Biomedical subjects

J M Hogle

Publications and source records attributed to J M Hogle.

At least 37 records · Page 2Linked to original sources

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

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.

Animals↗

PubMed1997

Ligand-induced conformational changes in poliovirus-antiviral drug complexes.

Crystal structures of the Mahoney strain of type 1 poliovirus complexed with the antiviral compounds R80633 and R77975 were determined at 2.9 A resolution. These compounds block infection by preventing conformational changes required for viral uncoating. In various drug-poliovirus complexes reported earlier, no significant conformational changes were found in the structures of the capsid proteins. In the structures reported here, the strain of virus is relatively insensitive to these antivirals. Correspondingly, significant conformational changes are necessary to accommodate the drug. These conformational changes affect both the immediate vicinity of the drug binding site, and more distant loops located near the fivefold axis. In addition, small but concerted shifts of the centers of mass of the major capsid proteins consistently have been detected whose magnitudes are correlated inversely with the effectiveness of the drugs. Collectively, the drug complexes appear to sample the conformational repertoire of poliovirus near equilibrium, and thus provide a possible model for the earliest stages of viral uncoating during infection.

Journal Article↗

PubMed1997

X-ray crystal structures of the S229A mutant and wild-type MurB in the presence of the substrate enolpyruvyl-UDP-N-acetylglucosamine at 1.8-A resolution.

MurB catalyzes the second committed step in the synthesis of peptidoglycan, a key component of the bacterial cell wall. The crystal structures of both a S229A mutant and wild-type MurB in the presence of the substrate enolpyruvyl-UDP-N-acetylglucosamine were solved and refined at 1.8 A resolution. The single point mutation of residue 229 from serine to alanine eliminated a hydroxyl group which has previously been proposed to play a critical role as a proton donor during the second half-reaction of MurB, namely, reoxidation of FADH2 and reduction of the enolpyruvyl substrate. The mutation also resulted in the loss of the water molecule-hydrogen bonded to the serine hydroxyl in the wild-type structure changing the hydrogen-bonding network with in the active site. Comparison of the wild-type and S229A mutant structures confirms that the dramatic kinetic defect of an approximately 10(7)-fold decrease observed for the Ser 229 Ala mutant in the second half-reaction [Benson, T.E., Walsh, C.T., & Massey, V. (1997) Biochemistry 36, 796-805] is a direct result of the loss of the serine hydroxyl moiety rather than other nonspecific active-site changes or general structural defects.

Binding Sites↗

PubMed1997

Use of the multiple copy simultaneous search (MCSS) method to design a new class of picornavirus capsid binding drugs.

A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) method and a simple scheme for joining MCSS functional group sites was applied to the binding pocket of P3/Sabin poliovirus and rhinovirus 14. The MCSS method determines where specific functional (chemical) groups have local potential energy minima in the binding site. Before the virus application, test calculations were run to determine the optimal set of input parameters to be used in evaluating the MCSS results. The MCSS minima are analysed and selected minima are connected with (CH2)n linkers to form candidate ligands, whose structures are optimized in the binding site. Estimates of the binding strength were made for the ligands and compared with those for known drugs. The results indicate that the proposed ligands should bind to P3/Sabin poliovirus at least as well as the best of the existing drugs, and that they should also bind to P1/Mahoney poliovirus and rhinovirus 14. A detailed comparison of the poliovirus and rhinovirus binding pockets and an analysis of drug binding specificity is presented.

Algorithms↗

PubMed1997

Structural studies of poliovirus mutants that overcome receptor defects.

In order to better understand the process of cell entry for non-enveloped viruses, we have solved the crystal structures of five poliovirus mutants which can infect cells expressing mutant poliovirus receptors. Four of these structures have been solved from frozen crystals using cryocrystallographic data collection methods. The mutations have a range of structural consequences, from small local perturbations to significant loop rearrangements. All of the mutant viruses are more labile to conversion to an apparent cell entry intermediate, suggesting that these mutant viruses could compensate for the suboptimal receptors by lowering the thermal energy required to undergo the receptor-mediated conformational change.

Capsid↗

PubMed1996

Poliovirus: new insights from an old paradigm.

A combination of structural and genetic studies of poliovirus suggests that the final stages of viral assembly lock the virus in a metastable structure primed to undergo the receptor-catalyzed conformational changes required for cell entry. Future studies promise to provide detailed insights into the conformational dynamics of the virion during its life cycle.

Membrane Proteins↗

PubMed1996

A pseudo-cell based approach to efficient crystallographic refinement of viruses.

Strategies have been developed for the inexpensive refinement of atomic models of viruses and of other highly symmetric structures. These methods, which have been used in the refinement of several strains of poliovirus, focus on an arbitrary-sized parallelepiped (termed the 'protomer' box) containing a single complete averaged copy of the structural motif which forms the protein capsid, together with the fragments of other symmetry-related copies of the motif which are located in its immediate neighborhood. The Fourier transform of the protomer box provides reference structure factors for stereochemically restrained crystallographic refinement of the atomic model parameters. The phases of the reference structure factors are based on the averaged map, and are not permitted to change during the refinement. It is demonstrated that models refined using the protomer box methods do not differ significantly from models refined by more expensive full-cell calculations.

