Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations.

A structural model of the transmembrane portion of the acetylcholine receptor was developed from sequences of all its subunits by using transfer energy calculations to locate transmembrane alpha-helices and to calculate which helical side chains should be in contact with water inside the channel, with portions of other transmembrane helices, or with lipid hydrocarbon chains. "Knobs-into-holes" side chain packing calculations were used with other factors to stack the transmembrane alpha-helices together. In the model each subunit has the following structures in order along the sequence from the NH2 terminus: a large extracellular domain of undetermined structure, a short apolar alpha-helix that lies on the extracellular lipid surface of the membrane; three apolar transmembrane alpha-helices (I, II, and III), a cytoplasmic domain of undetermined structure, an amphipathic transmembrane alpha-helix (L) that forms the channel lining, a short extracellular alpha-helix, another apolar transmembrane alpha-helix (IV), and a small cytoplasmic domain formed by the COOH-terminal end of the chain. Three concentric layers form the pore. A bundle of five amphipathic L helices forms the channel lining. This bundle is surrounded by a bundle of 10 alternating II and III helices. Helices I and IV cover portions of the outer surface of the bundle formed by helices II and III. Positions of disulfide bridges are predicted and a mechanism for opening and closing conformational changes is proposed that requires tilting transmembrane helices and possibly a thiol-disulfide interchange reaction.     

Refined structure of southern bean mosaic virus at 2.9 A resolution

The T = 3 capsid of southern bean mosaic virus is analyzed in detail. The beta-sheets of the beta-barrel folding motif that form the subunits show a high degree of twist, generated by several beta-bulges. Only 34 water molecules were identified in association with the three quasi-equivalent subunits, most of them on the external viral surface. Subunit contacts related by quasi-3-fold axes are similar, are dominated by polar interactions and have almost identical calcium binding sites. There is no metal ion on the quasi-3-fold axis, as previously reported. Subunits related by quasi-2-fold and icosahedral 2-fold axes have different contacts but nevertheless display almost identical interactions between the antiparallel helices alpha A. A dipole-dipole type interaction between these helices may produce an energetically stable hinge that allows two types of dimers in a T = 3 assembly. The temperature factor distribution, the hydrogen-bonding pattern, and the contacts across the icosahedral 2-fold axes suggest that one of the dimer types is present in the intact virion and probably also in solution; the other is produced only during capsid assembly. Interactions along the 5-fold axes are mainly polar and possibly form an ion channel. The beta-sheet structures of the three subunits can be superimposed with considerable precision. Significant relative distortions between quasi-equivalent subunits occur mainly in helices and loops. The two dimeric forms and the subunit distortions are the consequence of the non-equivalent subunit environments in the capsid.     

Structure of an insect virus at 3.0 A resolution

We report the first atomic resolution structure of an insect virus determined by single crystal X-ray diffraction. Black beetle virus has a bipartite RNA genome encapsulated in a single particle. The capsid contains 180 protomers arranged on a T = 3 surface lattice. The quaternary organization of the protomers is similar to that observed in the T = 3 plant virus structures. The protomers consist of a basic, crystallographically disordered amino terminus (64 residues), a beta-barrel as seen in other animal and plant virus subunits, an outer protrusion composed predominantly of beta-sheet and formed by three large insertions between strands of the barrel, and a carboxy terminal domain composed of two distorted helices lying inside the shell. The outer surfaces of quasi-threefold related protomers form trigonal pyramidyl protrusions. A cleavage site, located 44 residues from the carboxy terminus, lies within the central cavity of the protein shell. The structural motif observed in BBV (a shell composed of 180 eight-stranded antiparallel beta-barrels) is common to all nonsatellite spherical viruses whose structures have so far been solved. This highly conserved shell architecture suggests a common origin for the coat protein of spherical viruses, while the primitive genome structure of BBV suggests that this insect virus represents an early stage in the evolution of spherical viruses from cellular genes.     

Energy profiles in the acetylcholine receptor (AChR) channel. The MII-helix model and the role of the remaining helices

It is demonstrated by theoretical computations that no favorable energy profile for cation transfer can be obtained in a model of the AChR channel constructed with the sole five MII helices of the inner wall. A favorable profile is obtained upon including the effect of the remaining helices of the five subunits. The decisive role, for the exit of the ion, of the charged residues situated at the N-terminal of the MII segments, established before, is underlined further. The role of the other elements of the channel wall (peptide carbonyl oxygens, hydrocarbon residues and polar side chains) is analyzed.     

An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure.

