Diversity of Structural, Dynamic, and Environmental Effects Explain a Distinctive Functional Role of Transmembrane Domains in the Insulin Receptor Subfamily.

Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.

Activity-dependent conformational transitions of the insulin receptor-related receptor.

The insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor-related receptor (IRR) form a mini family of predimerized receptor-like tyrosine kinases. IR and IGF-1R bind to their peptide agonists triggering metabolic and cell growth responses. In contrast, IRR, despite sharing with them a strong sequence homology, has no peptide-like agonist but can be activated by mildly alkaline media. The spatial structure and activation mechanisms of IRR have not been established yet. The present work represents the first account of a structural analysis of a predimerized receptor-like tyrosine kinase by high-resolution atomic force microscopy in their basal and activated forms. Our data suggest that in neutral media, inactive IRR has two conformations, where one is symmetrical and highly similar to the inactive Λ/U-shape of IR and IGF-1R ectodomains, whereas the second is drop-like and asymmetrical resembling the IRR ectodomain in solution. We did not observe complexes of IRR intracellular catalytic domains of the inactive receptor forms. At pH 9.0, we detected two presumably active IRR conformations, Γ-shaped and T-shaped. Both of conformations demonstrated formation of the complex of their intracellular catalytic domains responsible for autophosphorylation. The existence of two active IRR forms correlates well with the previously described positive cooperativity of the IRR activation. In conclusion, our data provide structural insights into the molecular mechanisms of alkali-induced IRR activation under mild native conditions that could be valuable for interpretation of results of IR and IGF-IR structural studies.

Peptide-induced membrane elastic deformations decelerate gramicidin dimer-monomer equilibration.

Gramicidin A (gA) is a hydrophobic pentadecapeptide readily incorporating into a planar bilayer lipid membrane (BLM), thereby inducing a large macroscopic current across the BLM. This current results from ion-channel formation due to head-to-head transbilayer dimerization of gA monomers with rapidly established monomer-dimer equilibrium. Any disturbance of the equilibrium, e.g., by sensitized photoinactivation of a portion of gA monomers, causes relaxation toward a new equilibrium state. According to previous studies, the characteristic relaxation time of the gA-mediated electric current decreases as the current increases upon elevating the gA concentration in the membrane. Here, we report data on the current relaxation kinetics for gA analogs with N-terminal valine replaced by glycine or tyrosine. Surprisingly, the relaxation time increased rather than decreased upon elevation of the total membrane conductance induced by these gA analogs, thus contradicting the classical kinetic scheme. We developed a general theoretical model that accounts for lateral interaction of monomers and dimers mediated by membrane elastic deformations. The modified kinetic scheme of the gramicidin dimerization predicts the reverse dependence of the relaxation time on membrane conductance for gA analogs, with a decreased dimerization constant that is in a good agreement with our experimental data. The equilibration process may be also modulated by incorporation of other peptides ("impurities") into the lipid membrane.

The dimeric ectodomain of the alkali-sensing insulin receptor-related receptor (ectoIRR) has a droplike shape.

Insulin receptor-related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid-base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a "droplike" shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.

Monolayerwise application of linear elasticity theory well describes strongly deformed lipid membranes and the effect of solvent.

The theory of elasticity of lipid membranes is used widely to describe processes of cell membrane remodeling. Classically, the functional of a membrane's elastic energy is derived under assumption of small deformations; the membrane is considered as an infinitely thin film. This functional is quadratic on membrane surface curvature, with half of the splay modulus as its proportionality coefficient; it is generally applicable for small deformations only. Any validity of this functional for the regime of strong deformations should be verified experimentally. Recently, research using molecular dynamics simulations challenged the validity of this classic, linear model, i.e. the constancy of the splay modulus for strongly bent membranes. Here we demonstrate that the quadratic energy functional still can be applied for calculation of the elastic energy of strongly deformed membranes without introducing higher order terms with additional elastic moduli, but only if applied separately for each lipid monolayer. For cylindrical membranes, both classic and monolayerwise models yield equally accurate results. For cylindrical deformations we experimentally show that the elastic energy of lipid monolayers is additive: a low molecular weight solvent leads to an approximately twofold decrease in the membrane bending stiffness. Accumulation of solvent molecules in the inner monolayer of a membrane cylinder can explain these results, as the solvent partially prevents lipid molecules from splaying there. Thus, the linear theory of elasticity can be expanded through the range from weak to strong deformations-its simplicity and physical transparency describe various membrane phenomena.