Journal Article↗

PubMed1996

A genetic algorithm for the ab initio phasing of icosahedral viruses.

Genetic algorithms have been investigated as computational tools for the de novo phasing of low-resolution X-ray diffraction data from crystals of icosahedral viruses. Without advance knowledge of the shape of the virus and only approximate knowledge of its size, the virus can be modeled as the symmetry expansion of a short list of nearly tetrahedrally arranged lattice points which coarsely, but uniformly, sample the icosahedrally unique volume. The number of lattice points depends on an estimate of the non-redundant information content at the working resolution limit. This parameterization permits a simple matrix formulation of the model evaluation calculation, resulting in a highly efficient survey of the space of possible models. Initially, one bit per parameter is sufficient, since the assignment of ones and zeros to the lattice points yields a physically reasonable low-resolution image of the virus. The best candidate solutions identified by the survey are refined to relax the constraints imposed by the coarseness of the modeling, and then trials whose intensity-based statistics are comparatively good in all resolution ranges are chosen. This yields an acceptable starting point for symmetry-based direct phase extension about half the time. Improving efficiency by incorporating the selection criterion directly into the genetic algorithm's fitness function is discussed.

Journal Article↗

PubMed1996

(E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB).

UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP-methyl-N-acetylmuramic acid in the presence of NADPH. The X-ray crystal structure of the (E)-enolbutyryl-UDP-GlcNAc-MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc-MurB complex. In both structures the groups thought to be involved in hydride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond. The stereochemical outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O is thus predicted to yield a (2R,3R)-dideuterio product. This was validated by conversion of the 2,3-dideuterio-UDP-methyl-N-acetylmuramic acid product to 2,3-dideuterio-2-hydroxybutyrate, which was shown to be (2R) by enzymatic analysis and (3R) by NMR comparison to authentic (2R,3R)- and (2R,3S)-2,3-dideuterio-2-hydroxybutyrate. Remarkably, the (E)-enolbutyryl-UDP-GlcNAc was found to partition between reduction to UDP-methyl-N-acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site, indicative of a C2 carbanion/enol species that is sufficiently long-lived to rotate around the C2-C3 single bond during catalysis.

Binding Sites↗

PubMed1996

The structure of the substrate-free form of MurB, an essential enzyme for the synthesis of bacterial cell walls.

BACKGROUND: The repeating disaccharide and pentapeptide units of the bacterial peptidoglycan layer are connected by a lactyl ether bridge biosynthesized from UDP-N-acetylglucosamine and phosphoenolpyruvate in sequential enol ether transfer and reduction steps catalyzed by MurA and MurB respectively. Knowledge of the structure and mechanism of the MurB enzyme will permit analysis of this unusual enol ether reduction reaction and may facilitate the design of inhibitors as candidate next-generation antimicrobial agents. RESULTS: The crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase, MurB, has been solved at 3.0 A and compared with our previously reported structure of MurB complexed with its substrate enolpyruvyl-UDP-N- acetylglucosamine. Comparison of the liganded structure of MurB with this unliganded form reveals that the binding of substrate induces a substantial movement of domain 3 (residues 219-319) of the enzyme and a significant rearrangement of a loop within this domain. These ligand induced changes disrupt a stacking interaction between two tyrosines (Tyr190 and Tyr254) which lie at the side of the channel leading to the active site of the free enzyme. CONCLUSIONS: The conformational change induced by enolpyruvyl-UDP-N- acetylglucosamine binding to MurB results in the closure of the substrate-binding channel over the substrate. Tyr190 swings over the channel opening and establishes a hydrogen bond with an oxygen of the alpha-phosphate of the sugar nucleotide substrate which is critical to substrate binding.

Bacterial Proteins↗

PubMed1996

The poliovirus 135S particle is infectious.

The molecular mechanism of cell entry by unenveloped viruses is poorly understood. The picornaviruses poliovirus, human rhinovirus, and coxsackievirus convert to an altered form (the 135S or A particle) upon interaction with receptors on susceptible cells at 37 degrees C. The 135S particle is thought to be a necessary intermediate because it accumulates inside susceptible cells soon after infection and drugs which inhibit conversion of the virus to this form also prevent infection. However, since a variable fraction of the altered 135S particles is reported to elute unproductively from the surface of susceptible cells, their precise role remains unclear. We have found that poliovirus 135S particles can infect Chinese hamster ovary (CHO) and murine L cells, neither of which are susceptible to infection by native poliovirus. The infectivity of the particles in tissue culture appears to be between 10(3) to 10(5) times less than that of poliovirus on HeLa cells. The 135S particle infectivity was not sensitive to RNase but was greatly reduced by proteolytic treatment. Proteolysis specifically removed the newly exposed N terminus of VP1, a feature which has previously been shown to mediate interactions of the particle with lipid membranes. These results demonstrate that although the infectivity of the 135S particle appears to be receptor independent, it nonetheless requires some property associated with the protein coat. In particular, the N terminus of VP1 plays an important role in the infection process. Our findings are consistent with the hypothesis that the 135S particle is an intermediate in the normal cell entry pathway of poliovirus infection.

