Pathological laughter: common vs. unusual aetiology and presentation

Cell locomotion in amoeboid nematode sperm is generated by the vectorial assembly and bundling of filaments of the major sperm protein (MSP). MSP filaments are constructed from two helical subfilaments and here we describe the structure of putative MSP subfilament helices determined by X-ray crystallography at 3.3 A resolution. In addition to establishing the interfaces involved in polymerization, this structural model shows that the MSP helices are constructed from dimers and have no overall polarity, suggesting that it is unlikely that molecular motors play a direct role in the generation of protrusive force in these amoeboid cells.     

The motile major sperm protein (MSP) from Ascaris suum is a symmetric dimer in solution

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exist in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.     

2.5 A resolution crystal structure of the motile major sperm protein (MSP) of Ascaris suum

We have determined the structure of the Ascaris major sperm protein (MSP) to 2.5 A resolution using X-ray crystallography. The MSP polypeptide chain has an immunoglobulin-like fold based on a seven-stranded beta sandwich. In two strands, cis-proline residues impart distinctive kinks, and overall the structure most closely resembles that of the N-terminal domain of the bacterial chaperonin, PapD. In the C2 crystal form which we have solved here, two MSP chains are tightly associated in the asymmetric unit and are related by a non-crystallographic 2-fold rotation axis. This arrangement almost certainly represents the MSP dimer that is present in solution. Additionally, the arrangement of two MSP dimers at one of the crystallographic 2-fold axes in the 215 A unit cell suggests a possible mode for the assembly of MSP into the filaments which promote cell movement. This dimer-dimer association is based on a beta sheet extension mechanism between adjoining MSP monomers which resembles the interaction between PapD and its protein substrate.     

Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.     

Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy

Despite the development of vaccines, the hepatitis B virus remains a major cause of human liver disease. The virion consists of a lipoprotein envelope surrounding an icosahedral capsid composed of dimers of a 183-residue protein, 'core antigen' (HBcAg). Knowledge of its structure is important for the design of antiviral drugs, but it has yet to be determined. Residues 150-183 are known to form a protamine-like domain required for packaging RNA, and residues 1-149 form the 'assembly domain' that polymerizes into capsids and, unusually for a capsid protein, is highly alpha-helical. Density maps calculated from cryo-electron micrographs show that the assembly domain dimer is T-shaped: its stem constitutes the dimer interface and the tips of its arms make the polymerization contacts. By refining the procedures used to calculate the map, we have extended the resolution to 9 A, revealing major elements of secondary structure. In particular, the stem, which protrudes as a spike on the capsid's outer surface, is a 4-helix bundle, formed by the pairing of alpha-helical hairpins from both subunits.     

Structure and activity of the hairpin ribozyme in its natural junction conformation: effect of metal ions

The natural form of the hairpin ribozyme consists of a four-way RNA junction of which the single-stranded loop-carrying helices are adjacent arms. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. We find that the junction-based form of the hairpin ribozyme is active in magnesium, calcium, or strontium ions, but not in manganese, cadmium, or sodium ions. Using fluorescence resonance energy transfer experiments, we have investigated the global structure of the ribozyme. The basic folding of the construct is based on pairwise helical stacking, so that the two loop-carrying arms are located on opposite stacked helical pairs. In the presence of magnesium, calcium, or strontium ions, the junction of the ribozyme undergoes a rotation into a distorted antiparallel geometry, creating close physical contact between the two loops. Manganese ions induce the same global folding, but no catalytic activity; this change in global conformation is therefore necessary but not sufficient for catalytic activity. Fitting the dependence of the conformation on ionic concentration to a two-state model suggests that cooperative binding of two ions is required to bring about the folding. However, further ion binding is required for cleavage activity. Cobalt hexammine ions also bring about global folding, while spermidine generates a more symmetrical form of the antiparallel structure. Cadmium ions generate a different folded form, interpreted in terms of close loop-loop association while the junction is unfolded. Sodium ions were unable to induce any folding of the ribozyme, which remained slightly parallel. These results are consistent with a folding process induced by the binding of two group IIA metal ions, distributed between the junction and the loop interface.     

Sequential assignments and identification of secondary structure elements of the colicin E9 immunity protein in solution by homonuclear and heteronuclear NMR

1H-1H, 1H-15N, and 1H-1H-15N multidimensional NMR spectroscopic studies of the 86 amino acid protein that provides immunity against the DNase action of colicin E9 are reported. Through a combination of 2D NOESY and TOCSY and 3D TOCSY-HMQC, NOESY-HMQC, and HMQC-NOESY-HMQC experiments, almost complete 1H NMR and backbone 15N NMR assignments have been obtained, and the secondary structure of the protein has been partially elucidated. Approximately 50% of the protein forms three helices. The specificity determining region of the DNase immunity protein, identified from previously reported biochemical studies to include residues 32-40, is helical, indicating that the protein-protein interaction involves residues from at least one helix.     

