The beginnings of structural biology. Recollections, special section in honor of Max Perutz.
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England↗
Biomedical subjects
M G Rossmann
Publications and source records attributed to M G Rossmann.
Human rhinovirus 14 complexed with fragments of active antiviral compounds.
Crystallographic studies of human rhinovirus 14 (HRV14) crystals soaked with fragments of antiviral WIN compounds, at high concentrations (82-200 micrograms/ml), show the compounds bind into the hydrophobic beta-barrel (WIN pocket) of VP1. Two of these short compounds (5-[3,5-dimethyl-4-hydroxyphenyl]-2-methyltetrazole and phenol oxazoline) cause conformational changes in the virus similar to the active, longer WIN compounds. In addition, thermostabilization studies suggest these short WIN compounds provide some stability to the HRV14 capsid. We conclude that the short compounds appear to mimic the cellular cofactors observed in the hydrophobic pocket of VP1 for some picornaviruses. Both cofactors and short WIN compounds bind into the pocket, cause conformational changes in VP1, and provide a small degree of virion stabilization but are unlikely to inhibit attachment. Three specific binding sites for dimethyl sulfoxide (DMSO), used as solvent, were also identified. One of the DMSO molecules binds into the drug binding pocket near the pocket opening, while the other two bind in the canyon near the VP1 protomer-protomer interface.
Antiviral Agents↗
Crystallographic and cryo EM analysis of virion-receptor interactions.
Cryoelectron microscopy has been used to determine the first structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 (HRV16) complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule-1 (ICAM-1) shows that ICAM-1 binds into the 12 A deep "canyon" on the surface of the virus. This is consistent with the prediction that the viral receptor attachment site lies in a cavity inaccessible to the host's antibodies. The atomic structures of HRV14 and CD4, homologous to HRV16 and ICAM-1, showed excellent correspondence with observed density, thus establishing the virus-receptor interactions.
Cell Adhesion Molecules↗
Determination and refinement of the canine parvovirus empty-capsid structure.
The canine parvovirus (CPV) empty-capsid structure has been determined and refined to 3.0 A resolution in the tetragonal space group P4(3)2(1)2 with cell dimensions a = b = 254.5 and c = 795.0 A. The successful structure determination shows that reasonably good diffraction data were obtained in spite of the very long c axis. The structure was solved by molecular replacement using the electron density of CPV full particles in a monoclinic space group. The phases were refined by non-crystallographic symmetry averaging. The structure refinement was carried out by using the programs PROLSQ and X-PLOR. The final R factor for the structure that included 85 water molecules per icosahedral asymmetric unit was 21.1% for reflections between 6.0 and 3.0 A resolution with an r.m.s. deviation of bond lengths of 0.020 A from ideal values. The structure of CPV empty capsids showed conformational differences with respect to full capsids at a region where icosahedrally ordered DNA in full particles interacts with the capsid protein. It also confirmed the absence of density along the fivefold axis in the CPV empty-particle structure in contrast to the situation in CPV full particles.
Journal Article↗
A monoclinic crystal with R32 pseudo-symmetry: a preliminary report of Nodamura virus structure determination.
We have crystallized Nodamura virus, a T = 3 icosahedral virus that can infect both mammalian and insect hosts. Crystals are monoclinic, with two crystallographically independent virus molecules per asymmetric unit. Packing analysis reveals a pseudo-rhombohedral (pseudo-C2 in the monoclinic setting) arrangement of virus particles in the crystal lattice. Crystals differ from the R32 symmetry by rotational and translational deviations. The rhombohedral packing arrangement and its failure to describe the exact virus packing is analyzed in detail. The icosahedral threefold axis is rotated from the body diagonal of the pseudo-rhombohedral cell, breaking the rhombohedral symmetry. The C2 pseudo-symmetry breaks down rotationally and/or translationally.
Journal Article↗
The canine parvovirus empty capsid structure.
The structure of empty canine parvovirus capsids shows that residues 37 to the carboxy-terminal residue 584 (VP2 numbering) are ordered in each of the 60 subunits. The central structural motif of each subunit is the eight-stranded antiparallel beta-barrel that has been found in many other virus structures. Five beta-hairpin turns form a beta-cylindrical structure at each icosahedral 5-fold axis. The N-terminal glycine-rich sequence can be accommodated within this cylinder without excessive steric hindrance, consistent with the electron density distribution. By far the largest conformational differences between the full and empty virus were found in the region where some ordered DNA has been observed to bind in canine parvovirus full particles. Extensive interactions among 3-fold related subunits indicate that a trimeric subunit might be a viral assembly intermediate.