Animals↗

PubMed1995

Binding of the antiviral drug WIN51711 to the sabin strain of type 3 poliovirus: structural comparison with drug binding in rhinovirus 14.

The crystal structure of the Sabin strain of type 3 poliovirus (P3/Sabin) complexed with the antiviral drug WIN51711 has been determined at 2.9 A resolution. Drugs of this kind are known to inhibit the uncoating of the virus during infection, by stabilizing the capsid against receptor-induced conformational changes. The electron density for the bound drug is very well defined so that its position and orientation are unambiguous. The drug binds in a nearly extended conformation, slightly bent in the middle, in a blind pocket formed predominantly by hydrophobic residues in the core of the beta-barrel of capsid protein VP1. Comparisons between this structure, the corresponding drug complex in human rhinovirus 14 (HRV 14), and the native structures of both viruses demonstrate that the binding of WIN51711 has markedly different effects on the structures of these two viruses. Unlike HRV14, wherein large conformational changes are observed in the coat protein after drug binding, the binding of this drug in poliovirus does not induce any significant conformational changes in the structure of the capsid protein, though the drug has a greater inhibitory effect in P3/Sabin than in HRV14. The implications of this result for the mechanism of capsid stabilization are discussed.

Journal Article↗

PubMed1995

Structure of the complex between the Fab fragment of a neutralizing antibody for type 1 poliovirus and its viral epitope.

The crystal structure of the complex between the Fab fragment of C3, a neutralizing antibody for poliovirus, and a peptide corresponding to the viral epitope has been determined at 3.0 A resolution. Although this antibody was originally raised to heat inactivated (noninfectious) virus particles, it strongly neutralizes the Mahoney strain of type 1 poliovirus. Eleven peptide residues are well-defined in the electron-density map and form two type I beta-turns in series. At the carboxyl end, the peptide is bound snugly in the antibody-combining site and adopts a conformation that differs significantly from the structure of the corresponding residues in the virus. Structural comparisons between the peptide in the complex and the viral epitope suggests that on binding to infectious virions, this antibody may induce structural changes important for neutralization.

Amino Acid Sequence↗

PubMed1995

An enzyme-substrate complex involved in bacterial cell wall biosynthesis.

The crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase in the presence of its substrate, enolpyruvyl-UDP-N-acetylglucosamine, has been solved to 2.7 A resolution. This enzyme is responsible for the synthesis of UDP-N-acetylmuramic acid in bacterial cell wall biosynthesis and consequently provides an attractive target for the design of antibacterial agents. The structure reveals a novel flavin binding motif, shows a striking alignment of the flavin with the substrate, and suggests a catalytic mechanism for the reduction of this unusual enol ether.

Amino Acid Sequence↗

PubMed1994

Structures of poliovirus complexes with anti-viral drugs: implications for viral stability and drug design.

BACKGROUND: Picornaviruses, such as the structurally related polioviruses and rhinoviruses, are important human pathogens which have been the target of major drug development efforts. Receptor-mediated uncoating and thermal inactivation of poliovirus and rhinovirus are inhibited by agents that bind to each virus by inserting into a pocket in the beta barrel of the viral capsid protein, VP1. This pocket, which is normally empty in human rhinovirus-14 (HRV14), is occupied by an unknown natural ligand in poliovirus. Structural studies of HRV14-drug complexes have shown that drug binding causes large, localized changes in the conformation of VP1. RESULTS: We report the crystal structures of six complexes between poliovirus and capsid-binding, antiviral drugs, including complexes of four different drugs with the Sabin vaccine strain of type 3 poliovirus, and complexes of one of these drugs with two other poliovirus strains that contain sequence differences in the drug-binding site. In each complex, the changes in capsid structure associated with drug binding are limited to minor adjustments in the conformations of a few side chains lining the binding site. CONCLUSIONS: The minor structural changes caused by drug binding suggest a model of drug action in which it is the conformational changes prevented by the bound drug, rather than obvious conformational changes induced by drug binding, which exert the biological effect. Our results, along with additional structures of rhinovirus-drug complexes, suggest possible improvements in drug design, and provide important clues about the nature of the conformational changes that are involved in the uncoating process.

Amino Acids↗

PubMed1994

Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.

Bacterial Proteins↗

PubMed1994

Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution.

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 A resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VP0 to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N-terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N-terminal extension of VP1, as well as portions corresponding to VP4 and the N-terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP0 scissile bond is located some 20 A away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoil-shaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA-dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N-terminal network and in generating stable virions.

Capsid↗