Acyl-chain correlation in membrane fusion intermediates: x-ray diffraction from the rhombohedral lipid phase

We have studied the acyl-chain conformation in stalk phases of model membranes by x-ray diffraction from oriented samples. As an equilibrium lipid phase induced by dehydration, the stalk or rhombohedral phase exhibits lipidic passages (stalks) between adjacent bilayers, representing a presumed intermediate state in membrane fusion. From the detailed analysis of the acyl-chain correlation peak, we deduce the structural parameters of the acyl-chain fluid above, at, and below the transition from the lamellar to rhombohedral state, at the molecular level.     

Evidence of cholesterol accumulated in high curvature regions: implication to the curvature elastic energy for lipid mixtures

Recent experiments suggested that cholesterol and other lipid components of high negative spontaneous curvature facilitate membrane fusion. This is taken as evidence supporting the stalk-pore model of membrane fusion in which the lipid bilayers go through intermediate structures of high curvature. How do the high-curvature lipid components lower the free energy of the curved structure? Do the high-curvature lipid components modify the average spontaneous curvature of the relevant monolayer, thereby facilitate its bending, or do the lipid components redistribute in the curved structure so as to lower the free energy? This question is fundamental to the curvature elastic energy for lipid mixtures. Here we investigate the lipid distribution in a monolayer of a binary lipid mixture before and after bending, or more precisely in the lamellar, hexagonal, and distorted hexagonal phases. The lipid mixture is composed of 2:1 ratio of brominated di18:0PC and cholesterol. Using a newly developed procedure for the multiwavelength anomalous diffraction method, we are able to isolate the bromine distribution and reconstruct the electron density distribution of the lipid mixture in the three phases. We found that the lipid distribution is homogenous and uniform in the lamellar and hexagonal phases. But in the distorted hexagonal phase, the lipid monolayer has nonuniform curvature, and cholesterol almost entirely concentrates in the high curvature region. This finding demonstrates that the association energies between lipid molecules vary with the curvature of membrane. Thus, lipid components in a mixture may redistribute under conditions of nonuniform curvature, such as in the stalk structure. In such cases, the spontaneous curvature depends on the local lipid composition and the free energy minimum is determined by lipid distribution as well as curvature.     

Method of x-ray anomalous diffraction for lipid structures

The structures of the unit cells of lipid phases that exhibit long-range crystalline order but short-range liquid-like disorder are of biological interests. In particular, the recently discovered rhombohedral phase has a unit cell containing either the structure of a membrane fusion intermediate state or that of a peptide-induced transmembrane pore, depending on the lipid composition and participating peptides. Diffraction from such systems generally presents a difficult phase problem. The existing methods of phase determination all have their limitations. Therefore it is of general interest to develop a new phasing method. The method of multi-wavelength anomalous dispersion is routinely used in protein crystallography, but the same method is difficult for lipid systems for the practical reason that the commonly used lipid samples for diffraction do not have a well-defined thickness. Here we describe a practical approach to use the multi-wavelength anomalous dispersion method for lipid structures. The procedure is demonstrated with the lamellar phase of a brominated lipid. The method is general to all phases as long as anomalous diffraction is applicable.     

