Pf1 Inovirus. Electron density distribution calculated by a maximum entropy algorithm from native fibre diffraction data to 3 A resolution and single isomorphous replacement data to 5 A resolution

We have calculated the electron density distribution of the Pf1 strain of filamentous bacteriophage by a maximum entropy method. In the calculation we included native X-ray fibre diffraction data extending to 3 A resolution in the meridional direction on 60 layerlines that are resolved to 4 A in the equatorial direction, and lower resolution data from a single isomorphous derivative iodinated on the Tyr25 residue. The electron density map indicates that the 46-residue protein subunit is a single, gently curved stretch of alpha-helix with its axis at an angle of about 20 degrees to the axis of the virion. The alpha-helix subunit curves around the virion axis by about 1/6 turn, and decreases from about 27 A radius to about 13 A radius in the virion as the amino acid sequence of the subunit runs from the N terminus to the C terminus. Nearest-neighbour alpha-helical subunits are about 10 A apart along their length, and the axis of each subunit makes an unexpected negative angle with its nearest neighbours in the virion. To confirm the validity of the maximum entropy calculation, we have varied the constraints on the calculation. All variations result in either a map that is close to the original map or a map that cannot be interpreted in terms of secondary structure: we find only one map that makes structural sense.     

Structure of satellite tobacco necrosis virus after crystallographic refinement at 2.5 Å resolution

The structure of the protein subunit of satellite tobacco necrosis virus has been solved at 3.7 Å resolution. We have now crystallographically refined the original model and extended the resolution to 2.5 Å in order to get a model accurate enough to explain the details of the subunit interactions. The refinement was done with a novel method utilizing the icosahedral symmetry of the virus particle.The final model shows a complicated network of interactions, involving salt linkages, hydrogen bonds and hydrophobic contacts. In addition, we have located three different metal ion sites in the protein shell, linking the protein subunits together. These sites are probably occupied by calcium ions. One site is found in a general position near the icosahedral 3-fold axis of the virus. The ligands form an octahedral arrangement, with two main chain carbonyl oxygens (O-61 and O-64), one carboxylate oxygen (OD1 from Asp194) of the same subunit and a second carboxylate oxygen (OE1 of Glu25) from a 3-fold related subunit. Two water molecules complete the octahedral arrangement. A second site is on the icosahedral 3-fold axis and is liganded by the carboxylate oxygens of the 3-fold related Asp55 residues. The third metal ion site is found on the 5-fold axis, liganded by the five carbonyl oxygens of Thr138 and two water molecules.We are unable to locate the first 11 N-terminal amino acid residues, which point into the virus interior. No interpretable density for RNA has been found, indicating that the nucleic acid of the virus does not have a unique orientation in the crystal.     

Self-complementary motifs (SCM) in alpha-crystallin small heat shock proteins

Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein-protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved alpha-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in alpha-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of alpha-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of alpha-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.     

Structure-function relationship for the highly toxic crotoxin from Crotalus durissus terrificus

The three-dimensional structure of the highly toxic crotoxin from Crotalus durissus terrificus was modelled based on sequence analysis and the refined structure of calcium-free phospholipase of Crotalus atrox venom. Small-angle x-ray scattering experiments were performed on aqueous solutions of crotoxin. The radial distribution function derived from these scattering experiments and the one calculated from the model structure are in good agreement. Crotoxin consists of a basic and an acidic subunit. The model strongly suggests that the overall folding motif of phospholipases has been preserved in both subunits. The basic domain has an intact active site. The residues that are expected to contact the lipid tails of the phospholipid are different from other phospholipases, but they are all hydrophobic. The acidic domain consists of three independent chains interconnected by disulfide bonds. Compared to other phospholipases the active site for the greater part has been preserved in this domain, but it is not very well shielded from solvent. Most residues normally in contact with the lipid tails of the phospholipid are missing, which might explain the acidic subunit's lack of phospholipase activity. A homology between the third chain of the acidic domain and neurophysins suggests that the acidic domain may act as a chaperone for the basic domain. Correspondence to: Y P. Mascarenhas     

Crystal structure of tobacco necrosis virus at 2.25 A resolution

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.     

Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation.

