Tim Schmidt

Tje spherically averaged structures of cowpea mosaic virus components by X-ray solution scattering

The X-ray diffraction patterns of the four components of cowpea mosaic virus isolated from a cesium chloride gradient were measured, using film methods, to 30 A resolution. Diffraction patterns were analyzed by fitting computed two-shell spherical models to the observed data. The fitting procedure was applied to data to 80 A resolution to avoid the nonspherical contribution to the pattern observed at higher resolution. At pH 7.0 all four components displayed the same external spherically averaged radius of 140 +/-2 A. The lowest density component (top), which contains no RNA, was best modeled by an empty shell with an outer radius of 140 A and an inner radius of 101 +/- 3 A. The middle component, containing 27% RNA by weight, was modeled with a uniform electron density sphere. The bottom upper and bottom lower components, which are biologically identical but display different buoyant densities in cesium chloride solutions, were analyzed independently. The bottom upper component was best modeled with a 101 A inner (RNA containing) sphere of mean electron density 0.453e-/A(3) and a 101 to 140 A outer (protein containing) shell of electron density 0.410e-/A(3). The bottom lower component was fit with the same model except that the RNA containing region displayed a mean electron density of 0.459e-/A(3). The implications of the spherically averaged component structures for the protein structure, RNA and protein hydration, and cesium binding to RNA are discussed.

The Structure of Cowpea Mosaic Virus at 3.5 Å Resolution

There are nine groups of icosahedral, positive stranded RNA plant viruses (Francki et al., 1985) established on the basis of immunological relationships, genomic size, the number of RNA molecules composing the genome and the stability of the capsid. Eight of these groups are remarkably similar in both structural and functional properties while one group, the comoviruses, is quite different from the others (Goldbach and vanKammen, 1985). Cowpea mosaic virus (CPMV), the type member of the comovirus group, has only two properties in common with any of the other plant virus groups. The genome of CPMV is bipartite and a small protein (VpG) is linked to the 5′ terminus of each RNA molecule (Bruening, 1977; Daubert et al., 1979). In all its other properties CPMV is unique among the plant viruses. The RNA molecules of CPMV are polyadenylated at their 3′ termini while the genomes of the other virus groups are not. CPMV proteins are generated by the processing of two polyproteins, produced by translation of single open reading frames on each RNA molecule. The proteases that cleave the polyproteins are virally encoded and are initially part of the translation product of the larger of the two RNA molecules. The other plant virus groups utilize subgenomic RNA molecules as the messengers for protein synthesis with no posttranslational processing. The CPMV genome codes for a total of eight proteins, three (including the capsid proteins) derived from the small RNA molecule, and five from the large RNA.

Structural Fingerprinting: Subgrouping of Comoviruses by Structural Studies of Red Clover Mottle Virus to 2.4-Å Resolution and Comparisons with Other Comoviruses

ABSTRACT Red clover mottle virus (RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 Å. The overall structure of RCMV is similar to that of two other comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.

Comparative studies of T = 3 and T = 4 icosahedral RNA insect viruses

Crystallographic and molecular biological studies of T = 3 nodaviruses (180 identical subunits in the particle) and T = 4 tetraviruses (240 identical subunits in the particle) have revealed similarity in both the architecture of the particles and the strategy for maturation. The comparative studies provide a novel opportunity to examine an apparent evolution of particle size, from smaller (T = 3) to larger (T = 4), with both particles based on similar subunits. The BBV and FHV nodavirus structures are refined at 2.8 A and 3 A respectively, while the N omega V structure is at 6 A resolution. Nevertheless, the detailed comparisons of the noda and tetravirus X-ray electron density maps show that the same type of switching in subunit twofold contacts is used in the T = 3 and T = 4 capsids, although differences must exist between quasi and icosahedral threefold contacts in the T = 4 particle that have not yet been detected. The analyses of primary and tertiary structures of noda and tetraviruses show that N omega V subunits undergo a post assembly cleavage like that observed in nodaviruses and that the cleaved 76 C-terminal residues remain associated with the particle.