Amino Acid Sequence↗
A comparison of the anti-rhinoviral drug binding pocket in HRV14 and HRV1A.
The three-dimensional structures of two human rhinovirus serotypes (HRV14 and HRV1A) are compared when complexed with various antiviral agents. Although these agents all bind into the same hydrophobic pocket, the exact viral-drug interactions differ. In the absence of drugs, the pocket is occupied by a fatty acid in HRV1A, but is empty in HRV14 except for two water molecules. The conformation of each drug is dependent upon the shape of the hydrophobic pocket. In HRV14 the major residues determining the shape of the binding site are Y1128, P1174 and M1224, corresponding to I1125, M1169 and I1220 in HRV1A. When there is no cofactor or a drug in the pocket, the entrance to the pocket is open. However, the entrance is closed when the pocket is occupied by a cofactor or a drug. There are relatively small conformational changes when the agents displace the natural cofactor in HRV1A. In contrast, there are much larger conformational changes on binding a drug in HRV14. These differences cause an inhibition of viral attachment in HRV14 but not in HRV1A. Binding of the drugs results in three additional interprotomer hydrogen bonds in HRV14 and one in HRV1A. These hydrogen bonds and a potential loss of flexibility upon efficient packing of the pocket may contribute to the inhibition of uncoating in both serotypes.
Amino Acid Sequence↗
Refined structure of Sindbis virus core protein and comparison with other chymotrypsin-like serine proteinase structures.
Crystal forms 2 and 3 of Sindbis virus core protein have been refined to 2.8 A and 3.0 A resolution, respectively. The three independent molecular copies in the two crystal forms are essentially identical, except for regions where the molecules are involved in different crystal packing interactions. The overall polypeptide backbone fold of Sindbis virus core protein is similar to other chymotrypsin-like serine proteinase structures despite a lack of significant sequence homology. Detailed analysis revealed differences in the catalytic triad and the substrate binding pockets between the Sindbis virus core protein and the other serine proteinases. The catalytic aspartic acid residue (Asp163) and residue Asp214 (corresponding to Asp194 in chymotrypsin) are partially exposed to solvent in Sindbis virus core protein. Chymotrypsin Ser214, hydrogen bonded to the catalytic aspartic acid residue in all other serine proteinase structures, is changed to Leu231 in Sindbis virus core protein. Deletions in the loop regions on the surface of the protein account for the smaller size of the ordered part of Sindbis virus core protein (151 residues) as compared to chymotrypsin (236 residues), and permits the cis autocatalytic cleavage of the polyprotein to produce the viral capsid protein.
Amino Acid Sequence↗
Structure of a human rhinovirus complexed with its receptor molecule.
Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. The atomic structures of human rhinovirus 14 and CD4, homologous to human rhinovirus 16 and intercellular adhesion molecule 1, showed excellent correspondence with observed density, thus establishing the virus-receptor interactions.
Antigens, CD↗
Structure determination of feline panleukopenia virus empty particles.
Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 380.1 A, b = 379.3 A, and c = 350.9 A, has been determined to 3.3 A resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 A resolution and gradually extended to 3.3 A resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA.
Amino Acid Sequence↗
Structure, sequence, and function correlations among parvoviruses.
The capsid protein sequences of 10 representative parvoviruses were aligned against the sequence and three-dimensional structure of canine parvovirus (CPV). The structure of CPV was then analyzed after mapping onto it position-dependent sequence similarity scores and the locations of residues that are phenotypically important in other parvoviruses. Antigenicity is primarily associated with external exposed loops of high sequence variability. Amino acids in the canyon, a surface depression encircling each fivefold axis, are well conserved, but may have a function other than external receptor binding. Residues important to parvoviral cell specificity and erythrocyte binding are scattered near the rim of a less-conserved depression near the twofold axis, and on a shoulder of the threefold spike. The number of residues involved in various interactions and their conservation and properties suggest that uncoating may involve separation of fivefold and twofold related subunits before those related by threefold symmetry. The inner surface residues of the capsid are generally more highly conserved than those on the outer surface, presumably due to interactions with DNA, although the binding site that contains ordered DNA in the structure is not especially conserved.
Amino Acid Sequence↗
Comparison of surface properties of picornaviruses: strategies for hiding the receptor site from immune surveillance.