Cyclopentane rings in hydrophobic chains of a phospholipid enhance the bilayer stability to electric breakdown.

Archaeal lipids ensure unprecedented stability of archaea membranes in extreme environments. Here, we incorporate a characteristic structural feature of an archaeal lipid, the cyclopentane ring, into hydrocarbon chains of a short-chain (C12) phosphatidylcholine to explore whether the insertion would allow such a lipid (1,2-di-(3-(3-hexylcyclopentyl)-propanoate)-sn-glycero-3-phosphatidylcholine, diC12cp-PC) to form stable bilayers at room temperature. According to fluorescence-based assays, in water diC12cp-PC formed liquid-crystalline bilayers at room temperature. Liposomes produced from diC12cp-PC retained calcein for over a week when stored at +4 °C. diC12cp-PC could also form model bilayer lipid membranes that were by an order of magnitude more stable to electrical breakdown than egg PC membranes. Molecular dynamics simulation showed that the cyclopentane fragment fixes five carbon atoms (or four C-C bonds), which is compensated by the higher mobility of the rest of the chain. This was found to be the reason for the remarkable stability of the diC12cp-PC bilayer: restricted conformational mobility of a chain segment increases the membrane bending modulus (compared to a normal hydrocarbon chain of the same length). Here, higher stiffness practically does not affect the line tension of a membrane pore edge. Rather it makes it more difficult for diC12cp-PC to rearrange in order to line the edge of a hydrophilic pore; therefore, fewer pores are formed.

Ectodomain Pulling Combines with Fusion Peptide Inserting to Provide Cooperative Fusion for Influenza Virus and HIV.

Enveloped viruses include the most dangerous human and animal pathogens, in particular coronavirus, influenza virus, and human immunodeficiency virus (HIV). For these viruses, receptor binding and entry are accomplished by a single viral envelope protein (termed the fusion protein), the structural changes of which trigger the remodeling and merger of the viral and target cellular membranes. The number of fusion proteins required for fusion activity is still under debate, and several studies report this value to range from 1 to 9 for type I fusion proteins. Here, we consider the earliest stage of viral fusion based on the continuum theory of membrane elasticity. We demonstrate that membrane deformations induced by the oblique insertion of amphipathic fusion peptides mediate the lateral interaction of these peptides and drive them to form into a symmetric fusion rosette. The pulling force produced by the structural rearrangements of the fusion protein ectodomains gives additional torque, which deforms the membrane and additionally stabilizes the symmetric fusion rosette, thus allowing a reduction in the number of fusion peptides needed for fusion. These findings can resolve the large range of published cooperativity indices for HIV, influenza, and other type I fusion proteins.

Continuum Models of Membrane Fusion: Evolution of the Theory.

Starting from fertilization, through tissue growth, hormone secretion, synaptic transmission, and sometimes morbid events of carcinogenesis and viral infections, membrane fusion regulates the whole life of high organisms. Despite that, a lot of fusion processes still lack well-established models and even a list of main actors. A merger of membranes requires their topological rearrangements controlled by elastic properties of a lipid bilayer. That is why continuum models based on theories of membrane elasticity are actively applied for the construction of physical models of membrane fusion. Started from the view on the membrane as a structureless film with postulated geometry of fusion intermediates, they developed along with experimental and computational techniques to a powerful tool for prediction of the whole process with molecular accuracy. In the present review, focusing on fusion processes occurring in eukaryotic cells, we scrutinize the history of these models, their evolution and complication, as well as open questions and remaining theoretical problems. We show that modern approaches in this field allow continuum models of membrane fusion to stand shoulder to shoulder with molecular dynamics simulations, and provide the deepest understanding of this process in multiple biological systems.

Phosphatidylcholine Membrane Fusion Is pH-Dependent.

Membrane fusion mediates multiple vital processes in cell life. Specialized proteins mediate the fusion process, and a substantial part of their energy is used for topological rearrangement of the membrane lipid matrix. Therefore, the elastic parameters of lipid bilayers are of crucial importance for fusion processes and for determination of the energy barriers that have to be crossed for the process to take place. In the case of fusion of enveloped viruses (e.g., influenza) with endosomal membrane, the interacting membranes are in an acidic environment, which can affect the membrane's mechanical properties. This factor is often neglected in the analysis of virus-induced membrane fusion. In the present work, we demonstrate that even for membranes composed of zwitterionic lipids, changes of the environmental pH in the physiologically relevant range of 4.0 to 7.5 can affect the rate of the membrane fusion notably. Using a continual model, we demonstrated that the key factor defining the height of the energy barrier is the spontaneous curvature of the lipid monolayer. Changes of this parameter are likely to be caused by rearrangements of the polar part of lipid molecules in response to changes of the pH of the aqueous solution bathing the membrane.

