Membrane fusion: a structural perspective on the interplay of lipids and proteins

The fusion of biological membranes is governed by the carefully orchestrated interplay of membrane proteins and lipids. Recently determined structures of fusion proteins, individual domains of fusion proteins and their complexes with regulatory proteins and membrane lipids have yielded much suggestive insight into how viral and intracellular membrane fusion might proceed. These structures may be combined with new knowledge on the fusion of pure lipid bilayer membranes in an attempt to begin to piece together the complex puzzle of how biological membrane fusion machines operate on membranes.     

Octa-O-propanoyl-beta-maltose: crystal structure, acyl stacking, related structures, and conformational analysis

The crystal structure of beta-maltose octapropanoate (1) was solved to improve understanding of di-, oligo-, and polysaccharide conformations. The O6 and O6' atoms are in gg and gt orientations, respectively. Extrapolation of the coordinates of the non-reducing residue and observed linkage bond and torsion angles of 1 [Formula: see text] yields a left-handed helix similar to amylose triacetate I. The phi and psi values of 1 are also similar to those of other crystalline, acylated maltose compounds as well as some hydroxyl-bearing molecules. Acylated maltose moieties are often stabilized by stacking of the carbonyl groups and alpha-carbons on O3 and O2' as well as by the exo-anomeric effect. The conformation of 1 is within the 1-kcal/mol contour on a hybrid energy map built with a dielectric constant of 7.5, but corresponds to higher energies on maps made with lower dielectric constants. In one region of phi,psi space, both hydroxyl-bearing and derivatized maltose moieties are found but no inter-residue, intramolecular hydrogen-bonding occurs. In another region, only hydroxyl-bearing molecules crystallize and O2'...O3 hydrogen bonds are always found. In agreement with the energy surfaces, amylose helices extrapolated from available linkage geometries were almost all left-handed.     

Design of peptides: synthesis, crystal structure, molecular conformation, and conformational calculations of N-Boc-L-Phe-Dehydro-Ala-OCH3.

It is noteworthy that the dehydro-Ala residue adopts an extended conformation that is different than those observed in dehydro-Phe, dehydro-Leu, and dehydro-Abu. The peptide N-Boc-L-Phe-dehydro-Ala-OCH3 (C18H24N2O5) was synthesized by the usual workup procedure and finally by converting N-Boc-L-Phe-L-Ser-OCH3 to N-Boc-L-Phe-dehydro-Ala- OCH3. It was crystallized from its solution in a methanol-water mixture at room temperature. The crystals belong to the monoclonic space group P2(1), with a = 9.577(1) A, b = 5.195(3) A, c = 19.563(3) A, beta = 94.67(5) degrees, V = 970.1(6) A3, Z = 2, dm = 1.201(5) Mg m-3, dc = 1.197(5) Mg m-3. The structure was determined using direct method procedures. It was refined by a full-matrix least-squares procedure to an R value of 0.048 for 1370 observed reflections. The C2 alpha-C2 beta distance is 1.327(8) A, while the bond angles N2-C2 alpha-C2' and C1'-N2-C2 alpha are 109.8(5) degrees and 127.8(5) degrees, respectively. The backbone adopts a nonspecific conformation with dehydro-Ala in a fully extended conformation with the following torsion angles: theta 1 = 175.2(4) degrees, omega 0 = 170.2(4) degrees, phi 1 = 135.8(5) degrees, psi 1 = -22.6(6) degrees, omega 1 = 168.5(5) degrees, phi 2 = -170.3(5) degrees, psi 2T = -178.6(5) degrees, theta T = 178.4(7) degrees. The rigid planar and trans conformation of dehydro-Ala forces Phe to adopt a strained conformation. The Boc group has a trans-trans conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlations of conformational parameters and equilibrium conformational states in a variety of beta-D-arabinonucleosides and their analogues.

H nuclear magnetic resonance spectroscopy has been applied to a study of the conformations of a variety of purine and pyrimidine beta-D-arabinofuranosyl nucleosides. The experimental results, together with data collected from the literature, demonstrated the existence of reasonably good correlations between the coupling constants made it possible to define more accurately, than hitherto possible, the conformational states between which equilibria exist in solution. The equilibrium for the arabinonucleosides differs from that previously established for ribonucleosides; in particular, structural modifications and solvent effects may appreciably modify the conformational states between which equilibria exist. Preliminary measurements on some arabinosides in the syn conformation about the glycosidic bond indicated that these do not conform to the foregoing correlations, and will require separate study. A correlation has also been established between the conformation of the arabinose ring and that of the exocyclic 5'-CH2OH group. For both purine and pyrimidine arabinonucleosides, the conformational state 3E of the arabinose ring coexists to some extent with a gauche-gauche conformation of the exocyclic 5'-CH2OH, as in the case of pyrimidine (but not purine) ribonucleosides. Application of the foregoing to some biological problems is described.     