Intrastrand cross-linked actin between Gln-41 and Cys-374. II. Properties of cross-linked oligomers

Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s(o)20,w = 5.55 +/- 0.22 S), trimers (s(o)20,w = 6.93 +/- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment (S1) to cross-linked dimers in the presence of pyrenyl G-actin revealed about 2 orders of magnitude stronger binding of the first than that of the second S1 molecule to actin dimer. Under similar conditions the unpolymerized cross-linked actin species activated the MgATPase of S1 only severalfold compared to 70-fold activation by F-actin. The cross-linked dimers, trimers, and oligomers were polymerized into filaments by MgCl2 faster than un-cross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than un-cross-linked actin filaments. Small amounts of cross-linked actin dimers nucleated S1-induced polymerization of actin, but the polymerization by S1 was inhibited for pure populations of cross-linked dimers, trimers, and oligomers. The cross-linked dimers did not decrease the kinetic difference between the polymerization of actin by S1 isozymes S1(A1) and S1(A2). According to electron microscopy evidence, cross-linked actin oligomers polymerized by S1 yielded much shorter arrowhead structures than the un-cross-linked actin. These results indicate the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by S1.     

A buried polar interaction can direct the relative orientation of helices in a coiled coil

Coiled coils consist of bundles of two or more alpha-helices that are aligned in a parallel or an antiparallel relative orientation. The designed peptides, Acid-p1 and Base-p1, associate in solution to form a parallel, heterodimeric two-stranded coiled coil [O'Shea, E. K., Lumb, K. J., and Kim, P. S. (1993) Curr. Biol. 3, 658]. The buried interface of this complex is formed by hydrophobic Leu residues, with the exception of an Asn residue from each strand that is positioned to engage in a buried polar interaction. Substitution of these buried Asn residues by Leu residues results in a loss of structural uniqueness, as evidenced by a lack of a particular helix orientation in the Acid-Base coiled-coil complex [Lumb, K. J., and Kim, P. S. (1995) Biochemistry 34, 8642]. Here, we alter the positions of the Asn residues in the Acid and Base peptides such that a buried polar interaction is only expected to occur when the helices are in an antiparallel orientation. The resulting peptides, Acid-a1 and Base-a1, associate to form a helical heterodimer, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation. The helix orientation preference has been measured using covalently linked, disulfide-containing heterodimers in which the constituent peptides are constrained to interact in either a parallel or an antiparallel orientation. Although both the parallel and antiparallel heterodimers form stable, helical structures, the antiparallel heterodimer is the predominant species at equilibrium when the heterodimers are allowed to undergo thiol-disulfide exchange. In addition, the antiparallel heterodimer is more stable to chemical denaturation than the parallel counterpart by approximately 2.3 kcal/mol. These results demonstrate that a single buried polar interaction in the interface between the helices of a coiled coil is sufficient to determine the relative orientation of its constituent helices.     

Preservation of native conformation during aluminium-induced aggregation of tau protein

Aluminium exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27Al NMR spectroscopy. The CD studies revealed a five-fold increase in the observed elipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. 27Al NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits, the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for this process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.     

Characterization of the glycolipid associated with Alzheimer paired helical filaments

In the present study, analytical techniques including gas chromatography/mass spectrometry (GC/MS)-assisted carbohydrate linkage-analysis, one- and two-dimensional NMR, and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-MS) have been used to characterize the structure of the glycolipid associated with the paired helical filaments (PHF) isolated from the neurofibrillary tangles of Alzheimer's diseased brain. The 1H NMR spectrum of acid-hydrolyzed protein-resistant core PHF (prcPHF) displays resonances that can be assigned to fatty acid and glucose. There are no resonances present that would indicate the presence of protein, amino acids, or a sphingosine base. Using two-dimensional homonuclear correlated spectroscopy, homonuclear Hartmann-Hahn, and heteronuclear multiple quantum coherence experiments, resonances in the 1H and 13C NMR spectrum of native PHF were assigned to a nonreducing terminal alpha-1,6-glycosidically linked glucose, an internal alpha-1,6-linked glucose, and an alpha-1,2,6-linked glucose. The narrow line-widths observed for these residues suggest that they arise from glucose residues undergoing rapid segmental motion. The carbohydrate portion of the PHF-associated glycolipid was analyzed using GC/MS linkage analysis and confirmed the presence of terminal and internal alpha-1,6-linked glucose and alpha-1,2,6-linked glucose in a molar ratio of 2:1:1. Three components of the PHF-associated glycolipid fraction having masses 2,416, 2,325, and 2,237 Da were observed using MALDI-MS. The least abundant, heavier mass component (2,416 Da) was best fit to a structure with a tridecamer of glucose having a single esterified C20 fatty acid (Glc13 + C20 or Glc13 + C20:1), whereas the more abundant, lower mass components were best fit to noncovalently associated glycolipid dimers, each with a glucose pentamer or hexamer having two C14, C16, or C18 esterified fatty acids {D[(Glc5 + C18) + (Glc6 + C16)] or D[(Glc5 + C14) + (Glc6 + C14)]}. The ratio of glucose to fatty acid calculated from these best-fit structures of the more abundant mass components (5.5 +/- 1.1:1.0) is in reasonable agreement with the same ratio calculated from peak integrations in the NMR spectra of acid-hydrolyzed prcPHF (6.2 +/- 1.6). Structural similarities between PHF-associated glycolipid and other glycolipid amphiphiles known to form PHF-like filaments indirectly suggest that this unique glycolipid may be an integral component of the PHF suprastructure.     

