP2 with the F2F3 domain of the talin head, showing that PIP2 plays a critical role in the regulation of the autoinhibited form of talin and stimulates recruitment of talin to the membrane, which is essential for integrin inside-out signaling.

Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes.

Molecular dynamics simulations have been used to investigate the influence of the valency of counter-ions on the structure of freestanding bilayer membranes of the anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) lipid at 310 K and 1 atm. At this temperature, the membrane is in the fluid phase with a monovalent counter-ion and in the gel phase with a divalent counter-ion. The diffusion constant of water as a function of its depth in the membrane has been determined from mean-square-displacement calculations. Also, calculated incoherent quasielastic neutron scattering functions have been compared to experimental results and used to determine an average diffusion constant for all water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the electrostatic potential within phospholipid membranes imply an enormous electric field of 10(8)-10(9) V m(-1), which is likely to have great significance in controlling the conformation of translocating membrane proteins and in the transfer of ions and molecules across the membrane. We have calculated the membrane potential for DMPG bilayers and found ∼1 V (∼2 ⋅ 10(8) V m(-1)) when in the fluid phase with a monovalent counter-ion and ∼1.4 V (∼2.8 ⋅ 10(8) V m(-1)) when in the gel phase with a divalent counter-ion. The number of water molecules for a fully hydrated DMPG membrane has been estimated to be 9.7 molecules per lipid in the gel phase and 17.5 molecules in the fluid phase, considerably smaller than inferred experimentally for 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) membranes but comparable to the number inferred for 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) membranes. Some of the properties of the DMPG membrane are compared with those of the neutral zwitterionic DMPC bilayer membrane at 303 K and 1 atm, which is the same reduced temperature with respect to the gel-to-fluid transition temperature as 310 K is for the DMPG bilayer membrane.

The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.

The Membrane Lipid Composition Regulates The Activity of Talin

Integrins plays a central role in the dynamics of cell‐cell and cell to extra‐cellular matrix interactions and signaling. A key step in the activation of integrin receptors is the binding of the cytoplasmic domain of integrin subunits by talin. This interaction leads to separation of the integrin transmembrane domains and significant conformational changes in the extracellular domains, resulting in a dramatic increase in integrin's affinity for ligands. Talin is a 470 kDa adapter protein that consists of a N‐terminal head domain (THD) and a C‐terminal rod domain. THD is homologous to to the domain found in band 4.1/ezrin/radixin/moesin family of proteins (FERM domain) with uncanonical linear arrangement of F0–F3 domains. A second mechanism in the regulation of integrin activation is directly related to the fact that full‐length talin adopts an auto‐inhibited conformation. In the cytosol, the rod domain binds THD, preventing talin interactions with the membrane surface and the integrin cytoplasmic domain. The membrane bilayer also plays a critical role in the talin ‐integrin interaction, where anionic lipids are required for proper interaction, yet a detailed picture of the interplay between talin and the membrane has remained elusive. We describe a new fluorescence based assay for probing talin – membrane interactions with Nanodisc bilayers of controlled composition. We show that recruitment of THD to the membrane surface is governed by the absolute charge. Measurement of the donor‐acceptor distances reveals that anionic lipids promote a conformational change in the THD favoring a direct interaction of the F3 domain with the phospholipid bilayer. The magnitude of this conformational change is regulated by the identity of the phospholipid headgroup with phosphatidylinositides (e.g. PI4P and PIP2) promoting the largest conformational motions. In addition, the binding of THD to bilayers containing PIP2 is insensitive to the presence of the inhibitory segments of the talin rod domain, while the association of THD to other tested anionic lipid bilayers are strongly inhibited by purified talin rod domains. Furthermore, we have mapped the activation to the interaction of PIP2 with the F2–F3 domain of the THD. Thus, we show that PIP2 plays a central role in the converting talin to a conformation optimized for interactions with integrin cytoplasmic tails and regulation of the auto‐inhibited form of talin, which are essential for integrin inside‐out activation.Support or Funding InformationSupported by NIH grant GM101048.

X-ray Crystallographic Structure of Oligomers Formed by a Toxic β-Hairpin Derived from α-Synuclein: Trimers and Higher-Order Oligomers.

Oligomeric assemblies of the protein α-synuclein are thought to cause neurodegeneration in Parkinson’s disease and related synucleinopathies. Characterization of α-synuclein oligomers at high resolution is an outstanding challenge in the field of structural biology. The absence of high-resolution structures of oligomers formed by α-synuclein impedes understanding the synucleinopathies at the molecular level. This paper reports the X-ray crystallographic structure of oligomers formed by a peptide derived from residues 36–55 of α-synuclein. The peptide 1a adopts a β-hairpin structure, which assembles in a hierarchical fashion. Three β-hairpins assemble to form a triangular trimer. Three copies of the triangular trimer assemble to form a basket-shaped nonamer. Two nonamers pack to form an octadecamer. Molecular modeling suggests that full-length α-synuclein may also be able to assemble in this fashion. Circular dichroism spectroscopy demonstrates that peptide 1a interacts with anionic lipid bilayer membranes, like oligomers of full-length α-synuclein. LDH and MTT assays demonstrate that peptide 1a is toxic toward SH-SY5Y cells. Comparison of peptide 1a to homologues suggests that this toxicity results from nonspecific interactions with the cell membrane. The oligomers formed by peptide 1a are fundamentally different than the proposed models of the fibrils formed by α-synuclein and suggest that α-Syn36–55, rather than the NAC, may nucleate oligomer formation.