A stabilizing alpha/beta-hydrophobic core greatly contributes to hyperthermostability of archaeal [P62A]Ssh10b

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A 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.     

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.     

Molecular dynamics study of the proposed proton transport pathways in [FeFe]-hydrogenase

Possible proton transport pathways in Clostridium pasteurianum (CpI) [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to evaluate the functional pathway and provide insight into the hydrogen bonding features defining an active proton transport pathway. Three pathways were evaluated, two of which consist of water wires and one of predominantly amino acid residues. Our simulations suggest that protons are not transported through water wires. Instead, the five-residue motif (Glu282, Ser319, Glu279, H₂O, Cys299) was found to be the likely pathway, consistent with previously made experimental observations. The pathway was found to have a persistent hydrogen bonded core (residues Cys299 to Ser319), with less persistent hydrogen bonds at the ends of the pathway for both H₂ release and H₂ uptake. Single site mutations of the four residues have been shown experimentally to deactivate the enzyme. The theoretical evaluation of these mutations demonstrates redistribution of the hydrogen bonds in the pathway, resulting in enzyme deactivation. Finally, coupling between the protein dynamics near the proton transport pathway and the redox partner binding regions was also found as a function of H₂ uptake and H₂ release states, which may be indicative of a correlation between proton and electron movement within the enzyme.     

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.     

Liang Bua Homo floresiensis mandibles and mandibular teeth: a contribution to the comparative morphology of a new hominin species

In 2004, a new hominin species, Homo floresiensis, was described from Late Pleistocene cave deposits at Liang Bua, Flores. H. floresiensis was remarkable for its small body-size, endocranial volume in the chimpanzee range, limb proportions and skeletal robusticity similar to Pliocene Australopithecus, and a skeletal morphology with a distinctive combination of symplesiomorphic, derived, and unique traits. Critics of H. floresiensis as a novel species have argued that the Pleistocene skeletons from Liang Bua either fall within the range of living Australomelanesians, exhibit the attributes of growth disorders found in modern humans, or a combination of both. Here we describe the morphology of the LB1, LB2, and LB6 mandibles and mandibular teeth from Liang Bua. Morphological and metrical comparisons of the mandibles demonstrate that they share a distinctive suite of traits that place them outside both the H. sapiens and H. erectus ranges of variation. While having the derived molar size of later Homo, the symphyseal, corpus, ramus, and premolar morphologies share similarities with both Australopithecus and early Homo. When the mandibles are considered with the existing evidence for cranial and postcranial anatomy, limb proportions, and the functional anatomy of the wrist and shoulder, they are in many respects closer to African early Homo or Australopithecus than to later Homo. Taken together, this evidence suggests that the ancestors of H. floresiensis left Africa before the evolution of H. erectus, as defined by the Dmanisi and East African evidence.     

The finite element implementation of a K.P.P. equation for the simulation of tsetse control measures in the vicinity of a game reserve

An equation, strongly reminiscent of Fisher’s equation, is used to model the response of tsetse populations to proposed control measures in the vicinity of a game reserve. The model assumes movement is by diffusion and that growth is logistic. This logistic growth is dependent on an historical population, in contrast to Fisher’s equation which bases it on the present population. The model therefore takes into account the fact that new additions to the adult fly population are, in actual fact, the descendents of a population which existed one puparial duration ago, furthermore, that this puparial duration is temperature dependent. Artificially imposed mortality is modelled as a proportion at a constant rate. Fisher’s equation is also solved as a formality.The temporary imposition of a 2 % day⁻¹ mortality everywhere outside the reserve for a period of 2 years will have no lasting effect on the influence of the reserve on either the Glossina austeni or the G. brevipalpis populations, although it certainly will eradicate tsetse from poor habitat, outside the reserve. A 5 km-wide barrier with a minimum mortality of 4 % day⁻¹, throughout, will succeed in isolating a worst-case, G. austeni population and its associated trypanosomiasis from the surrounding areas. A more optimistic estimate of its mobility suggests a mortality of 2 % day⁻¹ will suffice. For a given target-related mortality, more mobile species are found to be more vulnerable to eradication than more sedentary species, while the opposite is true for containment.     

A region C-terminal to the proline-rich core of p47phox regulates activation of the phagocyte NADPH oxidase by interacting with the C-terminal SH3 domain of p67phox

Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47(phox) and p67(phox), each harboring two SH3 domains. p67(phox) interacts with p47(phox) via simultaneous binding of the p67(phox) C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360-369 and its C-terminally flanking region of p47(phox); the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47(phox)-p67(phox) interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47(phox) extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation.     