The Vpr gene product of human immunodeficiency virus type 1 is a virion-associated protein that is important for efficient viral replication in nondividing cells such as macrophages. At the cellular level, Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Incorporation of Vpr into viral particles requires a determinant within the p6 domain of the Gag precursor polyprotein Pr55gag. In the present study, we have used site-directed mutagenesis to identify a domain(s) of Vpr involved in virion incorporation and nuclear localization. Truncations of the carboxyl (C)-terminal domain, rich in basic residues, resulted in a less stable Vpr protein and in the impairment of both virion incorporation and nuclear localization. However, introduction of individual substitution mutations in this region did not impair Vpr nuclear localization and virion incorporation, suggesting that this region is necessary for the stability and/or optimal protein conformation relevant to these Vpr functions. In contrast, the substitution mutations within the amino (N)-terminal region of Vpr that is predicted to adopt an alpha-helical structure (extending from amino acids 16 to 34) impaired both virion incorporation and nuclear localization, suggesting that this structure may play a pivotal role in modulating both of these biological properties. These results are in agreement with a recent study showing that the introduction of proline residues in this predicted alpha-helical region abolished Vpr virion incorporation, presumably by disrupting this secondary structure (S. Mahalingam, S. A. Khan, R. Murali, M. A. Jabbar, C. E. Monken, R. G. Collman, and A. Srinivasan, Proc. Natl. Acad. Sci. USA 92:3794-3798, 1995). Interestingly, our results show that two Vpr mutants harboring single amino acid substitutions (L to F at position 23 [L23F] and A30F) on the hydrophobic face of the predicted helix coded for relatively stable proteins that retained their ability to translocate to the nucleus but exhibited dramatic reduction in Vpr incorporation, suggesting that this hydrophobic face might mediate protein-protein interactions required for Vpr virion incorporation but not nuclear localization. Furthermore, a single mutation (E25K) located on the hydrophilic face of this predicted alpha-helical structure affected not only virion incorporation but also nuclear localization of Vpr. The differential impairment of Vpr nuclear localization and virion incorporation by mutations in the predicted N-terminal alpha-helical region suggests that this region of Vpr plays a role in both of these biological functions of Vpr.     

TM2 but not TM4 of subunit c'' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking

The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c') relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c'. Subunit c' contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908-41913). We have now analyzed interaction of subunits a and c' using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c' centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c' does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.     

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.     

NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (alpha, epsilon, and theta). The theta subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the theta subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, theta was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3J(HN alpha) coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10-Asp19, Glu38-Glu43, and His47-Glu54), one short extended segment (Pro34-Ala37), and a long loop (Ala20-Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of theta is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the epsilon subunit have identified a loop region (Ala20-Glu29) in theta as the site of association with epsilon.     

ATP synthase from bovine mitochondria. The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs

ATP synthase from bovine mitochondria is a complex of 13 different polypeptides, whereas the Escherichia coli enzyme is simpler and contains eight subunits only. Two of the bovine subunits, b and d, which had not been characterized, have been isolated from the purified enzyme. Subunits with sizes corresponding to bovine subunits b and d are evident in preparations of the enzyme from mitochondria of other species. Partial protein sequences have been determined by direct methods. On the basis of some of this information, two oligonucleotide mixtures, 17 and 18 bases in length, have been synthesized and used as hybridization probes in the isolation of clones of the cognate cDNAs. The sequences of the two proteins have been deduced from their DNA sequences. Subunit b is 214 amino acid residues in length and has a free N terminus. Subunit d is 160 amino acid residues long. Its N-terminal alanine is blocked by an N-acetyl group, as demonstrated by fast atom bombardment mass spectrometry of N-terminal peptides. The sequence near the N terminus of the b subunit is made predominantly of hydrophobic residues, whereas the remainder of the protein is mainly hydrophilic. This N-terminal hydrophobic region may be folded into an alpha-helical structure spanning the lipid bilayer. In its distribution of hydrophobic residues, this protein resembles the b subunits of ATP synthase complexes in bacteria and chloroplasts. The b subunit in E. coli forms an important structural link between the extramembrane sector of the enzyme F1, and the intrinsic membrane domain, FO. It is proposed that the bovine mitochondrial subunit b serves a similar function. If this is so, the mitochondrial enzyme, as the chloroplast ATP synthase, contains equivalent subunits to all eight of those that constitute the E. coli enzyme. Subunit d has no extensive hydrophobic sequences, and is not apparently related to any subunit described in the simpler ATP synthases in bacteria and chloroplasts.     

Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein export.

The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.     

