Crystal structure of a novel-type archaeal rubisco with pentagonal symmetry.

BACKGROUND: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of the Calvin-Benson cycle and catalyzes the primary reaction of CO2 fixation in plants, algae, and bacteria. Rubiscos have been so far classified into two types. Type I is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L8S8), but type II is usually composed only of two L subunits (L2). Recently, some genuinely active Rubiscos of unknown physiological function have been reported from archaea. RESULTS: The crystal structure of Rubisco from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Rubisco) was determined at 2.8 A resolution. The enzyme is composed only of L subunits and showed a novel (L2)5 decameric structure. Compared to previously known type I enzymes, each L2 dimer is inclined approximately 16 degrees to form a toroid-shaped decamer with its unique L2-L2 interfaces. Differential scanning calorimetry (DSC), circular dichroism (CD), and gel permeation chromatography (GPC) showed that Tk-Rubisco maintains its secondary structure and decameric assembly even at high temperatures. CONCLUSIONS: The present study provides the first structure of an archaeal Rubisco, an unprecedented (L2)5 decamer. Biochemical studies indicate that Tk-Rubisco maintains its decameric structure at high temperatures. The structure is distinct from type I and type II Rubiscos and strongly supports that Tk-Rubisco should be classified as a novel type III Rubisco.     

The structure of an archaeal pilus

Bacterial pili are involved in a host of activities, including motility, adhesion, transformation, and immune escape. Structural studies of these pili have shown that several distinctly different classes exist, with no common origin. Remarkably, it is now known that the archaeal flagellar filament appears to have a common origin with the bacterial type IV pilus, and assembly in both systems involves hydrophobic N-terminal α-helices that form three-stranded coils in the center of these filaments. Recent work has identified further genes in archaea as being similar to bacterial type IV pilins, but the function or structures formed by such gene products was unknown. Using electron cryo-microscopy, we show that an archaeal pilus from Methanococcus maripaludis has a structure entirely different from that of any of the known bacterial pili. Two subunit packing arrangements were identified: one has rings of four subunits spaced by ∼ 44 Å and the other has a one-start helical symmetry with ∼ 2.6 subunits per turn of a ∼ 30 Å pitch helix. Remarkably, these schemes appear to coexist within the same filaments. For the segments composed of rings, the twist between adjacent rings is quite variable, while for the segments having a one-start helix there is a large variability in both the axial rise and the twist per subunit. Since this pilus appears to be assembled from a type IV pilin-like protein with a hydrophobic N-terminal helix, it provides yet another example of how different quaternary structures can be formed from similar building blocks. This result has many implications for understanding the evolutionary divergence of bacteria and archaea.     

The unique structure of archaeal 'hami', highly complex cell appendages with nano-grappling hooks

Proteinaceous, hair-like appendages known as fimbriae or pili commonly extend from the surface of prokaryotic cells and serve important functions such as cell adhesion, biofilm formation, motility and DNA transfer. Here we show that a novel group of archaea from cold, sulphidic springs has developed cell surface appendages of an unexpectedly high complexity with a well-defined base-to-top organization. It represents a new class of filamentous cell appendages, for which the term 'hamus' is proposed. Each archaeal cell is surrounded by a halo of about 100 hami, which mediate strong adhesion of the cells to surfaces of different chemical composition. The hami are mainly composed of 120 kDa subunits and remained stable in a broad temperature and pH range (0-70 degrees C; 0.5-11.5). Electron microscopy and cryo-electron tomography revealed that the hamus filament possesses a helical basic structure. At periodic distances, three prickles emanate from the filament, giving it the character of industrially produced barbwire. At its distal end the hami carry a tripartite, barbed grappling hook (60 nm in diameter). The architecture of this molecular hook is reminiscent of man-made fishhooks, grapples and anchors. It appears that nature has developed a perfect mechanical nano-tool in the course of biological evolution, which also might prove useful in the field of nanobiotechnology.     

