Use of MCSS to design small targeted libraries: application to picornavirus ligands.

Computational methods were used to design structure-based combinatorial libraries of antipicornaviral capsid-binding ligands. The multiple copy simultaneous search (MCSS) program was employed to calculate functionality maps for many diverse functional groups for both the poliovirus and rhinovirus capsid structures in the region of the known drug binding pocket. Based on the results of the MCSS calculations, small combinatorial libraries consisting of 10s or 100s of three-monomer compounds were designed and synthesized. Ligand binding was demonstrated by a noncell-based mass spectrometric assay, a functional immuno-precipitation assay, and crystallographic analysis of the complexes of the virus with two of the candidate ligands. The P1/Mahoney poliovirus strain was used in the experimental studies. A comparison showed that the MCSS calculations had correctly identified the observed binding site for all three monomer units in one ligand and for two out of three in the other ligand. The correct central monomer position in the second ligand was reproduced in calculations in which the several key residues lining the pocket were allowed to move. This study validates the computational methodology. It also illustrates that subtle changes in protein structure can lead to differences in docking results and points to the importance of including target flexibility, as well as ligand flexibility, in the design process.

Molecular genetic analysis of revertants from a poliovirus mutant that is specifically adapted to the mouse spinal cord.

SA virus, a mutant of the Mahoney strain of type 1 poliovirus (PV1/Mahoney), replicates specifically in the spinal cords of mice and causes paralysis, although the PV1/Mahoney strain does not show any mouse neurovirulence (Q. Jia, S. Ohka, K. Iwasaki, K. Tohyama, and A. Nomoto, J. Virol. 73:6041-6047, 1999). The key mutation site for the mouse neurovirulence of SA was mapped to nucleotide (nt) 928 of the genome (A to G), resulting in the amino acid substitution of Met for Ile at residue 62 within the capsid protein VP4 (VP4062). A small-plaque phenotype of SA appears to be indicative of its mouse-neurovirulent phenotype. To identify additional amino acid residues involved in the host range determination of PV, a total of 14 large-plaque (LP) variants were isolated from a single point mutant, Mah/I4062M, that showed the SA phenotype. All the LP variants no longer showed any mouse neurovirulence when delivered via an intraspinal inoculation route. Of these, 11 isolates had a back mutation at nt 928 (G to A) that restored the nucleotide of the PV1/Mahoney type. The reversions of the remaining three isolates (LP8, LP9, and LP14) were mediated by a second site mutation. Molecular genetic analysis involving recombinants between Mah/I4062M and the LP variants revealed that the mere substitution of an amino acid residue at position 107 in VP1 (Val to Leu) (LP9), position 33 in VP2 (Val to Ile) (LP14), or position 231 in VP3 (Ile to Thr) (LP8) was sufficient to restore the PV1/Mahoney phenotype. These amino acid residues are located either on the surface or inside of the virus particle. Our results indicate that the mouse neurovirulence of PV is determined by the virion surface structure, which is formed by all four capsid proteins.

Kinetic analysis of the effect of poliovirus receptor on viral uncoating: the receptor as a catalyst.

We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S or N particle) to an infectious intermediate (135S or A particle). The viral receptor behaves as a classic transition state theory catalyst, facilitating the N-to-A conversion by lowering the activation energy for the process by 50 kcal/mol. In contrast to earlier studies which demonstrated that capsid-binding drugs inhibit thermally mediated N-to-A conversion through entropic stabilization alone, capsid-binding drugs are shown to inhibit receptor-mediated N-to-A conversion through a combination of enthalpic and entropic effects.

Ab initio phasing of high-symmetry macromolecular complexes: successful phasing of authentic poliovirus data to 3.0 A resolution.