Lateral Membrane Heterogeneity Regulates Viral-Induced Membrane Fusion during HIV Entry.

Sphingomyelin- and cholesterol- enriched membrane domains, commonly referred to as "rafts" play a crucial role in a large number of intra- and intercellular processes. Recent experiments suggest that not only the volumetric inhomogeneity of lipid distribution in rafts, but also the arrangement of the 1D boundary between the raft and the surrounding membrane is important for the membrane-associated processes. The reason is that the boundary preferentially recruits different peptides, such as HIV (human immunodeficiency virus) fusion peptide. In the present work, we report a theoretical investigation of mechanisms of influence of the raft boundary arrangement upon virus-induced membrane fusion. We theoretically predict that the raft boundary can act as an attractor for viral fusion peptides, which preferentially distribute into the vicinity of the boundary, playing the role of 'line active components' of the membrane ('linactants'). We have calculated the height of the fusion energy barrier and demonstrated that, in the case of fusion between HIV membrane and the target cell, presence of the raft boundary in the vicinity of the fusion site facilitates fusion. The results we obtained can be further generalized to be applicable to other enveloped viruses.

Switching between Successful and Dead-End Intermediates in Membrane Fusion.

Fusion of cellular membranes during normal biological processes, including proliferation, or synaptic transmission, is mediated and controlled by sophisticated protein machinery ensuring the preservation of the vital barrier function of the membrane throughout the process. Fusion of virus particles with host cell membranes is more sparingly arranged and often mediated by a single fusion protein, and the virus can afford to be less discriminative towards the possible different outcomes of fusion attempts. Formation of leaky intermediates was recently observed in some fusion processes, and an alternative trajectory of the process involving formation of π-shaped structures was suggested. In this study, we apply the methods of elasticity theory and Lagrangian formalism augmented by phenomenological and molecular geometry constraints and boundary conditions to investigate the traits of this trajectory and the drivers behind the choice of one of the possible scenarios depending on the properties of the system. The alternative pathway proved to be a dead end, and, depending on the parameters of the participating membranes and fusion proteins, the system can either reversibly enter the corresponding "leaky" configuration or be trapped in it. A parametric study in the biologically relevant range of variables emphasized the fusion protein properties crucial for the choice of the fusion scenario.

Human Immunodeficiency Virus Type 1 Gag Polyprotein Modulates Membrane Physical Properties like a Surfactant: Potential Implications for Virus Assembly.

Human immunodeficiency virus (HIV) assembly at an infected cell's plasma membrane requires membrane deformation to organize the near-spherical shape of an immature virus. While the cellular expression of HIV Gag is sufficient to initiate budding of virus-like particles, how Gag generates membrane curvature is not fully understood. Using highly curved lipid nanotubes, we have investigated the physicochemical basis of the membrane activity of recombinant nonmyristoylated Gag-Δp6. Gag protein, upon adsorption onto the membrane, resulted in the shape changes of both charged and uncharged nanotubes. This shape change was more pronounced in the presence of charged lipids, especially phosphatidylinositol bisphosphate (PI(4,5)P2). We found that Gag modified the interfacial tension of phospholipid bilayer membranes, as judged by comparison with the effects of amphipathic peptides and nonionic detergent. Bioinformatic analysis demonstrated that a region of the capsid and SP1 domains junction of Gag is structurally similar to the amphipathic peptide magainin-1. This region accounts for integral changes in the physical properties of the membrane upon Gag adsorption, as we showed with the synthetic CA-SP1 junction peptide. Phenomenologically, membrane-adsorbed Gag could diminish the energetic cost of increasing the membrane area in a way similar to foam formation. We propose that Gag acts as a surface-active substance at the HIV budding site that softens the membrane at the place of Gag adsorption, lowering the energy for membrane bending. Finally, our experimental data and theoretical considerations give a lipid-centric view and common mechanism by which proteins could bend membranes, despite not having intrinsic curvature in their molecular surfaces or assemblies.

Structural Insights into Plant Viruses Revealed by Small-Angle X-ray Scattering and Atomic Force Microscopy.