Function of the p53-pRb-dependent path after exposure to DNA damaging agents in rats embryonal fibroblast cells, transformed by E1A and Ha-Ras oncogenes

The packing of the G-actin monomers within crystalline actin tubes was investigated at atomic detail. To achieve this, we have chosen an integrated structural approach which combines intermediate resolution electron microscopy based 3-D reconstruction and surface metal shadowing of crystalline actin tubes with atomic resolution X-ray data of the G-actin monomer. Distinct from the parallel, half-staggered packing of the actin subunits within F-actin filaments, the arrangement of actin monomers within the crystalline tubes involves antiparallel packing into dimers with p2 symmetry. Within the crystalline tubes, the actin monomers are oriented so that the filament axis runs parallel with the sheet plane and the intersubunit contacts in this direction are similar to those existing along the two long-pitch helical strands of the F-actin filament. The other intersubunit contacts within the crystalline tubes are not found in the actin filament. The ability of actin to form a variety of polymorphic oligomers is still not fully understood, and the functional implications of this variability have yet to be deciphered. Regularly packed actin assemblies such as sheets, tubes or ribbons may ultimately yield structural relationships to in vivo relevant actin oligomers such as, for example, the "lower dimer".     

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the "closed interface" but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the "open interface." The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.     

Molecular structure of the amyloid-forming protein kappa I Bre

The molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 A resolution. The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 A in diameter with a repeat of 77.56 A, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals. The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits. From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.     

Basis for monomer stabilization in Rhodopseudomonas palustris cytochrome c' derived from the crystal structure

The crystal structure of an unusual monomeric cytochrome c' from Rhodopseudomonas palustris (RPCP) has been determined at 2.3 A resolution. RPCP has the four-helix (helices A, B, C and D) bundle structure similar to dimeric cytochromes c'. However the amino acid composition of the surface of helices A and B in RPCP is remarkably different from that of the dimeric cytochromes c'. This surface forms the dimer interface in the latter proteins. RPCP has seven charged residues on this surface contrary to the dimeric cytochromes c', which have only two or three charged groups on the corresponding surface. Moreover, hydrophobic residues on this surface of RPCP are two to three times fewer than in dimeric cytochromes c'. As a result of the difference in amino acid composition, the A-B surface of RPCP is rather hydrophilic compared with dimeric cytochromes c'. We thus suggest that RPCP is monomeric in solution because of the hydrophilic nature of the A-B surface. The amino acid composition of the A-B surface is similar to that of Rhodobacter capsulatus cytochrome c' (RCCP), which is an equilibrium admixture of monomer and dimer. The charge distribution of the A-B surface in RCCP, however, is considerably different from that of RPCP. Due to the difference, RCCP can form dimers by both ionic and hydrophobic interactions. These dimers are quite different from those in proteins which form strong dimers such as in Chromatium vinosum, Rhodospirillum rubrum, Rhodospirillum molischianum and Alcaligenes. Cytochrome c' can be classified into two types. Type 1 cytochromes c' have hydrophobic A-B surfaces and they are globular. The A-B surface of type 2 cytochromes c' is hydrophilic and they take a monomeric or flattened dimeric form.     