A genetic algorithm-based computational method for the ab initio phasing of diffraction data from crystals of symmetric macromolecular structures, such as icosahedral viruses, has been implemented and applied to authentic data from the P1/Mahoney strain of poliovirus. Using only single-wavelength native diffraction data, the method is shown to be able to generate correct phases, and thus electron density, to 3.0 A resolution. Beginning with no advance knowledge of the shape of the virus and only approximate knowledge of its size, the method uses a genetic algorithm to determine coarse, low-resolution (here, 20.5 A) models of the virus that obey the known non-crystallographic symmetry (NCS) constraints. The best scoring of these models are subjected to refinement and NCS-averaging, with subsequent phase extension to high resolution (3.0 A). Initial difficulties in phase extension were overcome by measuring and including all low-resolution terms in the transform. With the low-resolution data included, the method was successful in generating essentially correct phases and electron density to 6.0 A in every one of ten trials from different models identified by the genetic algorithm. Retrospective analysis revealed that these correct high-resolution solutions converged from a range of significantly different low-resolution phase sets (average differences of 59.7 degrees below 24 A). This method represents an efficient way to determine phases for icosahedral viruses, and has the advantage of producing phases free from model bias. It is expected that the method can be extended to other protein systems with high NCS.

A structurally biased combinatorial approach for discovering new anti-picornaviral compounds.

BACKGROUND: Picornaviruses comprise a family of small, non-enveloped RNA viruses. A common feature amongst many picornaviruses is a hydrophobic pocket in the core of VP1, one of the viral capsid proteins. The pocket is normally occupied by a mixture of unidentified, fatty acid-like moieties, which can be competed out by a family of capsid-binding, antiviral compounds. Many members of the Picornaviridae family are pathogenic to both humans and livestock, yet no adequate therapeutics exist despite over a decade's worth of research in the field. To address this challenge, we developed a strategy for rapid identification of capsid-binding anti-picornaviral ligands. The approach we took involved synthesizing structurally biased combinatorial libraries that had been targeted to the VP1 pocket of poliovirus and rhinovirus. The libraries are screened for candidate ligands with a high throughput mass spectrometry assay. RESULTS: Using the mass spectrometry assay, we were able to identify eight compounds from a targeted library of 75 compounds. The antiviral activity of these candidates was assessed by (i) measuring the effect on the kinetics of viral uncoating and (ii) the protective effect of each drug in traditional cell-based assays. All eight of the candidates exhibited antiviral activity, but three of them were particularly effective against poliovirus and rhinovirus. CONCLUSIONS: The results illustrate the utility of combining structure-based design with combinatorial chemistry. The success of our approach suggests that assessment of small, targeted libraries, which query specific chemical properties, may be the best strategy for surveying all of chemical space for ideal anti-picornaviral compounds.

Functional group placement in protein binding sites: a comparison of GRID and MCSS.

One approach to combinatorial ligand design begins by determining optimal locations (i.e., local potential energy minima) for functional groups in the binding site of a target macromolecule. MCSS and GRID are two methods, based on significantly different algorithms, which are used for this purpose. A comparison of the two methods for the same functional groups is reported. Calculations were performed for nonpolar and polar functional groups in the internal hydrophobic pocket of the poliovirus capsid protein, and on the binding surface of the src SH3 domain. The two approaches are shown to agree qualitatively; i.e., the global characteristics of the functional group maps generated by MCSS and GRID are similar. However, there are significant differences in the relative interaction energies of the two sets of minima, a consequence of the different functional form used to evaluate polar interactions (electrostatics and hydrogen bonding) in the two methods. The single sphere representation used by GRID affords only positional information, supplemented by the identification of hydrogen bonding interactions. By contrast, the multi-atom representation of most MCSS groups yields in both positional and orientational information. The two methods are most similar for small functional groups, while for larger functional groups MCSS yields results consistent with GRID but superior in detail. These results are in accord with the somewhat different purposes for which the two methods were developed. GRID has been used mainly to introduce functionalities at specific positions in lead compounds, in which case the orientation is predetermined by the structure of the latter. The orientational information provided by MCSS is important for its use in the de novo design of large, multi-functional ligands, as well as for improving lead compounds.

The structure and oligomerization of the yeast arginine methyltransferase, Hmt1.

