Ralf Moll

Chemiosmotic H+ cycling across the plasma membrane of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

Transitory H+ ejection from Sulfolobus acidocaldarius cells induced by oxygen pulses, and anaerobic H+ backflow were investigated. Aerobic proton extrusion is inhibited by protonophores, by nigericin and by inhibitors of respiratory electron transport; it is stimulated by DCCD. In contrast, DCCD inhibits the rate of anaerobic H+ backflow. Aerobic proton extrusion is significantly enhanced by K+/valinomycin. Apparent H+/O ratios of 2.5–3 are measured. Proton extrusion generates large pH gradients (3–4 units) representing the major contribution to the total proton motive force across the plasma membrane of this thermoacidophilic archaebacterium.

The signal recognition particle of Archaea.

It is becoming increasingly clear that similarities exist in the manner in which extracytoplasmic proteins are targeted to complexes responsible for translocating these proteins across membranes in each of the three domains of life. In Eukarya and Bacteria, the signal recognition particle (SRP) directs nascent polypeptides to membrane-embedded translocation sites. In Archaea, the SRP protein targeting pathway apparently represents an intermediate between the bacterial and eukaryal systems. Understanding the archaeal SRP pathway could therefore reveal universal aspects of targeting not detected in current comparisons of the eukaryal and bacterial systems while possibly identifying aspects of the process either not previously reported or unique to Archaea.

The non-structural protein Nsp10 of mouse hepatitis virus binds zinc ions and nucleic acids

The non‐structural protein Nsp10 of coronaviruses is a small cleavage product of the viral replicase polyprotein that has been implicated in RNA synthesis. Nsp10 of mouse hepatitis virus (MHV) displays an apparent molecular mass of 13–16 kDa in reducing SDS–PAGE and analytical gel filtration, while dynamic light scattering suggests the existence of oligomeric forms. Atomic absorption spectroscopy reveals two metal ions per Nsp10 monomer, with a preference for Zn2+ over Fe2+/3+ and Co2+. These are probably bound by two Zn‐finger‐like motifs. Moreover, MHV Nsp10 interacts with tRNA, single‐stranded RNA, double‐stranded DNA and, to a lesser extent, single‐stranded DNA as shown by gel‐shift experiments. The K d for tRNA is 2.1 ± 0.2 μM.

Has Sulfolobus an Archaic Respiratory System? Structure, Function and Genes of its Components

Summary The electron transport system of Sulfolobus acidocaldarius has been shown to act as a respiration driven proton pump. Essentially it is composed of three functional units: the NADH-and substrate dehydrogenases, caldariella quinone as a pool of bound hydrogen, and one or two terminal oxidases catalyzing its reoxidation by molecular oxygen. The latter systems contain only FeS proteins, b- and a-type cytochromes; c-type cytochromes are absent. The cytochrome aa3 from Sulfolobus is the first heme-a containing terminal oxidase shown to act as a quinol oxidase. Its redox centers were investigated by redox potentiometry, EPR- and Raman-resonance spectroscopy, revealing a typical binuclear heme-a/Cu center, however displaying unusual structural interaction between the formyl substituents and the protein environment; the redox potentials of low and high spin centers were titrated. A cytochrome a587 atypical for any other species emerged to contain a low spin, low potential heme-a center, mimicing the function of a b-type cytochrome and likely to be the product of the SOX-C gene from the SOX operon (Lubben et al., 1992). The aa3 oxidase when reconstituted into liposomes is shown to generate a proton motive force. Though Sulfolobus contains no equivalent to the bc1 complex of classical respiratory chains, in addition to the caldariella quinol oxidase a Rieske-type FeS protein could be detected in its plasma membrane; it was isolated and characterized by EPR, strongly suggesting a participation in respiratory electron transport. Its redox potential displays significant pH dependence revealing two distinct pKs. It remains to be established whether it interacts with the aa3 oxidase or other electron carriers. A cytochrome b562 appears to be present constitutively in small amount, while cytochrome-b558 is the major heme-b containing compound in the membrane of Sulfolobus. A previously envisaged function as an alternate terminal oxidase is still questionable. Recently conditions were found allowing to modulate its expression dramatically, leading to almost complete suppression of cytochrome-b558. Based on differential spectroscopy and redox titrations a tentative scheme of the electron transport system from Sulfolobus is proposed. Sequence comparisons are discussed with regard to the question, as to whether it resembles a primitive “archaic” precursor form of more complex “modern” respiratory systems, or whether it evolved by aquisition and adaptation of “foreign” genes.

Variable Oligomerization Modes in Coronavirus Non-Structural Protein 9.

