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M G Rossmann

Publications and source records attributed to M G Rossmann.

At least 55 records · Page 3Linked to original sources
PubMed1997

Crystals of Rous sarcoma virus capsid protein show a helical arrangement of protein subunits.

Crystals of Rous sarcoma virus (RSV) capsid protein diffract X rays to 3.5 A resolution and belong to the monoclinic space group C2 with unit cell parameters a = 374.4 A, b = 128.1 A, c = 200.2 A, and beta = 121.8 degrees. One asymmetric unit of the crystal may contain between 28 and 35 molecules, based on reasonable crystal density assumptions. A self-rotation function and Patterson synthesis suggest that RSV capsid protein crystallizes as a helical array. The determinants of the viral particle morphology are not encoded in the capsid alone. The assembly of a helical array in the crystal reflects the absence of any conformational switching. However, it is expected that the subunit interactions seen in the crystal will be preferred and will relate to those found in the immature or mature virion.

Amino Acid Sequence↗
PubMed1997

Structure of a viral procapsid with molecular scaffolding.

The assembly of a macromolecular structure proceeds along an ordered morphogenetic pathway, and is accomplished by the switching of proteins between discrete conformations as they are added to the nascent assembly. Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. Although macromolecular assembly processes are fundamental to all biological systems, they have been characterized most thoroughly in viral systems, such as the icosahedral Escherichia coli bacteriophage phiX174. The phiX174 virion contains the proteins F, G, H and J. During assembly, two scaffoldingproteins B and D are required for the formation of a 108S, 360-A-diameter procapsid from pentameric precursors containing the F, G and H proteins. The procapsid contains 240 copies of protein D, forming an external scaffold, and 60 copies each of the internal scaffolding protein B, the capsid protein F, and the spike protein G. Maturation involves packaging of DNA and J proteins and loss of protein B, producing a 132S intermediate. Subsequent removal of the external scaffold yields the mature virion. Both the F and G proteins have the eight-stranded antiparallel beta-sandwich motif common to many plant and animal viruses. Here we describe the structure of a procapsid-like particle at 3.5-A resolution, showing how the scaffolding proteins coordinate assembly of the virus by interactions with the F and G proteins, and showing that the F protein undergoes conformational changes during capsid maturation.

Bacteriophage phi X 174↗
PubMed1997

Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.

BACKGROUND: Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. RESULTS: The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. CONCLUSIONS: The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal domain and the labile coiled-coil domain may be essential for the fibritin function and for the correct functioning of many other alpha-fibrous proteins.

Amino Acid Sequence↗
PubMed1997

The refined structure of human rhinovirus 16 at 2.15 A resolution: implications for the viral life cycle.

BACKGROUND: Rhinoviruses belong to the picornavirus family and are small, icosahedral, non-enveloped viruses containing one positive RNA strand. Human rhinovirus 16 (HRV16) belongs to the major receptor group of rhinoviruses, for which the cellular receptor is intercellular adhesion molecule-1 (ICAM-1). In many rhinoviruses, one of the viral coat proteins (VP1) contains a hydrophobic pocket which is occupied by a fatty acid-like molecule, or so-called 'pocket factor'. Antiviral agents have been shown to bind to the hydrophobic pocket in VP1, replacing the pocket factor. The presence of the antiviral compound blocks uncoating of the virus and in some cases inhibits receptor attachment. A refined, high-resolution structure would be expected to provide further information on the nature of the pocket factor and other features previously not clearly identified. RESULTS: The structure of native HRV16 has been refined to a resolution of 2.15 A. The hydrophobic pocket in VP1 is observed in two alternative conformations. In one of these, the pocket is filled by a pocket factor and the protein structure is similar to virus-antiviral compound complexes. In the other conformation, the hydrophobic pocket is collapsed and empty. RNA bases stack against both a tryptophan and a phenylalanine residue on the internal surface of the viral capsid. Site-directed mutagenesis of the tryptophan, which is conserved across the picornaviruses, to nonconservative residues results in non-viable virus. Five symmetry-related N termini of coat protein VP4 form a ten-stranded, antiparallel beta barrel around the base of the icosahedral fivefold axis. The N termini of VP1 are amphipathic alpha helices, which stack on the outside of this beta barrel. The N termini of VP1 and VP4 have not been observed previously in rhinovirus structures. CONCLUSIONS: The observation of a partially occupied hydrophobic pocket in HRV16 forms a missing link between HRV14, which is always observed with no pocket factor in the native form, and rhinovirus 1A and other picornaviruses (e.g. poliovirus, coxsackievirus) which contain pocket factors. The pocket factor molecules probably regulate viral entry, uncoating and assembly. Picornavirus assembly is known to proceed via pentamers, therefore, the interaction of RNA with the conserved tryptophan residues across twofold axes between pentamers may play a role in picornavirus assembly. The positioning of a cation on the icosahedral fivefold axes and the structure of the N termini of VP4 and VP1 around these axes suggest a mechanism for the uncoating of rhinoviruses.