Two conformational states in the crystal structure of the Homo sapiens cytoplasmic ribosomal decoding A site

The decoding A site of the small ribosomal subunit is an RNA molecular switch, which monitors codon–anticodon interactions to guarantee translation fidelity. We have solved the crystal structure of an RNA fragment containing two Homo sapiens cytoplasmic A sites. Each of the two A sites presents a different conformational state. In one state, adenines A1492 and A1493 are fully bulged-out with C1409 forming a wobble-like pair to A1491. In the second state, adenines A1492 and A1493 form non-Watson–Crick pairs with C1409 and G1408, respectively while A1491 bulges out. The first state of the eukaryotic A site is, thus, basically the same as in the bacterial A site with bulging A1492 and A1493. It is the state used for recognition of the codon/anticodon complex. On the contrary, the second state of the H.sapiens cytoplasmic A site is drastically different from any of those observed for the bacterial A site without bulging A1492 and A1493.     

Membrane lipids in plant-associated bacteria: their biosyntheses and possible functions

Membrane lipids in most bacteria generally consist of the glycerophospholipids phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine (PE). A subset of bacteria also possesses the methylated derivatives of PE, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine (PC). In Sinorhizobium meliloti, which can form a nitrogen-fixing root nodule symbiosis with Medicago spp., PC can be formed by two entirely different biosynthetic pathways, either the PE methylation pathway or the recently discovered PC synthase pathway. In the latter pathway, one of the building blocks for PC formation, choline, is obtained from the eukaryotic host. Under phosphorus-limiting conditions of growth, S. meliloti replaces its membrane phospholipids by membrane-forming lipids that do not contain phosphorus; namely, the sulfolipid sulfoquinovosyl diacylglycerol, ornithine-derived lipids, and diacylglyceryl-N,N,N-trimethylhomoserine. Although none of these phosphorus-free lipids is essential for growth in culture media rich in phosphorus or for the symbiotic interaction with the legume host, they are expected to have major roles under free-living conditions in environments poor in accessible phosphorus. In contrast, sinorhizobial mutants deficient in PC show severe growth defects and are completely unable to form nodules on their host plants. Even bradyrhizobial mutants with reduced PC biosynthesis can form only root nodules displaying reduced rates of nitrogen fixation. Therefore, in the cases of these microsymbionts, the ability to form sufficient bacterial PC is crucial for a successful interplay with their host plants.     

ApoA-I structure on discs and spheres. Variable helix registry and conformational states

Apolipoprotein A-I (apoA-I) readily forms discoidal high density lipoprotein (HDL) particles with phospholipids serving as an ideal transporter of plasma cholesterol. In the lipid-bound conformation, apoA-I activates the enzyme lecithin:cholesterol acyltransferase stimulating the formation of cholesterol esters from free cholesterol. As esterification proceeds cholesterol esters accumulate within the hydrophobic core of the discoidal phospholipid bilayer transforming it into a spherical HDL particle. To investigate the change in apoA-I conformation as it adapts to a spherical surface, fluorescence resonance energy transfer studies were performed. Discoidal rHDL particles containing two lipid-bound apoA-I molecules were prepared with acceptor and donor fluorescent probes attached to cysteine residues located at specific positions. Fluorescence quenching was measured for probe combinations located within repeats 5 and 5 (residue 132), repeats 5 and 6 (residues 132 and 154), and repeats 6 and 6 (residue 154). Results from these experiments indicated that each of the 2 molecules of discoidal bound apoA-I exists in multiple conformations and support the concept of a "variable registry" rather than a "fixed helix-helix registry." Additionally, discoidal rHDL were transformed in vitro to core-containing particles by incubation with lecithin:cholesterol acyltransferase. Compositional analysis showed that core-containing particles contained 11% less phospholipid and 633% more cholesterol ester and a total of 3 apoA-I molecules per particle. Spherical particles showed a lowering of acceptor to donor probe quenching when compared with starting rHDL. Therefore, we conclude that as lipid-bound apoA-I adjusts from a discoidal to a spherical surface its intermolecular interactions are significantly reduced presumably to cover the increased surface area of the particle.     