The structural study of plant viruses is of great importance to reduce the damage caused by these agricultural pathogens and to support their biotechnological applications. Nowadays, X-ray crystallography, NMR spectroscopy and cryo-electron microscopy are well accepted methods to obtain the 3D protein structure with the best resolution. However, for large and complex supramolecular structures such as plant viruses, especially flexible filamentous ones, there are a number of technical limitations to resolving their native structure in solution. In addition, they do not allow us to obtain structural information about dynamics and interactions with physiological partners. For these purposes, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are well established. In this review, we have outlined the main principles of these two methods and demonstrated their advantages for structural studies of plant viruses of different shapes with relatively high spatial resolution. In addition, we have demonstrated the ability of AFM to obtain information on the mechanical properties of the virus particles that are inaccessible to other experimental techniques. We believe that these under-appreciated approaches, especially when used in combination, are valuable tools for studying a wide variety of helical plant viruses, many of which cannot be resolved by classical structural methods.

Proton Migration on Top of Charged Membranes.

Proton relay between interfacial water molecules allows rapid two-dimensional diffusion. An energy barrier, ΔGr‡, opposes proton-surface-to-bulk release. The ΔGr‡-regulating mechanism thus far has remained unknown. Here, we explored the effect interfacial charges have on ΔGr‡'s enthalpic and entropic constituents, ΔGH‡ and ΔGS‡, respectively. A light flash illuminating a micrometer-sized membrane patch of a free-standing planar lipid bilayer released protons from an adsorbed hydrophobic caged compound. A lipid-anchored pH-sensitive dye reported protons' arrival at a distant membrane patch. Introducing net-negative charges to the bilayer doubled ΔGH‡, while positive net charges decreased ΔGH‡. The accompanying variations in ΔGS‡ compensated for the ΔGH‡ modifications so that ΔGr‡ was nearly constant. The increase in the entropic component of the barrier is most likely due to the lower number and strength of hydrogen bonds known to be formed by positively charged residues as compared to negatively charged moieties. The resulting high ΔGr‡ ensured interfacial proton diffusion for all measured membranes. The observation indicates that the variation in membrane surface charge alone is a poor regulator of proton traffic along the membrane surface.

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aß) peptides in the brain. The processing of amyloid precursor protein (APP) into Aß is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP672-726 (corresponding to Aß1-55) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP672-726 is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP672-726. In membranes without cholesterol APP672-726, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP672-726. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.

Electrostatic Potentials Caused by the Release of Protons from Photoactivated Compound Sodium 2-Methoxy-5-nitrophenyl Sulfate at the Surface of Bilayer Lipid Membrane.

Lateral transport and release of protons at the water-membrane interface play crucial roles in cell bioenergetics. Therefore, versatile techniques need to be developed for investigating as well as clarifying the main features of these processes at the molecular level. Here, we experimentally measured the kinetics of binding of protons released from the photoactivated compound sodium 2-methoxy-5-nitrophenyl sulfate (MNPS) at the surface of a bilayer lipid membrane (BLM). We developed a theoretical model of this process describing the damage of MNPS coupled with the release of the protons at the membrane surface, as well as the exchange of MNPS molecules and protons between the membrane and solution. We found that the total change in the boundary potential difference across the membrane, ∆ϕb, is the sum of opposing effects of adsorption of MNPS anions and release of protons at the membrane-water interface. Steady-state change in the ∆ϕb due to protons decreased with the concentration of the buffer and increased with the pH of the solution. The change in the concentration of protons evaluated from measurements of ∆ϕb was close to that in the unstirred water layer near the BLM. This result, as well as rate constants of the proton exchange between the membrane and the bulk solution, indicated that the rate-limiting step of the proton surface to bulk release is the change in the concentration of protons in the unstirred layer. This means that the protons released from MNPS remain in equilibrium between the BLM surface and an adjacent water layer.

Influenza A Virus M1 Protein Non-Specifically Deforms Charged Lipid Membranes and Specifically Interacts with the Raft Boundary.

