Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms.
Certain thermophiles are found to stabilize their proteins in extreme environments with additional disulfide bonds. A phylogenetic profile identifies a protein disulfide oxidoreductase critical to the stabilization process.
Archaea evoke interest among researchers for two enigmatic characteristics –a combination of bacterial and eukaryotic components in their molecular architectures and an enormous diversity in their life-style and metabolic capabilities. Despite considerable research efforts, lineage- specific/niche-specific molecular features of the whole archaeal world are yet to be fully unveiled. The study offers the first large-scale in silico proteome analysis of all archaeal species of known genome sequences with a special emphasis on methanogenic and sulphur-metabolising archaea.
Overall amino acid usage in archaea is dominated by GC-bias. But the environmental factors like oxygen requirement or thermal adaptation seem to play important roles in selection of residues with no GC-bias at the codon level. All methanogens, irrespective of their thermal/salt adaptation, show higher usage of Cys and have relatively acidic proteomes, while the proteomes of sulphur-metabolisers have higher aromaticity and more positive charges. Despite of exhibiting thermophilic life-style, korarchaeota possesses an acidic proteome. Among the distinct trends prevailing in COGs (Cluster of Orthologous Groups of proteins) distribution profiles, crenarchaeal organisms display higher intra-order variations in COGs repertoire, especially in the metabolic ones, as compared to euryarchaea. All methanogens are characterised by a presence of 22 exclusive COGs.
Divergences in amino acid usage, aromaticity/charge profiles and COG repertoire among methanogens and sulphur-metabolisers, aerobic and anaerobic archaea or korarchaeota and nanoarchaeota, as elucidated in the present study, point towards the presence of distinct molecular strategies for niche specialization in the archaeal world.
Amino acid usage; Isoelectric point; COG distribution; Methanogen; Sulphur metaboliser; Korarachaeota; Oxygen requirement
In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of β-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus.
IMPORTANCE This study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.
The Genomic Disulfide Analysis Program (GDAP) provides web access to computationally predicted protein disulfide bonds for over one hundred microbial genomes, including both bacterial and achaeal species. In the GDAP process, sequences of unknown structure are mapped, when possible, to known homologous Protein Data Bank (PDB) structures, after which specific distance criteria are applied to predict disulfide bonds. GDAP also accepts user-supplied protein sequences and subsequently queries the PDB sequence database for the best matches, scans for possible disulfide bonds and returns the results to the client. These predictions are useful for a variety of applications and have previously been used to show a dramatic preference in certain thermophilic archaea and bacteria for disulfide bonds within intracellular proteins. Given the central role these stabilizing, covalent bonds play in such organisms, the predictions available from GDAP provide a rich data source for designing site-directed mutants with more stable thermal profiles. The GDAP web application is a gateway to this information and can be used to understand the role disulfide bonds play in protein stability both in these unusual organisms and in sequences of interest to the individual researcher. The prediction server can be accessed at http://www.doe-mbi.ucla.edu/Services/GDAP.
Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process.
A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme.
The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.
α-Amylases; Evolutionary position; Site-directed mutagenesis; Thermostability; Domain C
A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 °C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6Å crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution.
disulfide bond; protein stability; catenane; citrate synthase; thermophile
Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.
Cystathionine β-synthase (CBS) domains are found in myriad proteins from organisms across the tree of life, and have been hypothesized to function as regulatory modules that sense the energy charge of the cell. Here we characterize the structure and stability of PAE2072, a dimeric, tandem CBS domain protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Crystal structures of the protein in unliganded and adenosine monophosphate (AMP)-bound forms, determined at resolutions of 2.10 Å and 2.35 Å respectively, reveal a remarkable conservation of key functional features seen in the γ subunit of the eukaryotic AMP-activated protein kinase (AMPK). The structures also confirm the presence of a suspected intermolecular disulfide bond between the two subunits that is shown to stabilize the protein. Our AMP-bound structure represents a first step in investigating the function of a large class of uncharacterized prokaryotic proteins. In addition, this work extends previous studies that have suggested that, in certain thermophilic microbes, disulfide bonds play a key role in stabilizing intracellular proteins and protein-protein complexes.
Cystathionine β-synthase; AMP-activated protein kinase; disulfide bond; protein stabilization; hyperthermophile
Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.
The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]1/2 at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys36-Cys49 and two disulfide bonds formed by two pair of consecutive cysteines, Cys22-Cys23 and Cys56-Cys57, a unique disulfide structure of polypeptide that has not been documented previously.
