Christian L. Schmidt

CMP-N-acetylneuraminic acid hydroxylase: the first cytosolic Rieske iron-sulphur protein to be described in Eukarya

Electron paramagnetic resonance (EPR) spectroscopy and analysis of the primary structure of the CMP‐N‐acetylneuraminic acid hydroxylase revealed that this enzyme is the first iron‐sulphur protein of the Rieske type to be found in the cytosol of Eukarya. The dithionite‐reduced hydroxylase exhibited an EPR signal known to be characteristic for a Rieske iron‐sulphur centre (2Fe‐2S), the g‐values being 1.78, 1.91 and 2.01, respectively. An analysis of the primary structure of the hydroxylase led to the identification of an amino acid sequence, known to be characteristic for Rieske proteins. Furthermore, possible binding sites for cytochrome b 5, the substrate CMP‐Neu5Ac and a mononuclear iron centre were also identified.

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cells response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.

The Structure of the Soluble Domain of an Archaeal Rieske Iron–Sulfur Protein at 1.1 Å Resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.

Arenaviruses and hantaviruses: From epidemiology and genomics to antivirals

The arenaviruses and hantaviruses are segmented genome RNA viruses that are hosted by rodents. Due to their association with rodents, they are globally widespread and can infect humans via direct or indirect routes of transmission, causing considerable human morbidity and mortality. Nevertheless, despite their obvious and emerging importance as pathogens, there are currently no effective antiviral drugs (except ribavirin which proved effective against Lassa virus) with which to treat humans infected by any of these viruses. The EU-funded VIZIER project (Comparative Structural Genomics of Viral Enzymes Involved in Replication) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the arenaviruses and bunyaviruses. This review highlights some of the major features of the arenaviruses and hantaviruses that have been investigated during recent years. After describing their classification and epidemiology, we review progress in understanding the genomics as well as the structure and function of replicative enzymes achieved under the VIZIER program and the development of new disease control strategies.

Two different respiratory Rieske proteins are expressed in the extreme thermoacidophilic crenarchaeon Sulfolobus acidocaldarius: cloning and sequencing of their genes

We have isolated two genes encoding Rieske iron sulfur proteins from the genomic DNA of the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639). One of the genes, named soxL, codes for the previously isolated novel Rieske‐I protein [1]. The second gene (soxF) [2] codes for the Rieske‐II protein associated with the second terminal oxidase of Sulfolobus [3]. Both proteins exhibit only 24% identical residues. The Rieske‐I protein shows a number of unusual features. (i) The distance between the two cluster binding sites is significantly larger than in all known proteins. (ii) An unexpected Pro → Asp exchange in one of the cluster binding sites. (iii) It shows some resemblance to the mitochondrial and plastidic Rieske proteins insofar as the soxL gene codes for a pre‐sequence which is no longer present in the mature Rieske‐I protein. Both proteins cluster together on a separate branch of the phylogenetic tree. To our knowledge this is the first proven case of two significantly different Rieske proteins in a prokaryote.

Heterogeneous Rieske proteins in the cytochrome b6f complex of Synechocystis PCC6803

The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3,encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein. Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystismembranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated cytochromeb 6 f complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous cytochromeb 6 f complexes inSynechocystis and suggests still to be established alternative electron transport routes.

Evidence for a Rieske-type FeS center in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

A high‐potential iron‐sulfur cluster with characteristics similar to a Rieske‐type center was detected in the plasma membrane of Sulfolobus acidocaldarius by EPR spectroscopy. In the reduced form this center has g‐values of g z = 2.031, g y = 1.890 and g x = 1.725 (g av = 1.88, rhombicity = 0.37) and its reduction potential at pH 7.4 was determined to be +325 ± 10 mV. The archaebacterial cluster exhibits some unique properties, in comparison to eubacterial and eukaryotic Rieske‐type centers. First, the reduction potential is pH‐dependent in the range from pH 6.7 to 8.2. Second, the typical inhibitor of Rieske FeS centers, DBMIB, had no effect on the g‐values of this cluster. The center is reducible by both NADH and succinate in the presence of cyanide, an inhibitor of the terminal oxidases. The possible role of a Rieske‐type center in an organism lacking any c‐type cytochromes is discussed.

Structure of the GTPase and GDI domains of FeoB, the ferrous iron transporter of Legionella pneumophila

Prokaryotic pathogens have developed specialized mechanisms for efficient uptake of ferrous iron (Fe2+) from the host. In Legionella pneumophila, the causative agent of Legionnaires’ disease, the transmembrane GTPase FeoB plays a key role in Fe2+ acquisition and virulence. FeoB consists of a membrane‐embedded core and an N‐terminal, cytosolic region (NFeoB). Here, we report the crystal structure of NFeoB from L. pneumophila, revealing a monomeric protein comprising two separate domains with GTPase and guanine‐nucleotide dissociation inhibitor (GDI) functions. The GDI domain displays a novel fold, whereas the overall structure of the GTPase domain resembles that of known G domains but is in the rarely observed nucleotide‐free state.

Synthesis and antiplasmodial activity of a cysteine protease-inhibiting biotinylated aziridine-2,3-dicarboxylate.

Abstract Cysteine proteases have been implicated in a variety of processes essential for the survival and progression of the malarial parasite Plasmodium falciparum. Here, we synthesized a cysteine protease inhibitor that contains the electrophilic aziridine-2,3-dicarboxylic acid as the reactive agent and biotin as a targeting label. Diethyl ester and dibenzyl ester derivatives of the inhibitor were active against cathepsin L and the plasmodial protease falcipain 2, but only the latter displayed potent antiplasmodial activity against viable parasites. The morphological changes observed during the intraerythrocytic life stages of Plasmodium suggest that degradation of hemoglobin of the host cell is seriously affected, eventually leading to growth arrest and cell death of the parasites. After incubation of infected erythrocytes with the compound plasmodial proteins were captured, with the biotinyl group of the inhibitor serving as an affinity tag. Among these the cysteine proteases falcipain 2 and falcipain 3 were identified as potential target proteins of the compound as evidenced by tandem mass spectrometry. Apparently, the compound gets access to intracellular compartments and therein targets plasmodial cysteine proteases. Accordingly, the reagent described here appears to be a valuable template to develop cell-permeable, nonradioactive reagents that selectively target enzymes involved in pathogenicity of the parasite.

Respiratory chains of archaea and extremophiles

Extremophilic organisms are adapted to harsh environmental conditions like high temperature, extremely acidic or alkaline pH, high salt, or a combination of those. With a few exceptions extremophilic bacteria are colonizing only moderately hot biotopes, whereas hyperthermophiles are found specifically among archaea (formerly archaebacteria) which can thrive at temperatures close to or even above the boiling point of water. It has been a challenging question whether the special properties of their proteins and membranes have been acquired by adaptation, or whether they might reflect early evolutionary states as suggested by their phylogenetic position at the lowest branches of the universal tree of life.