In vivo analysis of an essential archaeal signal recognition particle in its native host

The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.     

The dimerization of an alpha/beta-knotted protein is essential for structure and function

alpha/beta-Knotted proteins are an extraordinary example of biological self-assembly; they contain a deep topological trefoil knot formed by the backbone polypeptide chain. Evidence suggests that all are dimeric and function as methyltransferases, and the deep knot forms part of the active site. We investigated the significance of the dimeric structure of the alpha/beta-knot protein, YibK, from Haemophilus influenzae by the design and engineering of monomeric versions of the protein, followed by examination of their structural, functional, stability, and kinetic folding properties. Monomeric forms of YibK display similar characteristics to an intermediate species populated during the formation of the wild-type dimer. However, a notable loss in structure involving disruption to the active site, rendering it incapable of cofactor binding, is observed in monomeric YibK. Thus, dimerization is vital for preservation of the native structure and, therefore, activity of the protein.     

Crystal structure of an archaeal actin homolog

Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.     

The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion

The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.     

The relationship between the glucose oxidase subunit structure and its thermostability

The thermostability of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) at 60 degrees C has been studied as a function of its concentration in various media (pure water and pure deuterium oxide). In deuterium oxide, glucose oxidase is more stable than in water, and two kinds of stabilizing effect have been observed: the medium-organization effect and the enzyme-concentration effect. This effect has been related to the glucose oxidase subunit structure. This enzyme contains four forms of subunit: monomer, dimer, trimer, and tetramer, which are all composed of the identical monomer. The monomers of glucose oxidase subunits are linked by the non-covalent bond. Only dimer and trimer possess the enzymatic activity. During glucose oxidase denaturing, monomers assemble into dimer, trimer, or tetramer. This redistribution behavior depends on the enzyme concentration and the nature of the medium.     

The closed structure of an archaeal DNA ligase from Pyrococcus furiosus

DNA ligases join single-strand breaks in double-stranded DNA, and are essential to maintain genome integrity in DNA metabolism. Here, we report the 1.8 A resolution structure of Pyrococcus furiosus DNA ligase (PfuLig), which represents the first full-length atomic view of an ATP-dependent eukaryotic-type DNA ligase. The enzyme comprises the N-terminal DNA-binding domain, the middle adenylation domain, and the C-terminal OB-fold domain. The architecture of each domain resembles those of human DNA ligase I, but the domain arrangements differ strikingly between the two enzymes. The closed conformation of the two "catalytic core" domains at the carboxyl terminus in PfuLig creates a small compartment, which holds a non-covalently bound AMP molecule. This domain rearrangement results from the "domain-connecting" role of the helical extension conserved at the C termini in archaeal and eukaryotic DNA ligases. The DNA substrate in the human open-ligase is replaced by motif VI in the Pfu closed-ligase. Both the shapes and electrostatic distributions are similar between motif VI and the DNA substrate, suggesting that motif VI in the closed state mimics the incoming substrate DNA. Two basic residues (R531 and K534) in motif VI reside within the active site pocket and interact with the phosphate group of the bound AMP. The crystallographic and functional analyses of mutant enzymes revealed that these two residues within the RxDK sequence play essential and complementary roles in ATP processing. This sequence is also conserved exclusively among the covalent nucleotidyltransferases, even including mRNA-capping enzymes with similar helical extensions at the C termini.     

Characterization of an archaeal multidrug transporter with a unique amino acid composition