The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms

We studied the mechanism of the lamellar-to-inverted hexagonal (L alpha/H[II]) phase transition, using time-resolved cryotransmission electron microscopy (TRC-TEM), 31P-NMR, and differential scanning calorimetry. The transition was initiated in dispersions of large unilamellar vesicles of dipalmitoleoyl phosphatidylethanolamine (DiPoPE). We present evidence that the transition proceeds in three steps. First, many small connections form between apposed membranes. Second, the connections aggregate within the planes of the bilayers, forming arrays with hexagonal order in some projections. Third, these quasihexagonal structures elongate into small domains of H(II) phase, acquiring lipid molecules by diffusion from contiguous bilayers. A previously proposed membrane fusion mechanism rationalizes these results. The modified stalk theory predicts that the L alpha/H(II) phase transition involves some of the same intermediate structures as membrane fusion. The small interbilayer connections observed via TRC-TEM are compatible with the structure of a critical intermediate in the modified stalk mechanism: the trans monolayer contact (TMC). The theory predicts that 1) TMCs should form starting at tens of degrees below TH; 2) when TMCs become sufficiently numerous, they should aggregate into transient arrays like the quasihexagonal arrays observed here by TRC-TEM; and 3) these quasihexagonal arrays can then elongate directly into H(II) phase domains. These predictions rationalize the principal features of our data, which are incompatible with the other transition mechanisms proposed to date. Thus these results support the modified stalk mechanism for both membrane fusion and the L alpha/H(II) phase transition. We also discuss some implications of the modified stalk theory for fusion in protein-containing systems. Specifically, we point out that recent data on the effects of hydrophobic peptides and viral fusion peptides on lipid phase behavior are consistent with an effect of the peptides on TMC stability.     

A novel phase of compressed bilayers that models the prestalk transition state of membrane fusion

The force model of protein-mediated membrane fusion hypothesizes that fusion is driven by mechanical forces exerted on the membranes, but many details are unknown. Here, we investigated by x-ray diffraction the consequence of applying compressive force on a stack of membranes against the hydration barrier. We found that as the osmotic pressure increased, the lamellar phase transformed first to a new phase of tetragonal lattice (T-phase) over a narrow range of relative humidity, and then to a phase of rhombohedral lattice. The unit cell structure changed from parallel bilayers to a bent configuration with a point contact between adjacent bilayers and then to the stalk hemifusion configuration. The T-phase is discussed as a possible transition state in the membrane merging pathway of fusion. We estimate the work required to form the T-phase and the subsequent hemifusion-stalk-resembling R-phase. The work for the formation of a stalk is compatible with the energy estimated to be released by several SNARE complexes.     

New phases of phospholipids and implications to the membrane fusion problem

Membrane fusion is a ubiquitous process in eukaryotic cells. When two membranes fuse, lipid must undergo molecular rearrangements at the point of merging. To understand how lipid structure transitions occur, scientists studied the phase transition of lipid between the lamellar (L(alpha)) phase and the inverted hexagonal (H(II)) phase, based on the idea that lipid must undergo a similar rearrangement as in fusion. However, previous investigations on the system of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE) did not reveal intermediate phases between the L(alpha) and H(II) phases. Recently, we found a rhombohedral phase of diphytanoylphosphatidylcholine between its L(alpha) and H(II) phases using substrate-supported samples. Here we report the observation of two new phases in the DOPC-DOPE system: a rhombohedral phase and a distorted hexagonal phase. The rhombohedral phase confirms the stalk hypothesis for the L(alpha)-H(II) transition, but the phase of stable stalks exists only for a certain range of spontaneous curvature. The distorted hexagonal phase exists only in a lipid mixture. It implies that lipids may demix to adjust its local spontaneous curvature in order to achieve energy minimum under stress.     

On the theory of membrane fusion. The stalk mechanism

Based on literary data, conditions necessary for membrane fusion are discussed. It is proposed that fusion mechanisms should be classified according to the primary act involving a change in the membrane structure. Two principal fusion mechanisms are identified: the stalk mechanism, starting with the appearance of a stalk between approaching membranes, and the adhesion mechanism which involves bilayer reorganization as a result of a tight junction of the membranes. The origin and evolution of the monolayer and bilayer stalks between membranes are analysed. Using the expression for the elastic energy of the stalk it was possible to find the value of the spontaneous curvature of its membrane, Ks, at which the existence of a stalk is in principle possible. It is shown that, within the framework of the stalk mechanism, there exists a possibility of either the formation of a stalk of a finite radius, or complete fusion. The Ks values have been determined at which one of the variants occur. The energy barrier of the hydrophobic interaction and the elastic energy barrier, which have to be overcome by the membranes to form the stalk are analysed. The theoretical analysis of the stalk formation mechanism is supported by experimental data. It has been shown by freeze-fracture electron microscopy that the addition of Ca+2, Mg+2, Mn+2 or Cd+2 to suspensions of egg phosphatidylcholine and cardiolipin (1:1 or 3:1) leads to the formation of numerous intramembrane particles (imp's) and crater-like (stalk) structures.     