Protein methylation at arginines is ubiquitous in eukaryotes and affects signal transduction, gene expression and protein sorting. Hmt1/Rmt1, the major arginine methyltransferase in yeast, catalyzes methylation of arginine residues in several mRNA-binding proteins and facilitates their export from the nucleus. We now report the crystal structure of Hmt1 at 2.9 A resolution. Hmt1 forms a hexamer with approximate 32 symmetry. The surface of the oligomer is dominated by large acidic cavities at the dimer interfaces. Mutation of dimer contact sites eliminates activity of Hmt1 both in vivo and in vitro. Mutating residues in the acidic cavity significantly reduces binding and methylation of the substrate Npl3.

Is the 135S poliovirus particle an intermediate during cell entry?

Poliovirus binding to its receptor (PVR) on the cell surface induces a conformational transition which generates an altered particle with a sedimentation value of 135S versus the 160S of the native virion. A number of lines of evidence suggest that the 135S particle is a cell entry intermediate. However, the low infection efficiencies of the 135S particle and the absence of detectable 135S particles during infection at 26 degrees C by the cold-adapted mutants argue against a role for the 135S particle during the cell entry process. We show here that binding of 135S-antibody complexes to the Fc receptor (CDw32) increases the infectivity of these particles by 2 to 3 orders of magnitude. Thus, the low efficiency of infection by 135S particles is due in part to the low binding affinity of these particles. In addition, we show that there is an additional stage in the entry process that is associated with RNA release. This stage occurs after formation of the 135S particle, is rate limiting during infection at 37 degrees C, but not at 26 degrees C, and is PVR independent. The data also demonstrate that during infection at 26 degrees C, the rate-limiting step is the PVR-mediated conversion of wild-type 160S particles to 135S particles. This suggests that during infection at 26 degrees C by the cold-adapted viruses, 135S particles are formed, but they fail to accumulate to detectable levels because the subsequent post-135S particle events occur at a significantly faster rate than the initial conversion of 160S to 135S particles. These data support a model in which the 135S particle is an intermediate during poliovirus entry.

The crystal structure of an unusual processivity factor, herpes simplex virus UL42, bound to the C terminus of its cognate polymerase.

Herpes simplex virus DNA polymerase is a heterodimer composed of a catalytic subunit, Pol, and an unusual processivity subunit, UL42, which, unlike processivity factors such as PCNA, directly binds DNA. The crystal structure of a complex of the C-terminal 36 residues of Pol bound to residues 1-319 of UL42 reveals remarkable similarities between UL42 and PCNA despite contrasting biochemical properties and lack of sequence homology. Moreover, the Pol-UL42 interaction resembles the interaction between the cell cycle regulator p21 and PCNA. The structure and previous data suggest that the UL42 monomer interacts with DNA quite differently than does multimeric toroidal PCNA. The details of the structure lead to a model for the mechanism of UL42, provide the basis for drug design, and allow modeling of other proteins that lack sequence homology with UL42 or PCNA.

Stabilization of poliovirus by capsid-binding antiviral drugs is due to entropic effects.

When poliovirus attaches to its receptor or is heated in hypotonic buffers, the virion undergoes an irreversible conformational transition from the native 160 S (or N) particle to the 135 S (or A) particle, which is believed to mediate cell entry. The first-order rate constants for the thermally induced transition have been measured as a function of temperature for virus alone and for complexes of the virus with capsid-binding drugs that inhibit the receptor and thermally mediated conversion. Although the drugs have minimum inhibitory concentrations (MIC) that differ by almost three orders of magnitude, the activation energies for the N to A transition for the drug complexes (145 kcal/mol) were indistinguishable from each other or from that of the virus alone. We conclude that the antiviral activity of these drugs derives from a novel mechanism in which drug-binding stabilizes the virions through entropic effects.

Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function. Cofactor binding and substrate interactions.

Many eukaryotic RNA-binding proteins are modified by methylation of arginine residues. The yeast Saccharomyces cerevisiae contains one major arginine methyltransferase, Hmt1p/Rmt1p, which is not essential for normal cell growth. However, cells missing HMT1 and also bearing mutations in the mRNA-binding proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in which to study Hmt1p function. We now demonstrate that the catalytically active form of Hmt1p is required for its activity in vivo. Amino acid changes in the putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore growth to strains that require HMT1. In addition these mutations affect nucleocytoplasmic transport of Npl3p. A cold-sensitive mutant of Hmt1p was generated and showed reduced methylation of Npl3p, but not of other substrates, at 14 degrees C. These results define new aspects of Hmt1 and reveal the importance of its activity in vivo.