Asp79 makes a large, unfavorable contribution to the stability of RNase Sa

The two most buried carboxyl groups in ribonuclease Sa (RNase Sa) are Asp33 (99% buried; pK 2.4) and Asp79 (85% buried; pK 7.4). Above these pK values, the stability of the D33A variant is 6kcal/mol less than wild-type RNase Sa, and the stability of the D79A variant is 3.3kcal/mol greater than wild-type RNase Sa. The key structural difference between the carboxyl groups is that Asp33 forms three intramolecular hydrogen bonds, and Asp79 forms no intramolecular hydrogen bond. Here, we focus on Asp79 and describe studies of 11 Asp79 variants. Most of the variants were at least 2kcal/mol more stable than wild-type RNase Sa, and the most interesting was D79F. At pH 3, below the pK of Asp79, RNase Sa is 0.3kcal/mol more stable than the D79F variant. At pH 8.5, above the pK of Asp79, RNase Sa is 3.7kcal/mol less stable than the D79F variant. The unfavorable contribution of Asp79 to the stability appears to result from the Born self-energy of burying the charge and, more importantly, from unfavorable charge-charge interactions. To counteract the effect of the negative charge on Asp79, we prepared the Q94K variant and the crystal structure showed that the amino group of the Lys formed a hydrogen-bonded ion pair (distance, 2.71A; angle, 100 degrees ) with the carboxyl group of Asp79. The stability of the Q94K variant was about the same as the wild-type at pH 3, where Asp79 is uncharged, but 1kcal/mol greater than that of wild-type RNase Sa at pH 8.5, where Asp79 is charged. Differences in hydrophobicity, steric strain, Born self-energy, and electrostatic interactions all appear to contribute to the range of stabilities observed in the variants. When it is possible, replacing buried, non-hydrogen bonded, ionizable side-chains with non-polar side-chains is an excellent means of increasing protein stability.     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus epidermidis RP62A, a reference biofilm-positive strain

The ability to adhere to artificial surfaces and form biofilms is considered as a virulence factor of Staphylococcus epidermidis, one of the major causes of nocosomial infections, especially those related to implanted medical devices. Cell-wall teichoic acid is known to play an important role in biofilm formation of staphylococci. The structure of the cell wall and extracellular teichoic acids of S. epidermidis RP62A, a reference biofilm-positive strain, was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Their structures were found to be a (1-->3)-linked poly(glycerol phosphate), substituted at the 2-position of glycerol residues with alpha-Glc, alpha-GlcNAc, D-Ala and alpha-Glc6Ala. D-Alanyl acylation of a sugar hydroxyl group seems to be a novel structural feature of teichoic acids from staphylococci.     

Crystal structure of protein Ph1481p in complex with protein Ph1877p of archaeal RNase P from Pyrococcus horikoshii OT3: implication of dimer formation of the holoenzyme

Ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNA and five protein subunits. We previously determined crystal structures of four protein subunits. Ph1481p, an archaeal homologue for human hPop5, is the protein component of the P.horikoshii RNase P for which no structural information is available. Here we report the crystal structure of Ph1481p in complex with another protein subunit, Ph1877p, determined at 2.0 A resolution. Ph1481p consists of a five-stranded antiparallel beta-sheet and five helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. Ph1481p is, however, distinct from the typical RNP domain in that it has additional helices at the C terminus, which pack against one face of the beta-sheet. The presence of two complexes in the asymmetric unit, together with gel filtration chromatography indicates that the heterotetramer is stable in solution and represents a fundamental building block in the crystals. In the heterotetrameric structure (Ph1877p-(Ph1481p)(2)-Ph1877p), a homodimer of Ph1481p sits between two Ph1877p monomers. Ph1481p dimerizes through hydrogen bonding interaction from the loop between alpha1 and alpha2 helices, and each Ph1481p interacts with two Ph1877p molecules, where alpha2 and alpha3 in Ph1481p interact with alpha7 in one Ph1877p and alpha8 in the other Ph1877p molecule, respectively. Deletion of the alpha1-alpha2 loop in Ph1481p caused heterodimerization with Ph1877p, and abolished ability to homodimerize itself and heterotetramerize with Ph1877p. Furthermore, the reconstituted particle containing the deletion mutant Ph1481p (mPh1481p) exhibited significantly reduced nuclease activity. These results suggest the presence of the heterotetramer of Ph1481p and Ph1877p in P.horikoshii RNase P.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

Identification and comparative analysis of the Eriocheir sinensis microRNA transcriptome response to Spiroplasma eriocheiris infection using a deep sequencing approach

The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p < 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host’s innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.     

Significance of the extra C-terminal tail of CaLP, a novel calmodulin-like protein involved in oyster calcium metabolism

Oyster (Pinctada fucata) calmodulin-like protein (CaLP), containing a C-terminally extra hydrophilic tail (150D–161K), is a novel protein involved in the regulation of oyster calcium metabolism. To investigate the importance of the extra fragment to the Ca²⁺/Mg²⁺-dependent conformational changes in the intact CaLP molecule and the interactions between CaLP and its target proteins, a truncated CaLP mutant (M-CaLP) devoid of the extended C-terminus was constructed and overexpressed in Escherichia coli. The conformational characteristics of M-CaLP were studied by CD and fluorescence spectroscopy and compared with those of the oyster CaM and CaLP. The far-UV CD results reveal that the extra tail has a strong effect on the Ca²⁺-induced, but a relatively weak effect on the Mg²⁺-induced conformational changes in CaLP. However, upon Ca²⁺ or Mg²⁺ binding, only slight changes for intrinsic phenylalanine and tyrosine fluorescence spectra between M-CaLP and CaLP are observed. Our results also indicate that the extra tail can significantly decrease the exposure of the hydrophobic patches in CaLP. Additionally, affinity chromatography demonstrates that the target binding of CaLP is greatly influenced by its additional tail. All our results implicate that the extra tail may play some important roles in the interactions between CaLP and its targets in vivo.     

中国珍稀濒危孑遗植物珙桐种群的保护

珙桐(Davidia involucrata Baill.)为我国特有的珙桐科单型种,第三纪孑遗植物,素有"活化石"之称,是国家一级重点保护珍稀濒危植物,其特殊的分类地位决定它在全球生物多样性保育中具有不可替代的作用。从珙桐濒危状况和原因、保育技术、保护策略3个方面回顾和总结了珙桐种群保护的研究,并对未来研究方向提出一些建议。