The NS1 protein of influenza A virus has the unique property of binding to three apparently different RNAs: poly A; a stem-bulge in U6 small nuclear RNA; and double-stranded RNA. One of our major goals is to determine how the NS1 protein recognizes and binds to its several RNA targets. As the first step for conducting structural studies, we have succeeded in identifying a fragment of the NS1 protein that possesses all the RNA-binding activities of the full-length protein. The RNA-binding fragment consists of the 73 amino-terminal amino acids of the protein. We have developed procedures for obtaining large amounts of the polypeptide in pure form. This has enabled us to establish the RNA-binding properties of this polypeptide and to demonstrate that it retains the ability to dimerize exhibited by the full-length protein. In addition, far-UV CD spectroscopy indicates that this RNA-binding polypeptide is largely (approximately 80%) helical, suggesting that the mode of dimerization of the NS1 protein and of its interaction with RNA is mediated, at least in part, by helices.     

Solution structure of the Vts1 SAM domain in the presence of RNA

The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the "core" five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition.     

Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12

The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 Å resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.     

A molecular model for the bilayer helices of the acetylcholine receptor including an acetylcholine binding site

A molecular model for the bilayer helices of the acetylcholine receptor is constructed from the 7 channel elements and the 17 hydrophobic helices of the 5 protein subunits. The acetylcholine binding site and the opening to the ion channel are included.     

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.     

Partitivirus structure reveals a 120-subunit, helix-rich capsid with distinctive surface arches formed by quasisymmetric coat-protein dimers

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses.     

Structural refinement and analysis of Mengo virus

The structure of Mengo encephalomyelitis virus was refined at 3 A resolution with a final R-factor of 0.221 and a root-mean-square deviation from idealized bond lengths of 0.019 A for 10 A to 3 A data with F greater than or equal to 3 sigma(F). The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement averaging procedure and constrained by the icosahedral symmetry of the virus. The virus consists of 60 protomers each having three major subunits, VP1, VP2 and VP3, along with one smaller internal protein, VP4. The three major subunits form similar eight-stranded beta-barrel structures. Alterations in the original sequence were found at position 45 in VP1 (Arg to Ala) and at position 58 in VP3 (Met to Val). The residues in loops I and II of VP1 (82 to 102), the "FMDV loop" in VP1 (205 to 213), the flexible loop of VP3 in the putative receptor attachment site (175 to 185) as well as the terminal regions 260 to 268 in VP1, 253 to 256 in VP2 and 13 to 15 in VP4 were built or modified in regions of weak density. The variation in temperature factors at the end of the refinement is over a wide range (from 2 to 80 A2), with the disordered outer and inner regions showing high mobility. Four cis proline residues, 105 in VP1, 85 and 152 in VP2 and 59 in VP3, have been identified. The disulfide bridge Cys86 to Cys88 in VP3 has been characterized. One phosphate ion and 233 water positions were included in the refinement. It is suggested that this phosphate is associated with the receptor attachment site. There are two major hydrogen-bonding networks involving solvent atoms; one involving only the subunits of a protomer, and the other connecting the protomers in a pentamer. The distribution of atom types around the icosahedral symmetry axes shows that the 5-fold channel is more hydrophobic than that along the 3-fold axis and that there are more charged residues around the 2-fold axis. The analysis of contacts between the different subunits supports the assignment of the protomeric unit. The five protomers that form the pentameric unit are held together by interactions involving the smaller VP4 protein and the amino termini of VP1 and VP3. The pentamers are associated by means of the amino-terminal region of the VP2 subunits, the beta F strand of the VP3 subunits, the C terminus of the VP4 subunits and the electrostatic helical (alpha A) interactions of VP2 subunits across the icosahedral 2-fold axes. The superposition of the corresponding subunits of Mengo virus, human rhinovirus 14 and southern bean mosaic virus has provided an improved sequence alignment. The largest structural similarity is between the VP3 subunits of Mengo virus and rhinovirus, while the least similarity is between the VP1 subunits. The various specialized insertions in the different subunits can be associated with specific functional requirements.     

Npp106p, a Schizosaccharomyces pombe nucleoporin similar to Saccharomyces cerevisiae Nic96p, functionally interacts with Rae1p in mRNA export.

