Hyperstable stacked-disk structure of tobacco mosaic virus protein: electron cryomicroscopy image reconstruction related to atomic models

The stacked disk aggregate of tobacco mosaic virus protein is an intriguing object due to its high degree of stability, in spite of indications that the aggregate is held together to a great extent by water-mediated interactions between adjacent protein rings. Here, we present a set of models that were constructed using the atomic coordinates of the four-layer aggregate, and compare these with a three-dimensional reconstruction of the stacked disk obtained by means of cryoelectron microscopy and helical image processing. The comparison of the four possible models of the stacked disk with the data shows that there is a better correlation of the data with the left-handed model built from the A-A ring pair coordinates than with the two models involving the A-B ring pair, or with the right-handed model of the A-A ring pair. This establishes that the packing of the protein subunits in the stacked disk is different from that previously believed. We also note some differences between the observed structure and A-A ring pair model in the region of the flexible loop at small radius that might be an indication of conformational differences that give rise to the stability of the aggregate.     

Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

Background

Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination.

Methodology/Principal Findings

The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP¹⁹). Overall, N-His-TMV CP¹⁹ protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP¹⁹, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP¹⁹ layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle.

Conclusion

The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA.     

Conformation of a pentacosapeptide representing the RNA-binding N-terminus of cowpea chlorotic mottle virus coat protein in the presence of oligophosphates: a two-dimensional proton nuclear magnetic resonance and distance geometry study.

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. Two-dimensional proton NMR experiments were performed on the highly positively charged peptide containing six arginines and three lysines in the presence of an excess of monophosphates, tetra(poly)phosphates, or octadeca(poly)phosphates mimicking the phosphates of the RNA. The results show that the peptide alternates between various extended and helical structures in the presence of monophosphate and that this equilibrium shifts toward the helical structures (with the helical region situated between residues 10 and 20) in the presence of oligophosphates. Distance geometry calculations using distance constraints derived from a NOESY spectrum of the peptide in the presence of tetra(poly)phosphate resulted in eight structures belonging to two structure families. The first family consists of five structures with an alpha-helixlike conformation in the middle of the peptide, and the second family consists of three structures with a more open conformation. The propensity to form an alpha-helical conformation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.     

The structural relationship between the stacked disk and helical polymers of tobacco mosaic virus protein

An X-ray diffraction pattern from a well-oriented sol of the stacked disk rod aggregate of the protein of tobacco mosaic virus is presented. The helical parameters deduced from this are consistent with the stacked disk rod being a perturbed form of a stacked ring variant of the single helical polymer. Comparison of the intensity distributions in the X-ray diagrams of the two aggregates confirms their structural similarity.     

Variants of 3(10)-helices in proteins

An analysis of the shortest 3(10)-helices, containing three helical residues and two flanking capping residues that participate in two consecutive i + 3 --> i hydrogen bonds, shows that not all helices belong to the classic 3(10)-helix, where the three central residues adopt the right-handed helical conformation (alpha(R)). Three variants identified are: 3L10-helix with all residues in the left-handed helical region (alpha(L)), 3EL10-helix where the first residue is in the extended region followed by two residues in the alpha(L) conformation, and its mirror-image, the 3E'R10-helix. In the context of these helices, as well as the equivalent variants of alpha-helices, the length dependence of the handedness of secondary structures in protein structure is discussed. There are considerable differences in the amino acid preferences at different positions in the various types of 3(10)-helices. Each type of 3(10)-helix can be thought to be made up of an extension of a particular type of beta-turn (made up of residues i to i + 3) such that the (i + 3)th residue assumes the same conformation as the preceding residue. Distinct residue preferences at i and i + 3 positions seem to decide whether a particular stretch of four residues will be a beta-turn or a 3(10)-helix in the folded structure.     

The tobacco mosaic virus particle: structure and assembly

A short account is given of the physical and chemical studies that have led to an understanding of the structure of the tobacco mosaic virus particle and how it is assembled from its constituent coat protein and RNA. The assembly is a much more complex process than might have been expected from the simplicity of the helical design of the particle. The protein forms an obligatory intermediate (a cylindrical disk composed of two layers of protein units), which recognizes a specific RNA hairpin sequence. This extraordinary mechanism simultaneously fulfils the physical requirement for nucleating the growth of the helical particle and the biological requirement for specific recognition of the viral DNA.     

Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.     

The flagellar filament of Rhodobacter sphaeroides: pH-induced polymorphic transitions and analysis of the fliC gene

Flagellar motility in Rhodobacter sphaeroides is notably different from that in other bacteria. R. sphaeroides moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor. R. sphaeroides has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated R. sphaeroides filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the R. sphaeroides filament differs from that reported for Salmonella enterica serovar Typhimurium. The sequence of the R. sphaeroides fliC gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a sigma(28)-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of R. sphaeroides flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.     

Crystal structure of a recombinant form of the maltodextrin-binding protein carrying an inserted sequence of a B-cell epitope from the preS2 region of hepatitis B virus