Amino Acid Sequence↗
PubMed1997

Cations in human rhinoviruses.

All known crystal structures of rhinoviruses have some uninterpreted electron density on their fivefold axes at a distance of about 152 +/- 3 A from the viral center. This density had been assumed to be a Ca2+ ion, based on its shape, height, and the presence of Ca2+ ions in the crystallization solutions. Difference electron density maps between EGTA-soaked crystals of human rhinovirus 14 (HRV14), as well as HRV16, and their corresponding native structures show that this density is an EGTA-chelatable ion. Analysis of the coordination geometry indicates that the ions in HRV3, HRV14, and HRV1A could be Ca2+ and the ion in HRV16 might be Zn2+. These cations may play a role in regulation of rhinovirus stability, although the loss of the ion itself does not seem sufficient to lead to viral disassembly.

Calcium↗
PubMed1997

Structure determination of minute virus of mice.

The three-dimensional crystal structure of the single-stranded DNA-containing ('full') parvovirus, minute virus of mice (MVM), has been determined to 3.5 A resolution. Both full and empty particles of MVM were crystallized in the monoclinic space group C2 with cell dimensions of a = 448.7, b = 416.7, c = 305.3 A and beta = 95.8 degrees. Diffraction data were collected at the Cornell High Energy Synchrotron Source using an oscillation camera. The crystals have a pseudo higher R32 space group in which the particles are situated at two special positions with 32 point symmetry, separated by (1/2)c in the hexagonal setting. The self-rotation function showed that the particles are rotated with respect to each other by 60 degrees around the pseudo threefold axis. Subsequently, a more detailed analysis of the structure amplitudes demonstrated that the correct space-group symmetry is C2 as given above. Only one of the three twofold axes perpendicular to the threefold axis in the pseudo R32 space group is a 'true' crystallographic twofold axis; the other two are only 'local' non-crystallographic symmetry axes. The known canine parvovirus (CPV) structure was used as a phasing model to initiate real-space electron-density averaging phase improvement. The electron density was easily interpretable and clearly showed the amino-acid differences between MVM and CPV, although the final overall correlation coefficient was only 0.63. The structure of MVM has a large amount of icosahedrally ordered DNA, amounting to 22% of the viral genome, which is significantly more than that found in CPV.

Journal Article↗
PubMed1997

Structure of Semliki Forest virus core protein.

Alphaviruses are enveloped, insect-borne viruses, which contains a positive-sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin-like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy-terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 A and 3.3 A resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail-to-tail dimer contact, monomers B and C make a head-to-head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head-to-tail contacts found in the pentameric and hexameric associations within the virion utilize in the same monomer surface regions as found in the crystalline dimer interfaces.

Amino Acid Sequence↗
PubMed1997

The structure of alfalfa mosaic virus capsid protein assembled as a T=1 icosahedral particle at 4.0-A resolution.

K. Fukuyama, S. S. Abdel-Meguid, J. E. Johnson, and M. G. Rossmann (J. Mol. Biol. 167:873-984, 1983) reported the structure of alfalfa mosaic virus assembled from the capsid protein as a T=1 icosahedral empty particle at 4.5-A resolution. The information contained in the structure included the particle size, protein shell thickness, presence of wide holes at the icosahedral fivefold axes, and a proposal that the capsid protein adopts a beta-barrel structure. In the present work, the X-ray diffraction data of Fukuyama et al. as well as the data subsequently collected by I. Fita, Y. Hata, and M. G. Rossmann (unpublished) were reprocessed to 4.0-A resolution, and the structure was solved by molecular replacement. The current structure allowed the tracing of the polypeptide chain of the capsid protein confirming the beta-sandwich fold and provides information on intersubunit interactions in the particle. However, it was not possible to definitively assign the amino acid sequence to the side chain density at 4-A resolution. The particle structure was also determined by cryoelectron microscopy and image reconstruction methods and found to be in excellent agreement with the X-ray model.