Detection of a Trimeric Human Immunodeficiency Virus Type 1 Gag Intermediate Is Dependent on Sequences in the Matrix Protein, p17

Previous studies have shown that single amino acid changes in the amino-terminal matrix (MA) domain, p17, of the human immunodeficiency virus type 1 Gag precursor Pr55, can abrogate virion particle assembly. In the three-dimensional structure of MA such mutations lie in a single helix spanning residues 54 to 68, suggesting a key role for this helix in the assembly process. The fundamental nature of this involvement, however, remains poorly understood. In the present study, the essential features of the MA helix required for virus assembly have been investigated through the analysis of a further 15 site-directed mutants. With previous mutants that failed to assemble, residues mapped as critical for assembly were all located on the hydrophobic face of the helix and had a key role in stabilizing the trimeric interface. This implies a role for the MA trimer in virus assembly. We support this interpretation by showing that purified MA is trimeric in solution and that mutations that prevent virus assembly also prevent trimerization. Trimerization in solution was also a property of a larger MA-capsid (CA) Gag molecule, while under the same conditions CA only was a monomer. These data suggest that Gag trimerization driven by the MA domain is an intermediate stage in normal virion assembly and that it relies, in turn, on an MA conformation dependent on the hydrophobic core of the molecule.     

The top of the inserted-like domain of the integrin lymphocyte function-associated antigen-1 beta subunit contacts the alpha subunit beta -propeller domain near beta-sheet 3

We find that monoclonal antibody YTA-1 recognizes an epitope formed by a combination of the integrin alpha(L) and beta(2) subunits of LFA-1. Using human/mouse chimeras of the alpha(L) and beta(2) subunits, we determined that YTA-1 binds to the predicted inserted (I)-like domain of the beta(2) subunit and the predicted beta-propeller domain of the alpha(L) subunit. Substitution into mouse LFA-1 of human residues Ser(302) and Arg(303) of the beta(2) subunit and Pro(78), Thr(79), Asp(80), Ile(365), and Asn(367) of the alpha(L) subunit is sufficient to completely reconstitute YTA-1 reactivity. Antibodies that bind to epitopes that are nearby in models of the I-like and beta-propeller domains compete with YTA-1 monoclonal antibody for binding. The predicted beta-propeller domain of integrin alpha subunits contains seven beta-sheets arranged like blades of a propeller around a pseudosymmetry axis. The antigenic residues cluster on the bottom of this domain in the 1-2 loop of blade 2, and on the side of the domain in beta-strand 4 of blade 3. The I domain is inserted between these blades on the top of the beta-propeller domain. The antigenic residues in the beta subunit localize to the top of the I-like domain near the putative Mg(2+) ion binding site. Thus, the I-like domain contacts the bottom or side of the beta-propeller domain near beta-sheets 2 and 3. YTA-1 preferentially reacts with activated LFA-1 and is a function-blocking antibody, suggesting that conformational movements occur near the interface it defines between the LFA-1 alpha and beta subunits.     

Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions.

The influenza A virus M2 protein is an integral membrane protein of 97 amino acids that is expressed at the surface of infected cells with an extracellular N-terminal domain of 18 to 23 amino acid residues, an internal hydrophobic domain of approximately 19 residues, and a C-terminal cytoplasmic domain of 54 residues. To gain an understanding of the M2 protein function in the influenza virus replicative pathway, we produced and characterized a monoclonal antibody to M2. The antibody-binding site was located to the extracellular N terminus of M2 as shown by the loss of recognition after proteolysis at the infected-cell surface, which removes 18 N-terminal residues, and by the finding that the antibody recognizes M2 in cell surface fluorescence. The epitope was further defined to involve residues 11 and 14 by comparing the predicted amino acid sequences of M2 from several avian and human strains and the ability of the M2 protein to be recognized by the antibody. The M2-specific monoclonal antibody was used in a sensitive immunoblot assay to show that M2 protein could be detected in virion preparations. Quantitation of the amount of M2 associated with virions by two unrelated methods indicated that in the virion preparations used there are 14 to 68 molecules of M2 per virion. The monoclonal antibody, when included in a plaque assay overlay, considerably showed the growth of some influenza virus strains. This plaque size reduction is a specific effect for the M2 antibody as determined by an analysis of recombinants with defined genome composition and by the observation that competition by an N-terminal peptide prevents the antibody restriction of virus growth.     

Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography

The genomic RNA2s of nodaviruses encode a single gene, that of protein alpha, the precursor of virion proteins beta and gamma. We compared the sequences of the RNA2s of the nodaviruses, black beetle virus (BBV), flock house virus, boolarra virus and nodamura virus, with the objective of identifying homologies in the primary and secondary structure of these RNAs and in the structure of their encoded protein. The sequences of the four RNAs were found to be similar, so that homologous regions relating to translation and RNA replication were readily identified. However, the overall, secondary structures in solution, deduced from calculations of optimal Watson-Crick base-pairing configurations, were very different for the four RNAs. We conclude that a particular, overall, secondary structure in solution within host cells is not required for virus viability. The partially refined X-ray structure of BBV (R = 26.4% for the current model) was used as a framework for comparing the structure of the encoded proteins of the four viruses. Mapping of the four protein sequences onto the BBV capsid showed many amino acid differences on the outer surface, indicating that the exteriors of the four virions are substantially different. Mapping in the beta-barrel region showed an intermediate level of differences, indicating that some freedom in choice of amino acid residues is possible there although the basic framework of the capsids is evidently conserved. Mapping onto the interior surface of the BBV capsid showed a high degree of conservation of amino acid residues, particularly near the protein cleavage site, implying that that region is nearly identical in all four virions and has an essential role in virion maturation, and also suggests that all four capsid interior surfaces have similar surfaces exposed to the viral RNA. Apart from a small portion of the C promoter, the amino terminus of the BBV protein (residues 1 to 60) is crystallographically disordered and the amino acid residues in that region are not well conserved. The disordered portion of the BBV protein clearly projects from the capsid inner surface into the interior of the virion, the region occupied by the viral RNA. In all four viruses, residues 1 to 60 had a high proportion of basic residues, suggesting a virus-specific interaction of the amino terminus with the virion RNA.     

油桐尺蠖核型多角体病毒多角体蛋白的理化性质及二级结构预测

由SDS及梯度胶电泳测得油桐尺蠖核型多角体病毒（BsNPV）多角体蛋白天然状态及亚基分子量分别为363kD与31.5kD,从而推断此蛋白为十二聚体,亚基间无二硫键作用．BsNPV多角体蛋白的远紫外圆二色谱显示,它的二级结构含有31．7％的α螺旋,23．8％的β折叠及44．5％的无规卷曲,与二级结构预测结果相符．通过荧光光谱实验推知,BsNPV多角体蛋白的表面疏水性弱,其色氨酸残基位于蛋白疏水核内部．     

Structure of beef liver catalase

The three-dimensional structure of beef liver catalase has been determined to 2.5 å resolution by a combination of isomorphous and molecular replacement techniques. Heavy-atom positions were found using vector search and difference Fourier methods. The tetrameric catalase molecule has 222 symmetry with one of its dyads coincident with a crystallographic 2-fold axis. The known polypeptide sequence has been unambiguously fitted to the electron density map. The heme is well buried in a hydrophobic pocket, 20 Å below the surface of the molecule, and accessible through a hydrophobic channel. Residues that line the heme pocket belong to two different subunits. Tyr357 is the proximal heme ligand and the catalytically important residues on the distal side are residues His74 and Asnl47. The tertiary structure consists of four domains: an extended non-globular amino-terminal arm, which stabilizes the quaternary structure; an anti-parallel, eight-stranded β-barrel providing the residues on the distal side of the heme; a rather random “wrapping domain” around the subunit exterior including the proximal heme ligand; and a final λ-helical structure resembling the E, F, G and H helices of the globins.     

Differential localization of two epitopes of Escherichia coli ribosomal protein L2 on the large ribosomal subunit by immune electron microscopy using monoclonal antibodies.

Two monoclonal antibodies (mAb), directed toward different epitopes of Escherichia coli ribosomal protein L2, have been used as probes in immune electron microscopy. mAb 5-186 recognizes an epitope within residues 5-186 of protein L2; it is seen to bind to 50 S subunits at or near the peptidyl transferase center, beside the subunit head on the L1 shoulder. mAb 187-272 recognizes an epitope within residues 187-272. This antibody binds to the face of the 50 S subunit, below the head and slightly toward the side with the stalk; this site is near the translocation domain. Both antibodies can bind simultaneously to single subunits. This indicates that protein L2 is elongated, reaching from the peptidyl transferase center to below the subunit head and approaching the translocational domain. The different locations of the two epitopes are consistent with previous biochemical results with the two antibodies (Nag, B., Tewari, D. S., Etchison, J. R., Sommer, A., and Traut, R. R. (1986) J. Biol. Chem. 261, 13892-13897).     

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.     

The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.