Markus Perbandt

Crystal structure of vipoxin at 2.0 A: an example of regulation of a toxic function generated by molecular evolution.

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A2 (PLA2) and a non‐toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA2 activity is of great pharmacological interest, and the present structure will be a model for structure‐based drug design.

3C protease of enterovirus 68: Structure-based design of Michael acceptor inhibitors and their broad-spectrum antiviral effects against picornaviruses

ABSTRACT We have determined the cleavage specificity and the crystal structure of the 3C protease of enterovirus 68 (EV68 3Cpro). The protease exhibits a typical chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional structure is closely related to that of the 3Cpro of rhinovirus 2, as well as to that of poliovirus. The phylogenetic position of the EV68 3Cpro between the corresponding enzymes of rhinoviruses on the one hand and classical enteroviruses on the other prompted us to use the crystal structure for the design of irreversible inhibitors, with the goal of discovering broad-spectrum antiviral compounds. We synthesized a series of peptidic α,β-unsaturated ethyl esters of increasing length and for each inhibitor candidate, we determined a crystal structure of its complex with the EV68 3Cpro, which served as the basis for the next design round. To exhibit inhibitory activity, compounds must span at least P3 to P1′; the most potent inhibitors comprise P4 to P1′. Inhibitory activities were found against the purified 3C protease of EV68, as well as with replicons for poliovirus and EV71 (50% effective concentration [EC50] = 0.5 μM for the best compound). Antiviral activities were determined using cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus serotypes. The most potent inhibitor, SG85, exhibited activity with EC50s of ≈180 nM against EV71 and ≈60 nM against human rhinovirus 14 in a live virus–cell-based assay. Even the shorter SG75, spanning only P3 to P1′, displayed significant activity (EC50 = 2 to 5 μM) against various rhinoviruses.

Structure of the Lassa virus nucleoprotein revealed by X-ray crystallography, small-angle X-ray scattering, and electron microscopy.

Background: Nucleoprotein (NP) is an essential component of the virus replication complex. Results: The crystal and quaternary structures of Lassa virus NP were determined. Conclusion: Lassa virus NP assembles into a symmetric trimer in solution. Significance: The trimeric complex may have a biological function in the virus life cycle, and its assembly could be a target for antivirals. The nucleoprotein (NP) of Lassa virus (LASV) strain AV was expressed in a recombinant baculovirus system. The crystal structure of full-length NP was solved at a resolution of 2.45 Å. The overall fold corresponds to that of NP of LASV strain Josiah (Qi, X., Lan, S., Wang, W., Schelde, L. M., Dong, H., Wallat, G. D., Ly, H., Liang, Y., and Dong, C. (2010) Nature 468, 779–783) with a root mean square deviation of 0.67 Å for all atoms (6.3% difference in primary sequence). As the packing in the crystal offers two different trimer architectures for the biological assembly, the quaternary structure of NP in solution was determined by small-angle x-ray scattering and EM. After classification and averaging of >6000 EM raw images, trimeric centrosymmetric structures were obtained, which correspond in size and shape to one trimer in the crystal structure formed around a crystallographic 3-fold rotation axis (symmetric trimer). The symmetric trimer is also a good model for the small-angle x-ray scattering data and could be well embedded into the ab initio model. The N-terminal domain of NP contains a deep nucleotide-binding cavity that has been proposed to bind cellular cap structures for priming viral mRNA synthesis. All residues implicated in m7GpppN binding were exchanged, and the transcription/replication phenotype of the NP mutant was tested using a LASV replicon system. None of the mutants showed a specific defect in mRNA expression; most were globally defective in RNA synthesis. In conclusion, we describe the full-length crystal structure and the quaternary structure in solution of LASV NP. The nucleotide-binding pocket of NP could not be assigned a specific role in viral mRNA synthesis.

Trypanosoma brucei ATG8: structural insights into autophagic-like mechanisms in protozoa.

Bioinformatic searches of genome databases revealed that the number of autophagy-related genes (ATG) is considerably lower in trypanosomes than in higher eukaryotes and even in yeast. This raises the question of whether autophagy in this protozoan parasite is more primitive and represents a rudimentary paradigm due to its early branching off the evolutionary tree. We here present the crystal structure of TbATG8B. This molecule (MW 13.7 kDa) belongs to the ubiquitin-like proteins showing the typical ubiquitin fold and strong sequence homology to LC3, the human homologue. Due to its characteristic folding, it should readily bind to TbATG4.1 for being processed. This presumption was tested by molecular modeling approaches, docking TbATG8B to a homology model of TbATG4.1. Although exchanges of several amino acids are evident from sequence comparisons, the overall structure seems very much alike and the necessary catalytic triad (C-D-H) is well conserved in TbATG4.1. Thus membrane formation during appearance of the autophagic bodies seems very similar in trypanosomes and their higher eukaryotic counterpart.