Black beetle virus—crystallization and particle symmetry

Black beetle virus, propagated in cultured Drosophila cells, crystallized into rhombic dodecahedra which diffracted X rays to 3.0 A resolution. Center-to-center spacing of particles in the unit cell was 305 A, while the spherically averaged diameter obtained from small-angle X-ray scattering from virus in solution was 312 A. Low resolution diffraction patterns from single crystals showed that the protein subunits are distributed centrosymmetrically, while electron microscopy indicated the particles are icosahedral in shape. The size of the particle is sufficient to accommodate about 180 protein subunits (44 kDa) consistent with T = 3 quasisymmetry.

Crystallization and preliminary structure analysis of an insect virus with T=4 quasi-symmetry: Nudaurelia capensis ω virus

We report the crystallization of Nudaurelia capens& w virus, a pathogen of the pine emperor moth. The icosahedral particle has T= 4 quasi-equivalent sym

A Novel Role for Coenzyme A during Hydride Transfer in 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

In this study, we take advantage of the ability of HMG-CoA reductase (HMGR) from Pseudomonas mevalonii to remain active while in its crystallized form to study the changing interactions between the ligands and protein as the first reaction intermediate is created. HMG-CoA reductase catalyzes one of the few double oxidation-reduction reactions in intermediary metabolism that take place in a single active site. Our laboratory has undertaken an exploration of this reaction space using structures of HMG-CoA reductase complexed with various substrate, nucleotide, product, and inhibitor combinations. With a focus in this publication on the first hydride transfer, our structures follow this reduction reaction as the enzyme converts the HMG-CoA thioester from a flat sp(2)-like geometry to a pyramidal thiohemiacetal configuration consistent with a transition to an sp(3) orbital. This change in the geometry propagates through the coenzyme A (CoA) ligand whose first amide bond is rotated 180° where it anchors a web of hydrogen bonds that weave together the nucleotide, the reaction intermediate, the enzyme, and the catalytic residues. This creates a stable intermediate structure prepared for nucleotide exchange and the second reduction reaction within the HMG-CoA reductase active site. Identification of this reaction intermediate provides a template for the development of an inhibitor that would act as an antibiotic effective against the HMG-CoA reductase of methicillin-resistant Staphylococcus aureus.

Quaternary and Tertiary Structures of Isometric RNA Viruses

Small spherical RNA viruses infecting members of all five biological kingdoms have been subjects of biophysical studies for decades (Kaper, 1975; Argos and Johnson, 1984). Isolated from their hosts, these obligate parasites are homogeneous chemical entities that are now studied at atomic resolution using x-ray crystallography. In the crystal the virus exists in a resting or dormant state, however, particles released from dissolved crystals are fully infectious. Many viruses form crystalline inclusion bodies within their hosts (Martelli and Russo, 1977), suggesting that crystalline aggregates are a natural and stable state for storing virus particles. In the dormant state the viral capsid protects the nucleic acid from degradation and is essentially a storage protein. During other stages of the virus life cycle, the capsid protein participates in a variety of functions; some are listed in Table I. Although relatively few viruses have been investigated at atomic resolution (Table II), a clear pattern has emerged relating the quaternary structures of different virus capsids (Fig. 1) and the tertiary structures from different virus subunits (Fig. 2). Beyond the striking similarities there are differences in these virus structures that reflect unique strategies evolved for accomplishing required functions. In this paper the current understanding of the relationship between the structures of simple RNA viruses and their function will be discussed using, as examples, three structures recently determined in our laboratory (Hosur et al., 1987; Stauffacher et al., 1987; Chen et al., 1988). An introductory section on the structure determination of one of these viruses (beanpod mottle virus) will describe some of the modern methods of virus x-ray crystallography.