Structure of the alamethicin pore reconstructed by x-ray diffraction analysis

We reconstructed the electron density profile of the alamethicin-induced transmembrane pore by x-ray diffraction. We prepared fully hydrated multiple bilayers of alamethicin-lipid mixtures in a condition where pores were present, as established previously by neutron in-plane scattering in correlation with oriented circular dichroism. At dehydrated conditions, the interbilayer distance shortened and the interactions between bilayers caused the membrane pores to become long-ranged correlated and form a periodically ordered lattice of rhombohedral symmetry. To resolve the phase problem of diffraction, we used a brominated lipid and performed multiwavelength anomalous diffraction at the bromine K edge. The result unambiguously shows that the alamethicin pore is of the barrel-stave type consisting of eight alamethicin helices. This pore structure corresponds to the stable pores detected by neutron in-plane scattering in fully hydrated fluid bilayers at high peptide/lipid ratios, which are the conditions at which alamethicin was tested for its antibacterial activity.     

Stalk model of membrane fusion: solution of energy crisis.

Membrane fusion proceeds via formation of intermediate nonbilayer structures. The stalk model of fusion intermediate is commonly recognized to account for the major phenomenology of the fusion process. However, in its current form, the stalk model poses a challenge. On one hand, it is able to describe qualitatively the modulation of the fusion reaction by the lipid composition of the membranes. On the other, it predicts very large values of the stalk energy, so that the related energy barrier for fusion cannot be overcome by membranes within a biologically reasonable span of time. We suggest a new structure for the fusion stalk, which resolves the energy crisis of the model. Our approach is based on a combined deformation of the stalk membrane including bending of the membrane surface and tilt of the hydrocarbon chains of lipid molecules. We demonstrate that the energy of the fusion stalk is a few times smaller than those predicted previously and the stalks are feasible in real systems. We account quantitatively for the experimental results on dependence of the fusion reaction on the lipid composition of different membrane monolayers. We analyze the dependence of the stalk energy on the distance between the fusing membranes and provide the experimentally testable predictions for the structural features of the stalk intermediates.     

Gap junction structures: Analysis of the x-ray diffraction data

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.     

Membrane fusion: stalk model revisited.

Membrane fusion is believed to proceed via intermediate structures called stalks. Mathematical analysis of the stalk provided the elastic energy involved in this structure and predicted the possible evolution of the overall process, but the energies predicted by the original model were suspiciously high. This was due to an erroneous assumption, i.e., that the stalk has a figure of revolution of a circular arc. Here we abandon this assumption and calculate the correct shape of the stalk. We find that it can be made completely stress free and, hence, its energy, instead of being positive and high can become negative, thus facilitating the fusion process. Based on our new calculations, the energies of hemifusion, of complete fusion, and of the pore in a bilayer were analyzed. Implications for membrane fusion and lipid phase transitions are discussed.     

Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography

In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.     

Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.     

X-ray diffraction studies of retinal rods. I. Structure of the disc membrane,effect of illumination

The structure of the retinal rod disc membrane and its modifications upon bleaching have been studied by X-ray diffraction. Three types of preparations are used: functioning isolated frog retina, isolated rods from frog retina, oriented by a magnetic field, and stacked discs from cattle retina. X-rays are detected by a position-sensitive linear counter. Diffraction spectra are obtained in 10–100 s.The electron density profile favors models where the rhodopsin molecule spans the whole thickness of the membrane. Upon bleaching, a small increase of electron density appears instantly at the cytoplasmic edge of the membrane. In the intact retina this structural change is accompanied by disorder and slow swelling reactions which are not observed in the isolated rod outer segment.The diffraction signal arising from the protein distribution in the plane of the membrane has been reinvestigated carefully. Patterns identical to those of Blasie (Blasie (1969) J. Mol. Biol. 39, 407 and Blasie (1972) Biophys. J. 12, 191) can be obtained but these are shown to be dominated by artefacts. The actual signal is a single broad band around (55 Å)^-1, upon which bleaching has a negligible effect. No measurable displacement of rhodopsin in the thickness of the membrane occurs upon bleaching.Temperature effects on the protein distribution are found to be large only for disc membranes from cattle retina. In this material from a warm-blooded animal those effects are correlated with the occurrence, upon lowering the temperature, of a partial phase transition of the paraffin chains of the lipids. The position and the slope of the transition are not sensitive to bleaching.     