We report the crystal structure of MalE-B133, a recombinant form of the maltodextrin-binding protein (MBP) of Escherichia coli carrying an inserted amino-acid sequence of a B-cell epitope from the preS2 region of the hepatitis B virus (HBV). The structure was determined by molecular replacement methods and refined to 2.7 Å resolution. MalE-B133 is an insertion/deletion mutant of MBP in which residues from positions 134 to 142, an external α helix in the wild-type structure, are replaced by a foreign peptide segment of 19 amino acids. The inserted residues correspond to the preS2 sequence from positions 132 to 145 and five flanking residues that arise from the creation of restriction sites. The conformation of the recombinant protein, excluding the inserted segment, closely resembles that of wild-type MBP in the closed maltose-bound form. MalE-B133 was shown by previous studies to display certain immunogenic and antigenic properties of the hepatitis B surface antigen (HBsAg), which contains the preS2 region. The crystal structure reveals the conformation of the first nine epitope residues (preS2 positions 132 to 140) exposed on the surface of the molecule. The remaining five epitope residues (preS2 positions 141 to 145) are not visible in electron density maps. The path of the polypeptide chain in the visible portion of the insert differs from that of the deleted segment in the structure of wild-type MBP, displaying a helical conformation at positions 134 to 140 (preS2 sequence numbering). A tripeptide (Asp-Pro-Arg) at the N terminus of the helix forms a stable structural motif that may be implicated in the cross-reactivity of anti-HBsAg antibodies with the hybrid protein. Proteins 27:1–8 © 1997 Wiley-Liss, Inc.     

The sequence TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase displays structural ambivalence and interconverts between alpha-helical and beta-hairpin conformations mediated by collapsed conformational states.

The peptide TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase adopts a helical conformation in the crystal structure and is a site for two hydrated helical segments, which are thought to be helical folding intermediates. Overlapping sequences of four to five residues from the peptide, sample both helical and strand conformations in known protein structures, which are dissimilar to glyceraldehyde-3-phosphate dehydrogenase suggesting that the peptide may have a structural ambivalence. Molecular dynamics simulations of the peptide sequence performed for a total simulation time of 1.2 micros, starting from the various initial conformations using GROMOS96 force field under NVT conditions, show that the peptide samples a large number of conformational forms with transitions from alpha-helix to beta-hairpin and vice versa. The peptide, therefore, displays a structural ambivalence. The mechanism from alpha-helix to beta-hairpin transition and vice versa reveals that the compact bends and turns conformational forms mediate such conformational transitions. These compact structures including helices and hairpins have similar hydrophobic radius of gyration (Rgh) values suggesting that similar hydrophobic interactions govern these conformational forms. The distribution of conformational energies is Gaussian with helix sampling lowest energy followed by the hairpins and coil. The lowest potential energy of the full helix may enable the peptide to take up helical conformation in the crystal structure of the glyceraldehyde-3-phosphate dehydrogenase, even though the peptide has a preference for hairpin too. The relevance of folding and unfolding events observed in our simulations to hydrophobic collapse model of protein folding are discussed.     

A 31P NMR analysis of the helix to coil transition of natural DNA samples: evidence for the existence of different conformational states.

The helix to coil transitions of calf thymus and salmon sperm DNA were probed by ³¹P nuclear magnetic resonance spectroscopy. Both the helical and coil forms were observed in the melting region indicating slow exchange between the two forms with an estimated rate of interconversion ? 36 sec^?1 at 70°C. At least three different signals were also observed at temperatures significantly above the Tm, suggesting three classes of conformational states. One of these classes has a significantly lower T₁ than the other two indicating considerable residual structure in this form. The four only partially resolved signals indicate that the phosphate residues have very similar chemical shift environments. From the experimentally observed small chemical shift differences between the helical and coil forms, it is concluded that the gauche, gauche, conformation predominates in the coil form as has been found for the helix.     

Protein-dynamics of the putative HCV receptor CD81 large extracellular loop

The human CD81 protein is a likely receptor for the binding of hepatitis C virus (HCV) to hepatocytes and therefore a possible target for novel anti-HCV drugs. The two published X-ray structures of the HCV binding region of CD81 (1G8Q and 1IV5) have, particularly in a substructure that is formed by two helices, a slightly different conformation. The abovementioned substructure is a candidate target region for virtual screening approaches. We present here a molecular dynamics study of the two X-ray structures. Our results indicate that the conformation of the two helical regions in one of the X-ray structures (1G8Q) is affected by crystallographic contacts and most likely does not represent the native state of the protein.     

Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.     

The Vaccinia Virus 14-Kilodalton (A27L) Fusion Protein Forms a Triple Coiled-Coil Structure and Interacts with the 21-Kilodalton (A17L) Virus Membrane Protein through a C-Terminal α-Helix

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third α-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.     

The hand of the stacked-disk aggregate of tobacco mosaic virus protein

The hand of the helical arrangement of subunits in the stacked-disk form of tobacco mosaic virus protein has been determined from electron microscopy. This and previous structural results taken together indicate that the near-axial intersubunit contacts between disks are similar to those within a single disk but quite different from those in the virus. Discrepancies between the structures of single disks and stacked disks are attributed to cleavage of the protein within the stacked-disk rod.     

Circular dichroism studies on a synthetic peptide corresponding to the membrane-spanning region of vesicular stomatitis virus G protein and its fatty acyl derivative

The conformations of synthetic peptides Lys-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-OCH3 and Lys(epsilon-palmitoyl)-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-O CH3, which constitute a part of the membrane-spanning region of the vesicular stomatitis virus G protein, have been studied by circular dichroism (CD) spectroscopy. Secondary structural features are observed for both peptides in trifluoroethanol, methanol, aqueous mixtures of trifluoroethanol and methanol and in a micellar environment. In trifluoroethanol, the CD spectra indicate the presence of a helical conformation, whereas in aqueous mixtures of organic solvents, both helical and beta-conformations are observed. While fatty acid acylation does not directly modulate peptide conformation, it promotes self-association of the acylated peptide and association with micelles. In a micellar environment, the acylated peptide adopts an alpha-helical conformation.     

Protein-RNA interactions during TMV assembly

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-atom derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.     

Refined atomic model of the four-layer aggregate of the tobacco mosaic virus coat protein at 2.4-A resolution.

Previous x-ray studies (2.8-A resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-A resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long alpha-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90-110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.