Conformational stability of C3a; Denaturation of C3a; Unfolding of C3a; Method of disulfide scrambling; Scrambled isomers of C3a; Reductive unfolding of native C3a
The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of ∼60 kDa. These form, in various combinations depending on the species, a large structure or chaperonin complex sometimes called the thermosome. This multimolecular assembly is similar to the bacterial chaperonin complex GroEL/S, but it is made of only the large, double-ring oligomers each with eight (or nine) subunits instead of seven as in the bacterial complex. Like Hsp70(DnaK), the archaeal chaperonin subunits are remarkable for their evolution, but for a different reason. Ubiquitous among archaea, the chaperonins show a pattern of recurrent gene duplication—hetero-oligomeric chaperonin complexes appear to have evolved several times independently. The stress response and stress tolerance in the archaea involve chaperones, chaperonins, other heat shock (stress) proteins including sHsp, thermoprotectants, the proteasome, as yet incompletely understood thermoresistant features of many molecules, and formation of multicellular structures. The latter structures include single- and mixed-species (bacterial-archaeal) types. Many questions remain unanswered, and the field offers extraordinary opportunities owing to the diversity, genetic makeup, and phylogenetic position of archaea and the variety of ecosystems they inhabit. Specific aspects that deserve investigation are elucidation of the mechanism of action of the chaperonin complex at different temperatures, identification of the partners and substitutes for the Hsp70 chaperone machine, analysis of protein folding and refolding in hyperthermophiles, and determination of the molecular mechanisms involved in stress gene regulation in archaeal species that thrive under widely different conditions (temperature, pH, osmolarity, and barometric pressure). These studies are now possible with uni- and multicellular archaeal models and are relevant to various areas of basic and applied research, including exploration and conquest of ecosystems inhospitable to humans and many mammals and plants.
Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a _-hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N-and C-terminus contain a fluorescence donor (tryptophan) and acceptor (N_-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E°_ = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys—Gly—His—Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/KM = 1.7 _ 105 M–1M s–1, which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that catalysis of disulfide bond isomerization by PDI does not necessarily involve a cycle of substrate reduction/oxidation.
The hyperthermophilic endocellulase, EGPh (glycosyl hydrolase family 5) from Pyrococcus horikoshii possesses 4 cysteine residues forming 2 disulfide bonds, as identified by structural analysis. One of the disulfide bonds is located at the proximal region of the active site in EGPh, which exhibits a distinct pattern from that of the thermophilic endocellulase EGAc (glycosyl hydrolase family 5) of Acidothermus cellulolyticus despite the structural similarity between the two endocellulases. The structural similarity between EGPh and EGAc suggests that EGPh possesses a structure suitable for changing the position of the disulfide bond corresponding to that in EGAc. Introduction of this alternative disulfide bond in EGPh, while removing the original disulfide bond, did not result in a loss of enzymatic activity but the EGPh was no longer hyperthermostable. These results suggest that the contribution of disulfide bond to hyperthermostability at temperature higher than 100 °C is restrictive, and that its impact is dependent on the specific structural environment of the hyperthermophilic proteins. The data suggest that the structural position and environment of the disulfide bond has a greater effect on high-temperature thermostability of the enzyme than on the potential energy of the dihedral angle that contributes to disulfide bond cleavage.
Disulfide bond; Cellulase; Archaea; Thermostability; Crystal structure; Protein engineering
tRNA m1A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m1A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.
In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.
The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.
Many surface structures in archaea including various types of pili and the archaellum (archaeal flagellum) are homologous to bacterial type IV pili systems (T4P). The T4P consist of multiple proteins, often with poorly conserved sequences, complicating their identification in sequenced genomes. Here we report a comprehensive census of T4P encoded in archaeal genomes using sensitive methods for protein sequence comparison. This analysis confidently identifies as T4P components about 5000 archaeal gene products, 56% of which are currently annotated as hypothetical in public databases. Combining results of this analysis with a comprehensive comparison of genomic neighborhoods of the T4P, we present models of organization of 10 most abundant variants of archaeal T4P. In addition to the differentiation between major and minor pilins, these models include extra components, such as S-layer proteins, adhesins and other membrane and intracellular proteins. For most of these systems, dedicated major pilin families are identified including numerous stand alone major pilin genes of the PilA family. Evidence is presented that secretion ATPases of the T4P and cognate TadC proteins can interact with different pilin sets. Modular evolution of T4P results in combinatorial variability of these systems. Potential regulatory or modulating proteins for the T4P are identified including KaiC family ATPases, vWA domain-containing proteins and the associated MoxR/GvpN ATPase, TFIIB homologs and multiple unrelated transcription regulators some of which are associated specific T4P. Phylogenomic analysis suggests that at least one T4P system was present in the last common ancestor of the extant archaea. Multiple cases of horizontal transfer and lineage-specific duplication of T4P loci were detected. Generally, the T4P of the archaeal TACK superphylum are more diverse and evolve notably faster than those of euryarchaea. The abundance and enormous diversity of T4P in hyperthermophilic archaea present a major enigma. Apparently, fundamental aspects of the biology of hyperthermophiles remain to be elucidated.
type IV pili; archaea; evolution; comparative genomics; secretion ATPase
Two of the four conserved cysteine residues of an arsenic(III) S-adenosylmethionine methyltransferase bind aromatic arsenicals. The other two conserved cysteine residues form a disulfide bond.