Alfalfa mosaic virus↗
PubMed1996

Structural analysis of a mutation in canine parvovirus which controls antigenicity and host range.

A single mutation in canine parvovirus (CPV) of VP2 residue 300 from alanine to aspartic acid causes a loss of canine host range and alters the antigenic properties of the virus. The three-dimensional structure of this mutant has been solved to 3.25 A resolution. Crystals of full particles were triclinic, with cell dimensions of a = 267.6, b = 268.5, c = 274.3 A. alpha = 61.9, beta = 62.6, and gamma = 60.2 degrees. The native structure of CPV was used as an initial model. Phases were improved by real-space electron density averaging. In spite of the relative low percentage of observed reflections (32.5% of the data between 15.0 and 3.25 A resolution), the presence of 60-fold noncrystallographic redundancy allowed the averaging procedure to converge smoothly. The mutant aspartic acid at residue 300 forms a salt bridge with Arg81 in an icosahedrally threefold-related subunit, inducing local changes within the antigenic site B on the CPV surface. In addition, the loop between residues 359 and 374 adopts a conformation similar to that displayed by feline panleukopenia virus. The ability of the Ala300-->Asp mutant to evade antibody binding can be associated with the change of charge distribution and structure in the antigenic binding site. The variation in host range behavior may be due to the increased stability as a result of formation of the salt bridge between adjacent subunits.

Animals↗
PubMed1996

Human rhinovirus 3 at 3.0 A resolution.

BACKGROUND: The over 100 serotypes of human rhinoviruses (HRV) are major causative agents of the common cold in humans. These HRVs can be roughly divided into a major and minor group according to their cellular receptors. They can also be divided into two antiviral groups, A and B, based on their sensitivity to different capsid-binding antiviral compounds. The crystal structures of HRV14 and HRV16, major-receptor group rhinoviruses, as well as HRV1A, a minor-receptor group rhinovirus, were determined previously. Sequence comparisons had shown that HRV14 seemed to be an outlier among rhinoviruses. Furthermore, HRV14 was the only virus with no cellular 'pocket factor' in a hydrophobic pocket which is targeted by many capsid-binding antiviral compounds and is thought to regulate viral stability. HRV3, another major-receptor group virus, was chosen for study because it is one of a subset of serotypes that best represents the drug sensitivity of most rhinovirus serotypes. Both HRV3 and HRV14 belong to antiviral group A, while HRV16 and HRV1A belong to antiviral group B. RESULTS: HRV3 was found to be very similar to HRV14 in sequence and structure. Like HRV14, crystallized HRV3 also has no bound pocket factor. The structure of HRV3 complexed with an antiviral compound, WIN56291, was also determined and found to be similar to the same antiviral compound complexed with HRV14. CONCLUSIONS: The amino-acid sequence and structural similarity between HRV3 and HRV14 suggests that rhinoviruses in the same antiviral group have similar amino-acid sequences and structures. The similar amino-acid composition in the pocket region and the viral protein VP1 N termini in all known group B HRV sequences suggests that these viruses may all contain pocket factors and ordered N-terminal amphipathic helices in VP1. Both of these factors contribute to viral stability, which is consistent with the observations that group B rhinoviruses have a higher chance of successful transmission from one host to another and is a possible explanation for the observed higher pathogenicity of these rhinoviruses.

Antigens, Viral↗
PubMed1996

Structural analysis of Sindbis virus capsid mutants involving assembly and catalysis.