To identify components of the mRNA export machinery in Schizosaccharomyces pombe, a screen was developed to identify mutations that were synthetically lethal with the conditional mRNA export allele rae1-167. Mutations defining three complementation groups were isolated, and here we report the characterization of npp106 (for nuclear pore protein of 106 kDa). This gene encodes a predicted protein that has significant similarity to the Nic96p nucleoporin of Saccharomyces cerevisiae. Consistent with Npp106p being a nucleoporin, a functional green fluorescent protein (GFP)-tagged Npp106p localized to the nuclear periphery. In contrast to NIC96, the npp106 gene is not essential. Moreover, a delta npp106 mutant did not show cytoplasmic mislocalization of a simian virus 40 nuclear localization signal-GFP-LacZ reporter protein, and a fraction of cells had accumulation of poly(A)+ RNA in the nucleus. A consequence of the synthetic lethality between rae1-167 and npp106-1 was the accumulation of poly(A)+ RNA in the nucleus when cells were grown under synthetic lethal conditions. In addition to npp106-1, which is a nonsense mutation that truncates the protein at amino acid 292, the delta npp106 mutation was synthetically lethal with rae1-167, suggesting that the synthetic lethality is a consequence of the loss of a function of npp106. We further demonstrate that a region between amino acids 74 and 348 of Npp106p is required for complementation of the synthetic lethality. These results uncover a potential direct or indirect involvement of Npp106p in mRNA export.     

The selectivity filter of a ligand-gated ion channel. The helix-M2 model of the ion channel of the nicotinic acetylcholine receptor

Evidence from electrophysiology and biochemistry supports the hypothesis that the ion channel of the nicotinic acetylcholine receptor is formed by homologous amino acid sequences of all receptor subunits, called helices M2. A model of the ion channel is proposed and the selectivity filter is described as a ring of negatively-charged amino acid side chains [(1988) Nature 335, 645-648] which may undergo conformational changes upon permeation of the cation.     

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.     

Coordinated two-disk nucleation, growth and properties, of virus-like particles assembled from tobacco-mosaic-virus capsid protein with poly(A) or oligo(A) of different length

Assembly of nucleoprotein rods from tobacco mosaic virus (TMV) coat protein and poly(A) depends on the presence of 20S disks in a manner very similar to nucleation and growth of virions in reconstitution with TMV RNA. Products assembled with (A) approximately equal to 5000 appear to have the same buoyant density in CsCl, the same nucleotide/protein ratio and the same nuclease stability, as reconstituted and native TMV. Their rate of formation is very similar to the rate of reconstitution with TMV RNA when high-molecular-mass (A) approximately equal to 5000 is used, but becomes a function of chain length particularly with (A) less than or equal to 185. The composition of assembly products can be described sufficiently with the relation between number of capsid polypeptide monomers/particle, np, to the number of nucleotide residues/chain, nnt, of np = 1/3 (nnt + 50) with two important restrictions: (1) particles of less than four turns of helically arranged capsid subunits are unstable, and (2) particles with about 150 or less nucleotides per chain deviate in structure from mature virus and virus-like (= longer) assembly products. This is indicated by changes in both buoyant density in CsCl and optical properties, while 'dislocation' of the disk to the helical arrangement of capsid subunits ('helicalization') and nuclease stability already become established with chains as short as (A) approximately equal to 58 +/- 20. Consequently, we suggest that assembly proceeds through three distinct phases: (1) nucleation (resulting in helicalization) by interaction of nucleic acid with the first disk; (2) stabilization of the primary (unstable!) nucleation complex by addition of a second disk and formation of a four-turn virus-like and stable nucleoprotein helix, which is then fit for (3) elongation by addition of further disks. The question of what makes the TMV protein disk select specifically TMV RNA during virion assembly is discussed in some detail.     

Refined structure of desmodium yellow mottle tymovirus at 2.7 A resolution

Desmodium yellow mottle virus is a 28 nm diameter, T=3 icosahedral plant virus of the tymovirus group. Its structure has been solved to a resolution of 2.7 A using X-ray diffraction analysis based on molecular replacement and phase extension methods. The final R value was 0.151 (R(free)=0.159) for 134,454 independent reflections. The folding of the polypeptide backbone is nearly identical with that of turnip yellow mosaic virus, as is the arrangement of subunits in the virus capsid. However, a major difference in the disposition of the amino-terminal ends of the subunits was observed. In turnip yellow mosaic virus, those from the B and C subunits comprising the hexameric capsomeres formed an annulus about the interior of the capsomere, while the corresponding N termini of the pentameric capsomere A subunits were not visible at all in electron density maps. In Desmodium yellow mottle tymovirus, amino termini from the A and B subunits combine to form the annuli, thereby resulting in a much strengthened association between the two types of capsomeres and an, apparently, more stable capsid. The first 13 residues of the C subunit were invisible in electron density maps. Two ordered fragments of single-stranded RNA, seven and two nucleotides in length, were observed. The ordered water structure of the virus particle was delineated and required 95 solvent molecules per protein subunit.