Polar group interaction and molecular packing of membrane lipids: The crystal structure of lysophosphatidylethanolamine

The conformation and molecular packing of 3-palmitoyl-dl-glycerol-1-phosphoryl-ethanolamine has been determined by a single crystal analysis (R = 0.115); it crystallizes in the monoclinic space group $\text{P2}{}_1\text{a}$ with a unit cell of $\text{a = 7.66}\text{A}\text{?}\text{, b = 9.08}\text{A}\text{?}\text{, c = 37.08}\text{A}\text{?}\text{and}\text{β = 90.2 °}$, with four molecules per unit cell. The molecules exist as configurational and conformational enantiomers and pack in a bilayer arrangement. The phosphorylethanolamine groups have an orientation parallel to the layer surface. The hydrocarbon chains are arranged according to the T∥ chain packing mode and adopt an extreme tilt of 57.5 ° with respect to the layer normal. The free glycerol hydroxyl group forms an intramolecular hydrogen bond with, a phosphate oxygen and thus affects the conformation and orientation of the head group. The phosphorylethanolamine dipoles are oriented parallel to each other in double rows, while they are antiparallel and form a continuous network in dilauroylphosphatidylethanolamine (Elder et al., 1977). The area per molecule in 3-palmitoyl-dl-glycerol-1-phosphorylethanolamine (34.8 Ų) is less than in diacylphosphatidylethanolamine (38.6 Ų), indicating that in the latter the hydrocarbon chains determine the molecular cross-section. The significance of the interaction and space requirement of the phosphorylethanolamine group for the phase behaviour of phosphatidylethanolamine is discussed.     

Glycerol conformation and molecular packing of membrane lipids: The crystal structure of 2,3-dilauroyl-d-glycerol

The single-crystal structure of 2,3-dilauroyl-d-glycerol has been determined by Patterson rotation and translation methods and refined to R = 0.069. 2,3-dilauroyl-d-glycerol crystallizes in the monoclinic space group P2₁, with unit cell dimensions: $\text{a = 5.46}\text{A}\text{?}\text{, b = 7.59}\text{A}\text{?}\text{, c = 34.2}\text{A}\text{?}\text{and}\text{β = 93.1 °}$, and with two molecules per unit cell. The molecules have their hydrocarbon chains aligned parallel, and are arranged in a bilayer structure. The chain stacking is achieved by a bend in the fatty acid. The hydrocarbon chains pack according to the orthorhombic perpendicular chain packing mode, and are tilted 26.5 ° from the layer normal.The structural features of 2,3-dilauroyl-d-glycerol have been analysed with reference to the corresponding hydrophobic moieties in the crystal structures of different membrane lipids. The glycerol group in 2,3-dilauroyl-d-glycerol is oriented parallel to the layer plane, but changes to an approximately layer-perpendicular orientation when a polar group is attached. The molecular conformation of the glycerol-dicarboxylic ester group, however, is identical in both the absence and presence of a head group, indicating extensive conformational restrictions for this group due to both intrinsic properties and chain stacking. The gathered data provide detailed information on the structural properties of the hydrophobic moiety of membrane lipids.     

The electrical interplay between proteins and lipids in membranes

All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure.The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid–protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid–protein interactions.     

Calcium-binding to non-ionic lipids: crystal structure of a calcium chloride complex of geraniol.

X-ray diffraction data were used to determine the crystal structure of a calcium chloride complex of geraniol. The geraniol molecules assume a bilayer arrangement, with channels of calcium and chloride ions separating the bilayers. Each calcium ion is coordinated to the hydroxyl groups of two symmetry-related geraniol molecules and to four chloride ions. Our results demonstrate that hydrophobic interactions within a lipid bilayer can lead to an arrangement of hydroxyl groups suitable for binding calcium ions. Similar interactions may be involved in the calcium-binding sites on membrane surfaces.     

Calmodulin, conformational states, and calcium signaling. A single-molecule perspective

Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca2+-ATPase.     

The 1.6 A crystal structure of E. coli argininosuccinate synthetase suggests a conformational change during catalysis.