Sindbis virus core protein (SCP) has been isolated from virus and crystallized. The X-ray crystallographic structure showed that the amino-terminal 113 residues appeared to be either disordered or truncated during crystallization and that the carboxy-terminal residues 114 to 264 had a chymotrypsin-like structure. The carboxy-terminal residues 106 to 264 and 106 to 266 of SCP have now been expressed in Escherichia coli. Most crystal forms of the truncated proteins were isomorphous with those of the virally extracted protein. There are only small structural differences between the truncated recombinant protein and the ordered part of the wild-type virus-extracted protein. Hence, E. coli-expressed SCP can be used to study proteolytic properties and the contribution of SCP to nucleocapsid assembly, interaction with the E2 glycoprotein and interaction with RNA. The same dimer that was found in two different crystal forms of the virus-extracted SCP was present also in some of the crystals of the truncated recombinant protein. The monomer-monomer interface is maintained by two pairs of hydrogen bonds and by hydrophobic interactions. Removal of the hydrogen bonds by single substitutions did not prevent dimer formation. However, a mutation that reduced the hydrophobic contacts did inhibit dimer formation. The wild-type truncated SCP is active in E. coli, as evidenced by proteolytic processing of a series of progressively longer precursors that extend beyond residue 264. Unlike the virus-extracted capsid protein, the E. coli-expressed SCP described here is terminated following the carboxy-terminal residue and, therefore, does not require autocatalysis. Nevertheless, the E. coli-expressed protein folds with the carboxy-terminal tryptophan residue in the specificity pocket. Two crystallographically independent molecules of SCP(106 to 266), which had two additional downstream residues and had the essential S215 mutated to alanine, showed two distinct modes of binding the uncleaved carboxy-terminal residues. These may represent successive steps of binding substrate prior to catalytic cleavage. Refinement of the various crystal structures of SCP showed that the amino-terminal arm from residues 107 to 113 was not disordered, but is associated with neighboring molecules. Residues 108 to 111 bind into a hydrophobic pocket composed primarily of Y180, W247 and F166. It had been shown that the double mutant (Y180S; E183G), with the Y180S substitution in this pocket, produced a large number of non-infectious virions, possibly because of modification in the interaction of the glycoprotein spikes with core proteins. The crystal structure of this double mutant showed that there was a large positional change in the side-chain of W247, which moved into the space created by the replacement of Y180 with serine. These conformational changes may alter the stability of the virion and, thus, regulate its functional requirements during cell entry.

3C Viral Proteases↗
PubMed1996

Cryo-electron microscopy studies of empty capsids of human parvovirus B19 complexed with its cellular receptor.

The three-dimensional structures of human parvovirus B19 VP2 capsids, alone and complexed with its cellular receptor, globoside, have been determined to 26 resolution. The B19 capsid structure, reconstructed from cryo-electron micrographs of vitrified specimens, has depressions on the icosahedral 2-fold and 3-fold axes, as well as a canyon-like region around the 5-fold axes. Similar results had previously been found in an 8 angstrom resolution map derived from x-ray diffraction data. Other parvoviral structures have a cylindrical channel along the 5-fold icosahedral axes, whereas density covers the 5-fold axes in B19. The glycolipid receptor molecules bind into the depressions on the 3-fold axes of the B19:globoside complex. A model of the tetrasaccharide component of globoside, organized as a trimeric fiber, fits well into the difference density representing the globoside receptor. Escape mutations to neutralizing antibodies map onto th capsid surface at regions immediately surrounding the globoside attachment sites. The proximity of the antigenic epitopes to the receptor site suggests that neutralization of virus infectivity is caused by preventing attachment of viruses to cells.

Capsid↗
PubMed1996

Identification of a protein binding site on the surface of the alphavirus nucleocapsid and its implication in virus assembly.

BACKGROUND: Many enveloped viruses exit cells by budding from the plasma membrane. The driving force for budding is the interaction of an inner protein nucleocapsid core with transmembrane glycoprotein spikes. The molecular details of this process are ill defined. Alphaviruses, such as Sindbis virus (SINV) and Semliki Forest virus (SFV), represent some of the simplest enveloped viruses and have been well characterized by structural, genetic and biochemical techniques. Although a high-resolution structure of an alphavirus has not yet been attained, cryo-electron microscopy (cryo-EM) has been used to show the multilayer organization at 25 A resolution. In addition, atomic resolution studies are available of the C-terminal domain of the nucleocapsid protein and this has been modeled into the cryo-EM density. RESULTS: A recombinant form of Sindbis virus core protein (SCP) was crystallized and found to diffract much better than protein extracted from the virus (2.0 A versus 3.0 A resolution). The new structure showed that amino acids 108 to 111 bind to a specific hydrophobic pocket in neighboring molecules. Re-examination of the structures derived from virus-extracted protein also showed this 'N-terminal arm' binding to the same hydrophobic pocked in adjacent molecules. It is proposed that the binding of these capsid residues into the hydrophobic pocket of SCP mimics the binding of E2 (one of two glycoproteins that penetrate the lipid bilayer of the viral envelope) C-terminal residues in the pocket. Mutational studies of capsid residues 108 and 110 confirm their role in capsid assembly. CONCLUSIONS: Structural and mutational analyses of residues within the hydrophobic pocket suggest that budding results in a switch between two conformations of the capsid hydrophobic pocket. This is the first description of a viral budding mechanism in molecular detail.