Atomic structure of the Serratia marcescens endonuclease at 1.1 A resolution and the enzyme reaction mechanism.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

Comparative analysis of the human and chicken prion protein copper binding regions at pH 6.5

Recent experimental evidence supports the hypothesis that prion proteins (PrPs) are involved in the Cu(II) metabolism. Moreover, the copper binding region has been implicated in transmissible spongiform encephalopathies, which are caused by the infectious isoform of prion proteins (PrPSc). In contrast to mammalian PrP, avian prion proteins have a considerably different N-terminal copper binding region and, most interestingly, are not able to undergo the conversion process into an infectious isoform. Therefore, we applied x-ray absorption spectroscopy to analyze in detail the Cu(II) geometry of selected synthetic human PrP Cu(II) octapeptide complexes in comparison with the corresponding chicken PrP hexapeptide complexes at pH 6.5, which mimics the conditions in the endocytic compartments of neuronal cells. Our results revealed that structure and coordination of the human PrP copper binding sites are highly conserved in the pH 6.5–7.4 range, indicating that the reported pH dependence of copper binding to PrP becomes significant at lower pH values. Furthermore, the different chicken PrP hexarepeat motifs display homologous Cu(II) coordination at sub-stoichiometric copper concentrations. Regarding the fully cation-saturated prion proteins, however, a reduced copper coordination capability is supposed for the chicken prion protein based on the observation that chicken PrP is not able to form an intra-repeat Cu(II) binding site. These results provide new insights into the prion protein structure-function relationship and the conversion process of PrP.

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop.

The synthetic RNA fragment 5′‐CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79–97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 Å and refined to an R‐value of 24.1% also represents the first X‐ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.

Crystallization and Preliminary Diffraction Data of a Major Pollen Allergen CRYSTAL GROWTH SEPARATES A LOW MOLECULAR WEIGHT FORM WITH ELEVATED BIOLOGICAL ACTIVITY

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 Å, b = 87.7 Å, and c = 59.6 Å. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

Crystal structures of the complexes of a group IIA phospholipase A2 with two natural anti-inflammatory agents, anisic acid, and atropine reveal a similar mode of binding

Secretory low molecular weight phospholipase A2s (PLA2s) are believed to be involved in the release of arachidonic acid, a precursor for the biosynthesis of pro‐inflammatory eicosanoids. Therefore, the specific inhibitors of these enzymes may act as potent anti‐inflammatory agents. Similarly, the compounds with known anti‐inflammatory properties should act as specific inhibitors. Two plant compounds, (a) anisic acid (4‐methoxy benzoic acid) and (b) atropine (8‐methyl‐8‐azabicyclo oct‐3‐hydroxy‐2‐phenylpropanoate), have been used in various inflammatory disorders. Both compounds (a) and (b) have been found to inhibit PLA2 activity having binding constants of 4.5 × 10−5 M and 2.1 × 10−8 M, respectively. A group IIA PLA2 was isolated and purified from the venom of Daboia russelli pulchella (DRP) and its complexes were made with anisic acid and atropine. The crystal structures of the two complexes (i) and (ii) of PLA2 with compounds (a) and (b) have been determined at 1.3 and 1.2 Å resolutions, respectively. The high‐quality observed electron densities for the two compounds allowed the accurate determinations of their atomic positions. The structures revealed that these compounds bound to the enzyme at the substrate ‐ binding cleft and their positions were stabilized by networks of hydrogen bonds and hydrophobic interactions. The most characteristic interactions involving Asp 49 and His 48 were clearly observed in both complexes, although the residues that formed hydrophobic interactions with these compounds were not identical because their positions did not exactly superimpose in the large substrate‐binding hydrophobic channel. Owing to a relatively small size, the structure of anisic acid did not alter upon binding to PLA2, while that of atropine changed significantly when compared with its native crystal structure. The conformation of the protein also did not show notable changes upon the bindings of these ligands. The mode of binding of anisic acid to the present group II PLA2 is almost identical to its binding with bovine pancreatic PLA2 of group I. On the other hand, the binding of atropine to PLA2 is similar to that of another plant alkaloid aristolochic acid. Proteins 2006.

The first crystal structure of an RNA racemate.

The racemate of the RNA duplex r(CUGGGCGG).r(CCGCCUGG) from Thermus flavus 5S rRNA has been crystallized and examined by X-ray crystallography. The space group is P1(_) with approximate unit-cell parameters a = 26.5, b = 38.0, c = 45.4 Angstrom, alpha = 113.1, beta = 100.5, gamma = 93.3 degrees. The structure was solved by molecular replacement. There are four RNA duplexes in the unit cell. The crystal lattice consists of columns of RNA duplexes. The duplexes are stacked end-to-end and are stabilized by intermolecular base-stacking interactions. Within each column the L-duplexes and D-duplexes are stacked alternately. Every other duplex in each stack has two alternative conformations, approximately equally occupied, corresponding to molecules oriented in opposite directions. Neighbouring columns are related by the crystallographic centre of symmetry. The unit cell also contains approximately 250 ordered water molecules and six ordered calcium ions. A glycerol molecule is visible in the minor groove interacting with a guanosine residue.