The Gaussian curvature elastic energy of intermediates in membrane fusion

The Gaussian curvature elastic energy contribution to the energy of membrane fusion intermediates has usually been neglected because the Gaussian curvature elastic modulus, κ, was unknown. It is now possible to measure κ for phospholipids that form bicontinuous inverted cubic (Q_II) phases. Here, it is shown that one can estimate κ for lipids that do not form Q_II phases by studying the phase behavior of lipid mixtures. The method is used to estimate κ for several lipid compositions in excess water. The values of κ are used to compute the curvature elastic energies of stalks and catenoidal fusion pores according to recent models. The Gaussian curvature elastic contribution is positive and similar in magnitude to the bending energy contribution: it increases the total curvature energy of all the fusion intermediates by 100 units of k_BT or more. It is important to note that this contribution makes the predicted intermediate energies compatible with observed lipid phase behavior in excess water. An order-of-magnitude fusion rate equation is used to estimate whether the predicted stalk energies are consistent with the observed rates of stalk-mediated processes in pure lipid systems. The current theory predicts a stalk energy that is slightly too large, by ∼30 k_BT, to rationalize the observed rates of stalk-mediated processes in phosphatidylethanolamine or N-monomethylated dioleoylphosphatidylethanolamine systems. Despite this discrepancy, the results show that models of fusion intermediate energy are accurate enough to make semiquantitative predictions about how proteins mediate biomembrane fusion. The same rate model shows that for proteins to drive biomembrane fusion at observed rates, they have to perform mediating functions corresponding to a reduction in the energy of a purely lipidic stalk by several tens of k_BT. By binding particular peptide sequences to the monolayer surface, proteins could lower fusion intermediate energies by altering the elastic constants of the patches of lipid monolayer that form the stalk. Here, it is shown that if peptide binding changes κ or some other combinations of local elastic constants by only tens of percents, the stalk energy and the energy of catenoidal fusion pores would decrease by tens of k_BT relative to the pure lipid value. This is comparable to the required mediating effect. The curvature energies of stalks and catenoidal fusion pores have almost the same dependence on monolayer elastic constants as the curvature energies of the rhombohedral and Q_II phases; respectively. The effects of isolated fusion-relevant peptides on the energies of these intermediates can be determined by studying the effects of the peptides on the stability of rhombohedral and Q_II phases.     

Field theoretic study of bilayer membrane fusion. I. Hemifusion mechanism

Self-consistent field theory is used to determine structural and energetic properties of metastable intermediates and unstable transition states involved in the standard stalk mechanism of bilayer membrane fusion. A microscopic model of flexible amphiphilic chains dissolved in hydrophilic solvent is employed to describe these self-assembled structures. We find that the barrier to formation of the initial stalk is much smaller than previously estimated by phenomenological theories. Therefore its creation it is not the rate-limiting process. The relevant barrier is associated with the rather limited radial expansion of the stalk into a hemifusion diaphragm. It is strongly affected by the architecture of the amphiphile, decreasing as the effective spontaneous curvature of the amphiphile is made more negative. It is also reduced when the tension is increased. At high tension the fusion pore, created when a hole forms in the hemifusion diaphragm, expands without bound. At very low membrane tension, small fusion pores can be trapped in a flickering metastable state. Successful fusion is severely limited by the architecture of the lipids. If the effective spontaneous curvature is not sufficiently negative, fusion does not occur because metastable stalks, whose existence is a seemingly necessary prerequisite, do not form at all. However if the spontaneous curvature is too negative, stalks are so stable that fusion does not occur because the system is unstable either to a phase of stable radial stalks, or to an inverted-hexagonal phase induced by stable linear stalks. Our results on the architecture and tension needed for successful fusion are summarized in a phase diagram.     