Effects of covalent dimerization on the structure and function of the carboxy-terminal fragment of neuropeptide Y

To determine whether or not the dimeric structure of neuropeptide Y (NPY) that is found in solution is necessary for its function, we investigated the effects of covalent dimerization on the structure and function of NPY using the carboxy-terminal fragment, NPY(12-36), in which residues 12 and 31 (located at both ends of alpha-helical region) were replaced by Cys residues. Among the three species (the parallel dimer, the anti-parallel dimer, and the intramolecularly cross-linked monomer) obtained by oxidation of the fragment, the anti-parallel dimer was predominant. NMR analysis showed that both parallel and anti-parallel dimers had alpha-helices similar to that of intact NPY, suggesting that covalent dimerization might have little effect on the helical structure. A binding assay with Y2 receptors on porcine hippocampal membranes revealed that the IC50 value of the anti-parallel dimer was almost the same as that of NPY (13-36), which is known as a Y2-specific ligand. By contrast, the binding by the parallel dimer was weaker by more than one order of magnitude. Our results suggest that the formation of dimers of NPY is not essential for binding to the receptor.     

Helix-stabilizing nonpolar interactions between tyrosine and leucine in aqueous and TFE solutions: 2D-1H NMR and CD studies in alanine-lysine peptides

Interactions between side chains spaced (i,i + 3) and (i,i + 4) may explain the context dependence of helix propensities observed in different systems. Nonpolar residues with these spacings occur frequently in protein helices and stabilize isolated peptide helices. Here (i,i + 3) and (i,i + 4) nonpolar interactions between Tyr and Leu in different solution conditions are studied in detail in alanine-based peptides using 2D 1H NMR and CD spectroscopy. Helix contents analyzed using current models for helix-coil transitions yield interaction energies which demonstrate significant helix stabilization in aqueous 1 M NaCl solutions by Tyr-Leu or Leu-Tyr pairs when spaced (i,i + 4) and, to a smaller extent, when spaced (i,i + 3), comparable to those estimated for other residue pairs. The interactions persist in solutions containing TFE, a helix-stabilizing solvent believed to diminish hydrophobic interactions, but not in helix-destabilizing 6 M urea. 1H NMR resonances for all peptides and solution conditions except in 6 M urea were completely assigned. NMR data indicate that the N-terminal residues are more helical and that the N-acetyl group participates in helix formation. The two (i,i + 4) spaced pairs show the same pattern of NOE cross-peaks between the Tyr and Leu side chains, as do the two (i,i + 3) pairs in 1 M NaCl as well in TFE solutions, and correspond well with that expected for the specific Tyr-Leu pair with side-chain contacts in protein helices.     

A mathematical approach to cytoskeletal assembly

The cytoskeleton is a fundamental and important part of cell's structure, and is known to play a large role in controlling the shape, function, division, and motility of the cell. In recent years, the traditional biological and biophysical experimental work on the cytoskeleton has been enhanced by a variety of theoretical, physical and mathematical approaches. Many of these approaches have been developed in the traditional frameworks of physicochemical and statistical mechanics or equilibrium thermodynamic principles. An alternative is to use kinetic modelling and couch the analysis in terms of differential equations which describe mean field properties of cytoskeletal networks or assemblies. This paper describes two such recent efforts. In the first part of the paper, a summary of work on the kinetics of polymerization, fragmentation, and dynamics of actin and polymers in the presence of gelsolin (which nulceates, fragments, and caps the filaments) is given. In the second part, some of the kinetic models aimed at elucidating the spatio-angular density distribution of actin filaments interacting via crosslinks is described. This model given insight into effects that govern the formation of clusters and bundles of actin filaments, and their spatial distribution.     

Solution structure of human neuropeptide Y

The three-dimensional structure of synthetic human neuropeptide Y in aqueous solution at pH 3.2 and 37 degrees C was determined from two-dimensional 1H NMR data recorded at 600 MHz. A restraint set consisting of 440 interproton distance restraints inferred from NOEs and 11 backbone and 4 side-chain dihedral angle restraints derived from spin-spin coupling constants was used as input for distance geometry calculations on DIANA and simulated annealing and restrained energy minimization in X-PLOR. The final set of 26 structures is well defined in the region of residues 11-36, with a mean pairwise rmsd of 0.51 A for the backbone heavy atoms (N, C alpha and C) and 1.34 A for all heavy atoms. Residues 13-36 form an amphipathic alpha-helix. The N-terminal 10 residues are poorly defined relative to the helical region, although some elements of local structure are apparent. At least one of the three prolines in the N-terminal region co-exists in both cis and trans conformations. An additional set of 24 distances was interpreted as intermolecular distances within a dimer. A combination of distance geometry and restrained simulated annealing yielded a model of the dimer having antiparallel packing of two helical units, whose hydrophobic faces form a well-defined core. Sedimentation equilibrium experiments confirm the observation that neuropeptide Y associates to form dimers and higher aggregates under the conditions of the NMR experiments. Our results therefore support the structural features reported for porcine neuropeptide Y [Cowley, D.J. et al. (1992) Eur. J. Biochem., 205, 1099-1106] rather than the 'aPP' fold described previously for human neuropeptide Y [Darbon, H. et al. (1992) Eur. J. Biochem., 209, 765-771].