The surface topology and sequence conservation of different picornaviruses have been compared using molecular graphics and statistical analyses. The comparisons suggest that the canyons, surface depressions encircling the fivefold axes, are the sites of receptor attachment of enteroviruses as well as human rhinovirus (HRV). In HRV14, the receptor binding footprint extends beyond the canyon (Olson et al. (1993), Proc. Natl. Acad. Sci. USA 90, 507-511), but these more exposed regions are not conserved in polioviruses or minor receptor group rhinoviruses, suggesting some differences in receptor binding. When rhinoviruses are compared to other picorna- and parvoviruses, the surface sequence similarity relative to the similarity of buried residues is about the same. Thus, the effect of host immune surveillance on surface residues is similar. Therefore, it is probable that some means of protecting a conserved binding site from immune detection (such as a canyon) will be an essential requirement in other viruses. However, there may be other strategies for achieving this result. For instance, as in foot-and-mouth disease virus, the receptor attachment site of Mengo virus is associated with a flexible structural component which may inhibit the production of neutralizing antibodies that are able to recognize only one explicit conformation.
Picornaviridae↗
Conformationally restricted analogues of disoxaril: a comparison of the activity against human rhinovirus types 14 and 1A.
A series of conformationally restricted analogs of disoxaril has been synthesized and evaluated against human rhinovirus types (HRV) 14 and 1A. The sensitivity of these serotypes to this series varied and was dependent upon the length of the molecule as well as upon the flexibility of the aliphatic chain. Minimum energy conformations of these compounds were overlaid with the X-ray structure of a closely related analog 9 bound to the capsid protein of both HRV-14 and -1A and then modeled in the compound-binding site of both serotypes. A comparative sweep volume of these compounds about the isoxazole ring revealed an inaccessible region of space for the cis-olefin 8b, which is not the case for either the trans-olefin 8a or the acetylene 5. This region may be important to the binding of the compounds to the HRV-14 site particularly during entry into the pocket.
Antiviral Agents↗
Acid-induced structural changes in human rhinovirus 14: possible role in uncoating.
X-ray diffraction data were collected from human rhinovirus 14 crystals a few minutes after exposure to acid vapor and prior to excessive crystalline disorder. Conformational changes occurred (i) in the GH loop of viral protein (VP) 1, (ii) at the ion binding site on the outer surface of the pentamer center, and (iii) in VP3 and VP4 on the virion's interior in the vicinity of the fivefold axis. Amino acid substitutions in mutants resistant to low pH, or to drugs that inhibit uncoating, were concentrated in the vicinity of the GH loop. It is proposed that the acid-induced changes reflect processes that trigger uncoating.
Amino Acid Sequence↗
Structure determination of the bacteriophage phiX174.
The structure of the single-stranded DNA phage phiX174 has been determined to 3.4 A resolution. The crystal space group was P2(1) with one icosahedral particle per asymmetric unit, giving 60-fold noncrystallographic redundancy. Oscillation diffraction photographs were collected using synchrotron radiation at various wavelengths. The particle orientations in the unit cell were determined with a rotation function. Because cowpea mosaic virus has a similar external envelope to phiX174, it was used as a search model to find the approximately positions of the phiX174 particles in the unit cell relative to the crystallographic symmetry axes. An initial phase set to 12 A resolution was then based on the cowpea mosaic virus atomic structure. These phases were improved by 20 cycles of real-space molecular replacement averaging. The phase information was gradually extended to 3.4 A resolution by molecular replacement electron density averaging. One reciprocal lattice point was used for each extension followed by four cycles of averaging. The unusual particle capsid, with its 12 pentameric spikes, required the careful determination of a precise molecular envelope. This was redetermined at regular intervals, as was the particle center. The resultant electron density map was readily interpreted in terms of the F, G and J polypeptides in the capsid. A difference electron density map between full and partially empty particles showed some ordered DNA structure.
Bacteriophage phi X 174↗
The structure determination of Sindbis virus core protein using isomorphous replacement and molecular replacement averaging between two crystal forms.
The structure of Sindbis virus core protein has been determined by a combination of multiple isomorphous replacement and molecular replacement averaging techniques. The multiple isomorphous replacement phase determinations were made for two crystal forms (P2(1) and P4(3)2(1)2) of the core protein. The real-space molecular replacement averaging was subsequently carried out between two copies of the protein per asymmetric unit in the monoclinic form and one copy in the tetragonal form. This greatly improved the quality of the electron density maps. The Sindbis virus core protein polypeptide could be traced and related to the known amino acid sequence. The averaging procedure between different crystal forms, as described in this paper, should be generally applicable to other systems.
Crystallization↗
Preliminary X-ray crystallographic analysis of intercellular adhesion molecule-1.
Crystals of the two amino-terminal domains of intercellular adhesion molecule-1, the receptor for the major group of human rhinovirus serotypes, diffract to 3.0 A resolution. The crystals are trigonal in space group P3(1)21 or P3(2)21 with cell dimensions of a = b = 55.7 A, c = 166.3 A, with probably six molecules per unit cell.
Antigens, CD↗