BACKGROUND: Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. RESULTS: The crystal structure of E. coli AS (EAS) has been determined by the use of selenomethionine incorporation and MAD phasing. The structure has been refined at 1.6 A resolution in the absence of its substrates and at 2.0 A in the presence of aspartate and citrulline (EAS*CIT+ASP). Each monomer of this tetrameric protein has two structural domains: a nucleotide binding domain similar to that of the "N-type" ATP pyrophosphatase class of enzymes, and a novel catalytic/multimerization domain. The EAS*CIT+ASP structure clearly describes the binding of citrulline at the cleft between the two domains and of aspartate to a loop of the nucleotide binding domain, whereas homology modeling with the N-type ATP pyrophosphatases has provided the location of ATP binding. CONCLUSIONS: The first three-dimensional structures of AS are reported. The fold of the nucleotide binding domain confirms AS as the fourth structurally defined member of the N-type ATP pyrophosphatases. The structures identify catalytically important residues and suggest the requirement for a conformational change during the catalytic cycle. Sequence similarity between the bacterial and human enzymes has been used for providing insight into the structural and functional effects of observed clinical mutations.     

The conformational states of cytochrome oxidase in the mitochondrion.

THE Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized" oxidase. With increasing activity the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.     

Fission of biological membranes: interplay between dynamin and lipids

Membrane budding and fission are the key stages of ubiquitous processes of formation of intracellular transport vesicles. We present a theoretical consideration of one of the most important types of fission machinery, which is mediated by GTPase dynamin and controlled by lipid composition of the membrane. We suggest a mechanism for collapse of a membrane neck driven by interplay between the dynamin collar and the bending elastic energy of the neck membrane. The collar plays a role of a rigid external skeleton, which imposes mechanical constraints on the neck. We show that in certain conditions the membrane of the neck loses its stability and collapses. Collapse can result from: (i) shifting of the spontaneous curvature of the neck membrane towards negative values, (ii) stretching of the dynamin collar, (iii) tightening of the dynamin collar. The three factors can act separately or concertedly. The suggested model accounts for the major experimental knowledge on membrane fission mediated by dynamin. It includes the elements of all previous models of dynamin action based on different sets of experimental results [Sever et al., Traffic 2000; 1: 385-392]. It reconciles, at least partially, the apparent contradictions between the existing alternative views on biomembrane fission machinery.     

Conformation and packing properties of membrane lipids: the crystal structure of sodium dimyristoylphosphatidylglycerol

The conformation and molecular packing of sodium 1,2-dimyristoyl-sn-glycero-phospho-rac-glycerol (DMPG) have been determined by single crystal analysis (R = 0.098). The lipid crystallizes in the monoclinic spacegroup P2(1) with the unit cell dimensions a = 10.4, b = 8.5, c = 45.5 A and beta = 95.2 degrees. There are two independent molecules (A and B) in the asymmetric unit which with respect to configuration and conformation of their glycerol headgroup are mirror images. The molecules pack tail to tail in a bilayer structure. The phosphoglycerol headgroups have a layer-parallel orientation giving the molecules an L-shape. At the bilayer surface the (-) phosphoglycerol groups are arranged in rows which are separated by rows of (+) sodium ions. Laterally the polar groups interact by an extensive network of hydrogen, ionic and coordination bonds. The packing cross-section per molecule is 44.0 A2. The hydrocarbon chains are tilted (29 degrees) and have opposite inclination in the two bilayer halves. In the chain matrix the chain planes are arranged according to a so far unknown hybride packing mode which combines the features of T parallel and O perpendicular subcells. The two fatty acid substituted glycerol oxygens have mutually a - synclinal rather than the more common + synclinal conformation. The conformation of the diacylglycerol part of molecule A and B is distinguished by an axial displacement of the two hydrocarbon chains by four methylene units. This results in a reorientation of the glycerol back bone and a change in the conformation and stacking of the hydrocarbon chains. In molecule A the beta-chain is straight and the gamma-chain is bent while in molecule B the chain conformation is reversed.     

The conformational states of oxidized and oxygenated beef-heart cytochrome c oxidase.

1. The "oxygenated" form of cytochrome has been generated by treatment of the enzyme with ascorbic acid. 2. "Oxygenated oxidase" so generated is stable over long periods (24 h). 3. Sedimentation velocity experiments have shown the "oxygenated" oxidase to be a less compact molecule than the oxidized.