Topological rearrangements of biological membranes, such as fusion and fission, often require a sophisticated interplay between different proteins and cellular membranes. However, in the case of fusion proteins of enveloped viruses, even one molecule can execute membrane restructurings. Growing evidence indicates that matrix proteins of enveloped viruses can solely trigger the membrane bending required for another crucial step in virogenesis, the budding of progeny virions. For the case of the influenza A virus matrix protein M1, different studies report both in favor and against M1 being able to produce virus-like particles without other viral proteins. Here, we investigated the physicochemical mechanisms of M1 membrane activity on giant unilamellar vesicles of different lipid compositions using fluorescent confocal microscopy. We confirmed that M1 predominantly interacts electrostatically with the membrane, and its ability to deform the lipid bilayer is non-specific and typical for membrane-binding proteins and polypeptides. However, in the case of phase-separating membranes, M1 demonstrates a unique ability to induce macro-phase separation, probably due to the high affinity of M1's amphipathic helices to the raft boundary. Thus, we suggest that M1 is tailored to deform charged membranes with a specific activity in the case of phase-separating membranes.

Antimicrobial activity of photosensitizers: arrangement in bacterial membrane matters.

Porphyrins are well-known photosensitizers (PSs) for antibacterial photodynamic therapy (aPDT), which is still an underestimated antibiotic-free method to kill bacteria, viruses, and fungi. In the present work, we developed a comprehensive tool for predicting the structure and assessment of the photodynamic efficacy of PS molecules for their application in aPDT. We checked it on a series of water-soluble phosphorus(V) porphyrin molecules with OH or ethoxy axial ligands and phenyl/pyridyl peripheral substituents. First, we used biophysical approaches to show the effect of PSs on membrane structure and their photodynamic activity in the lipid environment. Second, we developed a force field for studying phosphorus(V) porphyrins and performed all-atom molecular dynamics simulations of their interactions with bacterial lipid membranes. Finally, we obtained the structure-activity relationship for the antimicrobial activity of PSs and tested our predictions on two models of Gram-negative bacteria, Escherichia coli and Acinetobacter baumannii. Our approach allowed us to propose a new PS molecule, whose MIC50 values after an extremely low light dose of 5 J/cm2 (5.0 ± 0.4 µg/mL for E. coli and 4.9 ± 0.8 µg/mL for A. baumannii) exceeded those for common antibiotics, making it a prospective antimicrobial agent.

Lysolipids regulate raft size distribution.

The lipid matrix of cellular membranes, directly and indirectly, regulates many vital functions of the cell. The diversity of lipids in membranes leads to the formation of ordered domains called rafts, which play a crucial role in signal transduction, protein sorting and other cellular processes. Rafts are believed to impact the development of different neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's ones, amyotrophic lateral sclerosis, some types of cancer, etc. These diseases correlate with the change in the membrane lipid composition resulting from an oxidative stress, age-related processes, dysfunction of proteins, and many others. In particular, a lot of studies report a significant rise in the level of lysolipids. Physicochemical properties of rafts are determined by membrane composition, in particular, by the content of lysolipids. Lysolipids may thus regulate raft-involving processes. However, the exact mechanism of such regulation is unknown. Although studying rafts in vivo still seems to be rather complicated, liquid-ordered domains are well observed in model systems. In the present study, we used atomic force microscopy (AFM) to examine how lysophospholipids influence the liquid-ordered domains in model ternary membranes. We demonstrated that even a small amount of lysolipids in a membrane significantly impacts domain size depending on the saturation of the lysolipid hydrocarbon tails and the amount of cholesterol. The mixture with the bigger relative fraction of cholesterol was more susceptible to the action of lysolipids. This data helped us to generalize our previous theoretical model of the domain size regulation by lipids with particular molecular shape expanding it to the case of lysolipids and dioleoylglycerol.

Peripheral Groups of Dicationic Pyrazinoporphyrins Regulate Lipid Membrane Binding.

Photodynamic therapy (PDT) is a widely used technique for skin cancer treatment and antimicrobial therapy. An improvement in PDT efficiency requires not only an increase in quantum yield of photosensitizer (PS) molecules but also their applicability for biological systems. Recently, we demonstrated that the activity of porphyrin-based PSs in the lipid membrane environment depends on the nature of the cation in the macrocycle due to its interactions with the lipid phosphate moiety, as well as the orientation of the PS molecules inside the membrane. Here, we report the synthesis, membrane binding properties and photodynamic efficiency of novel dicationic free-base, Ni(II) and Zn(II) pyrazinoporphyrins with terminal tetraalkylammonium units (2H-1, Ni-1 and Zn-1), to show the possibility to enhance the membrane binding of PS molecules, regardless of the central cation. All of these substances adsorb at the lipid membrane, while free-base and Zn(II) porphyrins actively generate singlet oxygen (SO) in the membranes. Thus, this study reveals a new way to tune the PDT activity of PSs in biological membranes through designing the structure of the peripheral groups in the macrocyclic photosensitizer.