Alphavirus↗
PubMed1996

Preliminary crystallographic studies of bacteriophage T4 fibritin confirm a trimeric coiled-coil structure.

Fibritin, a 52-kDa product of gene wac of bacteriophage T4, forms fibrous "whiskers" that connect to the phage tail and facilitate the later stages of phage assembly. Preliminary experiments suggest that fibritin is a trimer, and its predominant central part has a parallel alpha-helical coiled-coil structure. To investigate the oligomerization and function of fibritin, we have designed and studied two related deletion mutants, denoted M and E, that consist of its last 75 and 120 amino acids, respectively. Both proteins contain part of the coiled-coil region and the 29 amino acid carboxy-terminal domain essential for the trimerization of fibritin. The proteins are expressed as a soluble product in an Escherichia coli system. We have obtained crystals of fibritins M and E. Complete native X-ray diffraction data sets have been collected to 1.85 and 2.7 A resolution, respectively. The crystals have space group P3 with a=44.3 A, c=91.3 A (fibritin M) and R32 with a=41.2 A, b=358.7 A (fibritin E) in the hexagonal setting. Symmetry and packing considerations show that fibritin is a triple coiled coil.

Amino Acid Sequence↗
PubMed1996

Phase combination.

In 1961 Rossmann & Blow published a simple procedure for analytically combining the phase probabilities derived from various isomorphous derivatives or other phase-determining procedures [Rossmann & Blow (1961). Acta Cryst. 14, 641-647]. However, they found it necessary to make an approximation in obtaining the expression for the lack of closure (epsilon) of the phase triangle. In 1970 Hendrickson & Lattman [Hendrickson & Lattman (1970). Acta Cryst. B26, 136-143] suggested an alternative method of defining the lack of closure of the phase triangle which did not require any approximation in deriving the same analytical expression for the phase-probability function. It is now shown that it is possible to avoid the Rossmann-Blow approximation and thereby maintain the original meaning of the lack of closure as defined by Blow & Crick [(1959). Acta Cryst. 12, 794-802] and Dickerson, Kendrew & Strandberg [(1961). Acta Cryst. 14, 1188-1195].

Journal Article↗
PubMed1996

Atomic structure of the degraded procapsid particle of the bacteriophage G4: induced structural changes in the presence of calcium ions and functional implications.

Bacteriophage G4 and phiX174 are members of the Microviridae family. The degree of similarity of the structural proteins ranges from 66% identity of the F protein to 40% identity of the G protein. The atomic structure of the phiX174 virion had previously been determined by X-ray crystallography. Bacteriophage G4 procapsids, consisting of the structural proteins F, G, D, B, H, and small traces of J but no DNA, were set up for crystallization. However, the resultant crystals were of degraded procapsid particles, which had lost the assembly scaffolding proteins D and B, resulting in particles that resembled empty virions. The structure of the degraded G4 procapsid has been determined to 3.0 angstrom resolution. The particles crystallized in the hexagonal space group P6(3)22 with unit cell dimensions a=b=414.2(5) angstrom and c=263.0(3) angstrom. The diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) on film and image plates using oscillation photography. Packing considerations indicated there were two particles per unit cell. A self-rotation function confirmed that the particles were positioned on 32 point group special positions in the unit cell. Initial phases were calculated to 6 angstrom resolution, based on the known phiX174 virion model. Phase information was then extended in steps to 3.0 angstrom resolution by molecular replacement electron density modification and particle envelope generation. The resulting electron density map was readily interpretable in terms of the F and G polypeptides, as occur in the mature capsid of phiX174. In a few regions of the electron density map there were inconsistencies between the density and the published amino acid sequence. Redetermining the amino acid sequence confirmed that the density was correct. The r.m.s. deviation between the Calpha backbone of the mature capsid of phiX174 and the degraded G4 procapsid was 0.36 angstrom for the F protein and 1.38 angstrom for the G protein. This is consistent with the greater conservation of the F protein compared to the G protein sequences among members of the Microviridae family. Functionally important features between phiX174 and G4 had greater conservation. Calcium ions (Ca2+) were shown to bind to G4 at a general site located near the icosahedral 3-fold axis on the F protein capsid, equivalent to sites found previously in phiX174. Binding of Ca2+ also caused the ordering of the conserved region of the DNA binding protein J, which was present in the degraded procapsid particle in the absence of DNA.

Amino Acid Sequence↗