The Smr family of multidrug transporters consists of small membrane proteins that extrude various drugs in exchange with protons rendering cells resistant to these drugs. Smr proteins identified to date have been found only in Eubacteria. In this work we present the cloning and characterization of an Smr protein from the archaeon Halobacterium salinarum, the first Smr in the archaeal kingdom. The protein, named Hsmr, was identified through sequence similarity to the Smr family, and the DNA sequence was cloned into an Escherichia coli expression system. Hsmr is heterologously expressed in a functional form despite the difference in lipid composition of the membrane and the lower salt in the cell and its environment. Cells harboring the Hsmr plasmid transport ethidium bromide in an uncoupler-sensitive process and gain resistance to ethidium bromide and acriflavine. Hsmr binds tetraphenylphosphonium (TPP(+)) with a relatively low affinity (K(D) approximately 200 nm) at low salt concentration that increases (K(D) approximately 40 nm) upon the addition of 2 m of either NaCl or KCl. The Hsmr protein contains many of the signature sequence elements of the Smr family and also a high content of negative residues in the loops, characteristic of extreme halophiles. Strikingly, Hsmr is composed of over 40% valine and alanine residues. These residues are clustered at certain regions of the protein in domains that are not important for activity, as judged from lack of conservation and from previous studies with other Smr proteins. We suggest that this high content of alanine and valine residues is a reflection of a "natural" alanine and valine scanning necessitated by the high GC content of the gene. This phenomenon reveals significant sequence elements in small multidrug transporters.     

The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.     

A unique spacer domain of synaptotagmin IV is essential for Golgi localization

Synaptotagmin (Syt) family members consist of six separate domains: a short amino terminus, a single transmembrane domain, a spacer domain, a C2A domain, a C2B domain and a short carboxyl (C) terminus. Despite sharing the same domain structures, several synaptotagmin isoforms show distinct subcellular localization. Syt IV is mainly localized at the Golgi, while Syt I, a possible Ca(2+)-sensor for secretory vesicles, is localized at dense-core vesicles and synaptic-like microvesicles in PC12 cells. In this study, we sought to identify the region responsible for the Golgi localization of Syt IV by immunocytochemical and biochemical analyses as a means of defining the distinct subcellular localization of the synaptotagmin family. We found that the unique C-terminus of the spacer domain (amino acid residues 73-144) between the transmembrane domain and the C2A domain is essential for the Golgi localization of Syt IV. In addition, the short C-terminus is probably involved in proper folding of the protein, especially the C2B domain. Without the C-terminus, Syt IVdeltaC proteins are not targeted to the Golgi and seem to colocalize with an endoplasmic reticulum (ER) marker (i.e. induce crystalloid ER-like structures). On the basis of these results, we propose that the divergent spacer domain among synaptotagmin isoforms may contain certain signals that determine the final destination of each isoform.     

The structure of an archaeal homodimeric ligase which has RNA circularization activity

The genome of Pyrococcus abyssi contains two open reading frames encoding proteins which had been previously predicted to be DNA ligases, Pab2002 and Pab1020. We show that while the former is indeed a DNA ligase, Pab1020 had no effect on the substrate deoxyoligo-ribonucleotides tested. Instead, Pab1020 catalyzes the nucleotidylation of oligo-ribonucleotides in an ATP-dependent reaction, suggesting that it is an RNA ligase. We have solved the structure of Pab1020 in complex with the ATP analog AMPPNP by single-wavelength anomalous dispersion (SAD), elucidating a structure with high structural similarity to the catalytic domains of two RNA ligases from the bacteriophage T4. Additional carboxy-terminal domains are also present, and one of these mediates contacts with a second protomer, which is related by noncrystallographic symmetry, generating a homodimeric structure. These C-terminal domains are terminated by short domain swaps which themselves end within 5 Å of the active sites of the partner molecules. Additionally, we show that the protein is indeed capable of circularizing RNA molecules in an ATP-dependent reaction. These structural and biochemical results provide an insight into the potential physiological roles of Pab1020.     

The archaeal exosome core is a hexameric ring structure with three catalytic subunits

The exosome is a 3' --> 5' exoribonuclease complex involved in RNA processing. We report the crystal structure of the RNase PH core complex of the Sulfolobus solfataricus exosome determined at a resolution of 2.8 A. The structure reveals a hexameric ring-like arrangement of three Rrp41-Rrp42 heterodimers, where both subunits adopt the RNase PH fold common to phosphorolytic exoribonucleases. Structure-guided mutagenesis reveals that the activity of the complex resides within the active sites of the Rrp41 subunits, all three of which face the same side of the hexameric structure. The Rrp42 subunit is inactive but contributes to the structuring of the Rrp41 active site. The high sequence similarity of this archaeal exosome to eukaryotic exosomes and its high structural similarity to the bacterial mRNA-degrading PNPase support a common basis for RNA-degrading machineries in all three domains of life.     