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.     

Molecular Dynamics Simulation Analysis of Membrane Defects and Pore Propensity of Hemifusion Diaphragms

Membrane fusion often exhibits slow dynamics in electrophysiological experiments, involving prespike foot and fusion pore-flickering, but the structural basis of such phenomena remains unclear. Hemifusion intermediates have been implicated in the early phase of membrane fusion. To elucidate the dynamics of formation of membrane defects and pores within the hemifusion diaphragm (HD), atomistic and coarse-grained models of hemifusion intermediates were constructed using dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine membranes. The work necessary to displace a lipid molecule to the hydrophobic core of the bilayer was measured. For a lipid within the HD with radius of 4 nm, the work was ∼80 kJ/mol, similar to that in a planar bilayer. The work was much less (∼40 kJ/mol) when the HD was surrounded by a steep stalk, i.e., stalk wings forming a large angle at the junction of three bilayers. In the latter case, the lipid displacement engendered formation of a pore contacting the HD rim. The work was similarly small (40 kJ/mol) for a small HD of 1.5 nm radius, where a pore formed and grew rapidly, quickly generating a toroidal structure (<40 ns). Combining the steep stalk and the small HD decreased the work further, although quantitative analysis was difficult because the latter system was not in a stable equilibrium state. Results suggest that fine tuning of fusion dynamics requires strict control of the HD size and the angle between the expanded stalk and HD. In additional free simulations, the steep stalk facilitated widening of a preformed pore contacting the HD rim.     

Normal-mode refinement of anisotropic thermal parameters for potassium channel KcsA at 3.2 A crystallographic resolution

We report a normal-mode method for anisotropic refinement of membrane-protein structures, based on a hypothesis that the global near-native-state disordering of membrane proteins in crystals follows low-frequency normal modes. Thus, a small set of modes is sufficient to represent the anisotropic thermal motions in X-ray crystallographic refinement. By applying the method to potassium channel KcsA at 3.2 A, we obtained a structural model with an improved fit with the diffraction data. Moreover, the improved electron density maps allowed for large structural adjustments for 12 residues in each subunit, including the rebuilding of 3 missing side chains. Overall, the anisotropic KcsA structure at 3.2 A was systematically closer to a 2.0 A KcsA structure, especially in the selectivity filter. Furthermore, the anisotropic thermal ellipsoids from the refinement revealed functionally relevant structural flexibility. We expect this method to be a valuable tool for structural refinement of many membrane proteins with moderate-resolution diffraction data.     

X-Ray Diffraction Study in Water of Lipids Extracted from Human Erythrocytes: The Position of Cholesterol in the Lipid Lamellae

Lipids, carefully extracted from fresh human erythrocytes, form liquid-crystalline structures in water. A phase diagram of this system was constructed, characterizing, by X-ray diffraction, the structures which form as a function of concentration of lipid and temperature. One extended range of concentration of the phase diagram, in which a single lamellar phase exists, permitted further analysis of the diffraction data. This phase consists of lipid layers of constant thickness separated by water layers of varying thickness according to the water content of the system. The distribution of the electron density is precisely analyzed and the amplitude of the reflections is, at all concentrations, proportional to the Fourier Transform of an isolated lipid layer. This shows that the lipid layer is filled with the hydro-carbon chains of the phospholipids and is covered on both sides by their hydrophilic groups. Cholesterol, present in high concentration in erythrocyte membranes, is located so that part of its steroid nucleus is between the polar groups of the phospholipid molecules while the rest of the molecule extends into the inner hydrocarbon layer.