Abstract Non-structural protein 9 (Nsp9) of coronaviruses is believed to bind single-stranded RNA in the viral replication complex. The crystal structure of Nsp9 of human coronavirus (HCoV) 229E reveals a novel disulfide-linked homodimer, which is very different from the previously reported Nsp9 dimer of SARS coronavirus. In contrast, the structure of the Cys69Ala mutant of HCoV-229E Nsp9 shows the same dimer organization as the SARS-CoV protein. In the crystal, the wild-type HCoV-229E protein forms a trimer of dimers, whereas the mutant and SARS-CoV Nsp9 are organized in rod-like polymers. Chemical cross-linking suggests similar modes of aggregation in solution. In zone-interference gel electrophoresis assays and surface plasmon resonance experiments, the HCoV-229E wild-type protein is found to bind oligonucleotides with relatively high affinity, whereas binding by the Cys69Ala and Cys69Ser mutants is observed only for the longest oligonucleotides. The corresponding mutations in SARS-CoV Nsp9 do not hamper nucleic acid binding. From the crystal structures, a model for single-stranded RNA binding by Nsp9 is deduced. We propose that both forms of the Nsp9 dimer are biologically relevant; the occurrence of the disulfide-bonded form may be correlated with oxidative stress induced in the host cell by the viral infection.

Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens.

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP-RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic supernatants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic supernatants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

The signal recognition particle receptor α subunit of the hyperthermophilic archaeon Acidianus ambivalens exhibits an intrinsic GTP-hydrolyzing activity

Two adjacent genes of the acidophilic and hyperthermophilic crenarchaeon Acidianus ambivalens were cloned and sequenced. The 1.6 kb genomic nucleotide sequence under investigation consists of the 1.12 kb SRa gene encoding the putative signal recognition particle receptor alpha subunit (SR alpha, 42.2 kDa) and the 186 basepair secE gene coding for the putative secretory component secE subunit (6800 Da). The SR alpha protein is structured by three distinct regions: the N-terminal hydrophilic H-region, the following X-region and the C-terminal GTP-binding domain. A polyclonal anti-E. coli lacZ/A. ambivalens SR alpha antiserum detects a 51 kDa cell protein (p51) on immunoblots. Proteolysis of the recombinant SR alpha protein by Proteinase K produces a 31.6 kDa protease-resistant protein fragment comprising X-region and G-domain. The protein binds tightly to the GTP-agarose affinity matrix in a temperature-dependent manner. It hydrolyzes GTP readily at higher temperatures only in the presence of Mg2+. Point mutations (T326N) and (D329A) in the G-4 element of A. ambivalens SR alpha G-domain diminish the GTPase activity significantly. In contrast, the deletion mutant protein SR alpha (delta1-92) lacking the hydrophilic H-region displays a higher GTP-hydrolyzing activity when compared to the unmodified recombinant protein. Addition of GDP greatly inhibits GTP hydrolysis in mutant and unmodified A. ambivalens SR alpha.

[30] Respiratory enzymes from Sulfolobus acidocaldarius

Publisher Summary Sulfolobus is one of the best investigated thermoacidophilic aerobic organisms from the archaeal domain. The genus was first described by Brock et al . It belongs to the order Sulfolobales, which also includes the genus Acidianus (previously termed Desulfurolobus). Members of the genus Sulfolobus are S. acidocaldarius, S. solfataricus, S. brierleyi, S. shibatae, S. metallicus, and Sulfolobus sp. strain 7, which is likely to be a S. solfataricus strain. Respiratory complexes have been studied mainly from S. acidocaldarius. A highly glycosylated b -type cytochrome is expressed in S. acidocaldarius as an integral membrane protein under specific growth conditions; its function in cellular respiration is not yet known but it has been proposed that it links periplasmic redox reactions to the respiratory chain. The preparation and properties of membrane-residing respiratory complexes and enzymes from S. acidocaldarius (DSM 639) are described in detail.

Nucleotide sequence of a gene cluster encoding ribosomal proteins in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

A 1.6 kb genomic DNA fragment derived from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius (DSM 639) comprises four open reading frames. The sequence contains three genes encoding crenarchaeal ribosomal proteins with apparent molecular masses of 6.3 kDa, 15.2 kDa and 9.9 kDa, which all represent strongly basic properties. These were identified by sequence comparison as RL46, RL31 and RL33. One open reading frame encodes a new polypeptide (22.1 kDa, pI = 7.3) with no homology to known proteins. The latter is transcribed as a common mRNA with RL46 and RL31. This gene cluster immediately precedes another cluster including genes encoding the putative SRP receptor alpha subunit as well as the putative secEp.

Crystallization and preliminary X-ray diffraction studies on the conserved GTPase domain of the signal recognition particle from Acidianus ambivalens

The signal recognition particle (SRP) of bacteria consists of only one protein, known as Ffh or the SRP54 homologue, which forms a complex with 4.5S RNA. It also binds to signal peptides and contains a GTPase which displays interesting differences to Ras GTPases. The conserved NG-domain of Ffh from the archaebacterium Acidianus ambivalens was cloned and overexpressed with a C-terminal His tag in Escherichia coli. Crystallization experiments of the native protein as well as of the Thr112Ala mutant, which is deficient in GTP hydrolysis, resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 64.5, b = 128.3, c = 72.0 A. At cryogenic temperatures, the crystals diffracted to a resolution limit of 2.8 A using a rotating-anode generator and contain one molecule per asymmetric unit. A native data set has been collected using synchrotron radiation to around 2.0 A resolution. Selenomethionine protein was produced; its crystals diffract in-house to about 2.8 A resolution.