The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological processes, including adaptive immunity, innate immunity, and bone metabolism. Importantly, it is essential for activating NF-kappaB signaling pathway in response to interleukin-1 and Toll-like receptor ligands. Previously, we characterized TRAF6 to be a ubiquitin ligase. In combination with the ubiquitin conjugating enzyme complex Ubc13/Uev1A, TRAF6 could catalyze the formation on itself of unique Lys-63 linked polyubiquitin chain that positively regulated NF-kappaB signaling pathway. However, it remains unknown how this auto-ubiquitination process is regulated. In this study, we found that the coiled-coil domain of TRAF6 was essential for its auto-ubiquitination and activating NF-kappaB signaling pathway. This domain served not as the specific target where the polyubiquitin chain was linked, but as a specific bridge to recruit Ubc13/Uev1A.     

The oligomeric integrity of toposome is essential for its morphogenetic function

Sea urchin embryos are uniquely suitable for the study of morphogenetic cell interactions. Efforts to identify the molecules responsible for morphogenetic cell adhesion led to the isolation of a 22S glycoprotein complex from Paracentrotus lividus sea urchin embryo, that has been called toposome. The biological activity of toposome in mediating cellular adhesion has been fully documented. Its function in determining positional guidance during the development of the sea urchin embryo has been proposed. Here studies on the molecular structure of toposome are reported showing that, under non-reducing conditions, it is resolved in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in a major band with an apparent molecular weight of 260 kDa, a doublet of 180-160 kDa and a lower band of 80 kDa. Digestion with EndoH endoglycosidase reduced the molecular sizes of the bands of 10%, 20% and 40%, respectively. In order to establish if the oligomeric integrity of toposome was essential for its function, the biological activity of each subunit on cells dissociated from sea urchin blastula embryos was tested. The resulting swimming embryoids were lacking skeleton, while reaggregating cells supplemented with native toposome developed into pluteus-like structures with skeletal elements.     

Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI

Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are classified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymmetric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 A crystal structure of the archaeal PI-PfuI intein from Pyroccocus furiosus. The structure reveals a unique domain, designated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a catalytic center for protein splicing, which involves both N and C-terminal residues. The endonuclease domain, which is inserted into the Hint domain, consists of two copies of substructure related by an internal pseudo 2-fold axis. In contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-binding cleft formed by a pair of four-stranded beta-sheets. DNase I footprinting experiments showed that PI-PfuI covers more than 30 bp of the substrate asymmetrically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompassing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, the two strands in the DNA duplex were cleaved by PI-PfuI with different efficiencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites.     

The quaternary structure of Escherichia coli inorganic pyrophosphatase is essential for phosphorylation

The hexameric inorganic pyrophosphatase (PPase) is irreversibly inactivated by phosphoric acid monoesters. The inactivation kinetics are consistent with the formation of a dissociable complex of the phosphoric acid monoester with the enzyme, followed by phosphorylation of the dicarboxylic amino acid of its active site. PPi and its analogues, binding at the regulatory site, release the inhibitor from the active site and thus restore PPase activity. Chemically identical subunits in the hexameric PPase interact, promoting their cooperativity in a reaction with phosphoric acid monoesters. The trimeric and monomeric PPase, exhibiting full catalytic activity, form a dissociable complex with the phosphoric acid monoesters but, in contrast to the hexameric PPase, do not form a covalent bond with them. This indicates that the native hexameric structure is essential for the irreversible inactivation of Escherichia coli PPase by phosphoric acid monoesters. Possible nontraditional pathways for activity regulation of PPase are discussed.