Computational design of D-peptide inhibitors of hepatitis delta antigen dimerization.

Hepatitis delta virus (HDV) encodes a single polypeptide called hepatitis delta antigen (DAg). Dimerization of DAg is required for viral replication. The structure of the dimerization region, residues 12 to 60, consists of an anti-parallel coiled coil [Zuccola et al., Structure, 6(1998)821]. Multiple Copy Simultaneous Searches (MCSS) of the hydrophobic core region formed by the bend in the helix of one monomer of this structure were carried out for many diverse functional groups. Six critical interaction sites were identified. The Protein Data Bank was searched for backbone templates to use in the subsequent design process by matching to these sites. A 14 residue helix expected to bind to the D-isomer of the target structure was selected as the template. Over 200,000 mutant sequences of this peptide were generated based on the MCSS results. A secondary structure prediction algorithm was used to screen all sequences. and in general only those that were predicted to be highly helical were retained. Approximately 100 of these 14-mers were model built as D-peptides and docked with the L-isomer of the target monomer. Based on calculated interaction energies, predicted helicity, and intrahelical salt bridge patterns, a small number of peptides were selected as the most promising candidates. The ligand design approach presented here is the computational analogue of mirror image phage display. The results have been used to characterize the interactions responsible for formation of this model anti-parallel coiled coil and to suggest potential ligands to disrupt it.

Three-dimensional structure of poliovirus receptor bound to poliovirus.

Poliovirus initiates infection by binding to its cellular receptor (Pvr). We have studied this interaction by using cryoelectron microscopy to determine the structure, at 21-A resolution, of poliovirus complexed with a soluble form of its receptor (sPvr). This density map aided construction of a homology-based model of sPvr and, in conjunction with the known crystal structure of the virus, allowed delineation of the binding site. The virion does not change significantly in structure on binding sPvr in short incubations at 4 degrees C. We infer that the binding configuration visualized represents the initial interaction that is followed by structural changes in the virion as infection proceeds. sPvr is segmented into three well-defined Ig-like domains. The two domains closest to the virion (domains 1 and 2) are aligned and rigidly connected, whereas domain 3 diverges at an angle of approximately 60 degrees. Two nodules of density on domain 2 are identified as glycosylation sites. Domain 1 penetrates the "canyon" that surrounds the 5-fold protrusion on the capsid surface, and its binding site involves all three major capsid proteins. The inferred pattern of virus-sPvr interactions accounts for most mutations that affect the binding of Pvr to poliovirus.

Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus.

Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at approximately 22-A resolution by cryo-electron microscopy. The 135S and 80S particles are both approximately 4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 A were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Poliovirus mutants at histidine 195 of VP2 do not cleave VP0 into VP2 and VP4.

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.

Crystal structure of a 14 bp RNA duplex with non-symmetrical tandem GxU wobble base pairs.

Adjacent GxU wobble base pairs are frequently found in rRNA. Atomic structures of small RNA motifs help to provide a better understanding of the effects of various tandem mismatches on duplex structure and stability, thereby providing better rules for RNA structure prediction and validation. The crystal structure of an RNA duplex containing the sequence r(GGUAUUGC-GGUACC)2 has been solved at 2.1 A resolution using experimental phases. Novel refinement strategies were needed for building the correct solvent model. At present, this is the only short RNA duplex structure containing 5'-U-U-3'/3'-G-G-5' non-symmetric tandem GxU wobble base pairs. In the 14mer duplex, the six central base pairs are all displaced away from the helix axis, yielding significant changes in local backbone conformation, helix parameters and charge distribution that may provide specific recognition sites for biologically relevant ligand binding. The greatest deviations from A-form helix occur where the guanine of a wobble base pair stacks over a purine from the opposite strand. In this vicinity, the intra-strand phosphate distances increase significantly, and the major groove width increases up to 3 A. Structural comparisons with other short duplexes containing symmetrical tandem GxU or GxT wobble base pairs show that nearest-neighbor sequence dependencies govern helical twist and the occurrence of cross-strand purine stacks.

The poliovirus empty capsid specifically recognizes the poliovirus receptor and undergoes some, but not all, of the transitions associated with cell entry.

Experimental results presented here demonstrate that the poliovirus empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with mature virus for binding sites on cells. Hence, induced changes in the structure and/or stability of the particle by RNA encapsidation and virus maturation are not necessary for recognition by receptor. In mature virus, heat-induced rearrangements mimic those induced by receptor at physiological temperatures in several important respects, namely, expulsion of VP4 and externalization of the VP1 N-terminal arm. It is shown here that in the empty capsid the VP1 N-terminal arm is externalized but the VP4 portion of VP0 is not. Thus, these two hallmark rearrangements associated with cell entry can be uncoupled.

Structural basis of the oligomerization of hepatitis delta antigen.

BACKGROUND: The hepatitis D virus (HDV) is a small satellite virus of hepatitis B virus (HBV). Coinfection with HBV and HDV causes severe liver disease in humans. The small 195 amino-acid form of the hepatitis delta antigen (HDAg) functions as a trans activator of HDV replication. A larger form of the protein containing a 19 amino acid C-terminal extension inhibits viral replication. Both of these functions are mediated in part by a stretch of amino acids predicted to form a coiled coil (residues 13-48) that is common to both forms. It is believed that HDAg forms dimers and higher ordered structures through this coiled-coil region. RESULTS: The high-resolution crystal structure of a synthetic peptide corresponding to residues 12 to 60 of HDAg has been solved. The peptide forms an antiparallel coiled coil, with hydrophobic residues near the termini of each peptide forming an extensive hydrophobic core with residues C-terminal to the coiled-coil domain in the dimer protein. The structure shows how HDAg forms dimers, but also shows the dimers forming an octamer that forms a 50 A ring lined with basic sidechains. This is confirmed by cross-linking studies of full-length recombinant small HDAg. CONCLUSIONS: HDAg dimerizes through an antiparallel coiled coil. Dimers then associate further to form octamers through residues in the coiled-coil domain and residues C-terminal to this region. Our findings suggest that the structure of HDAg represents a previously unseen organization of a nucleocapsid protein and raise the possibility that the N terminus may play a role in binding the viral RNA.

The crystal structure of Dps, a ferritin homolog that binds and protects DNA.

The crystal structure of Dps, a DNA-binding protein from starved E. coli that protects DNA from oxidative damage, has been solved at 1.6 A resolution. The Dps monomer has essentially the same fold as ferritin, which forms a 24-mer with 432 symmetry, a hollow core and pores at the three-fold axes. Dps forms a dodecamer with 23 (tetrahedral) point group symmetry which also has a hollow core and pores at the three-folds. The structure suggests a novel DNA-binding motif and a mechanism for DNA protection based on the sequestration of Fe ions.

Structure determination of echovirus 1.

The atomic structure of echovirus 1 (a member of the enterovirus genus of the picornavirus family) has been determined using cryo-crystallography and refined to 3.55 A resolution. Echovirus 1 crystallizes in space group P22121 with a = 352.45, b = 472.15 and c = 483.20 A. The crystals contain one full virus particle in the asymmetric unit allowing for 60-fold noncrystallographic symmetry averaging. The diffraction pattern shows strong pseudo-B-centering with reflections with h + l = 2n + 1 being systematically weak or absent below about 6 A resolution. The size of the unit cell and presence of pseudo-B-centering placed strong constraints on the allowed packing of the icosahedral particle in the crystal lattice. These constraints greatly facilitated the determination of the orientation and position of the virus by reducing the dimensionality of the search, but interactions between the crystallographic and noncrystallographic symmetries rendered the choice of space group ambiguous until very late in the structure determination. This structure determination provides a striking example of the power of packing analysis in molecular replacement and illustrates how subtle interactions between crystallographic and noncrystallographic symmetries can be resolved.