Methylation of the toxic metalloid arsenic is widespread in nature. Members of every kingdom have arsenic(III) S-adenosylmethionine (SAM) methyltransferase enzymes, which are termed ArsM in microbes and AS3MT in animals, including humans. Trivalent arsenic(III) is methylated up to three times to form methylarsenite [MAs(III)], dimethylarsenite [DMAs(III)] and the volatile trimethylarsine [TMAs(III)]. In microbes, arsenic methylation is a detoxification process. In humans, MAs(III) and DMAs(III) are more toxic and carcinogenic than either inorganic arsenate or arsenite. Here, new crystal structures are reported of ArsM from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508 (CmArsM) with the bound aromatic arsenicals phenylarsenite [PhAs(III)] at 1.80 Å resolution and reduced roxarsone [Rox(III)] at 2.25 Å resolution. These organoarsenicals are bound to two of four conserved cysteine residues: Cys174 and Cys224. The electron density extends the structure to include a newly identified conserved cysteine residue, Cys44, which is disulfide-bonded to the fourth conserved cysteine residue, Cys72. A second disulfide bond between Cys72 and Cys174 had been observed previously in a structure with bound SAM. The loop containing Cys44 and Cys72 shifts by nearly 6.5 Å in the arsenic(III)-bound structures compared with the SAM-bound structure, which suggests that this movement leads to formation of the Cys72–Cys174 disulfide bond. A model is proposed for the catalytic mechanism of arsenic(III) SAM methyltransferases in which a disulfide-bond cascade maintains the products in the trivalent state.
arsenic(III) S-adenosylmethionine methyltransferase; AS3MT; phenylarsenite; roxarsone; disulfide-bond cascade
The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol. 81:8868–8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.
During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.
IMPORTANCE Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of Salmonella enterica serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example, Escherichia coli, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family Enterobacteriaceae. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmid-encoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the S. enterica serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not. pefA and srgA were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in pef gene expression are also involved in srgA expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of S. enterica serovar Typhimurium.
Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors.
The human pathogen Pseudomonas aeruginosa causes life-threatening infections in immunodepressed and cystic fibrosis patients. The emergence of P. aeruginosa strains resistant to all of the available antibacterial agents calls for the urgent development of new antibiotics active against this bacterium. The pathogenic power of P. aeruginosa is mediated by an arsenal of extracellular virulence factors, most of which are stabilized by disulfide bonds. Thus, targeting the machinery that introduces disulfide bonds appears to be a promising strategy to combat P. aeruginosa. Here, we unraveled the oxidative protein folding system of P. aeruginosa in full detail. The system uniquely consists of two membrane proteins that generate disulfide bonds de novo to deliver them to P. aeruginosa DsbA1 (PaDsbA1), a soluble oxidoreductase. PaDsbA1 in turn donates disulfide bonds to secreted proteins, including virulence factors. Disruption of the disulfide bond formation machinery dramatically decreases P. aeruginosa virulence, confirming that disulfide formation systems are valid targets for the design of antimicrobial drugs.
Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published.
Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations.
The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.
Thermophily; Comparative genomics; Protein engineering; Eukaryotes; Fungi
Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.
Here we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possible
Our results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.
The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).
To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.
These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.
The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23°22′ N, 44°57′ W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20°C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the ɛ subclass of the Proteobacteria (ɛ-Proteobacteria) (40%), and the genus Desulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the ɛ-Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S0, SO4, thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.
Disulfide bonds play an important role in protein folding and structure stability. Accurately predicting disulfide bonds from protein sequences is important for modeling the structural and functional characteristics of many proteins.
In this work, we introduce an approach of enhancing disulfide bonding prediction accuracy by taking advantage of context-based features. We firstly derive the first-order and second-order mean-force potentials according to the amino acid environment around the cysteine residues from large number of cysteine samples. The mean-force potentials are integrated as context-based scores to estimate the favorability of a cysteine residue in disulfide bonding state as well as a cysteine pair in disulfide bond connectivity. These context-based scores are then incorporated as features together with other sequence and evolutionary information to train neural networks for disulfide bonding state prediction and connectivity prediction.
The 10-fold cross validated accuracy is 90.8% at residue-level and 85.6% at protein-level in classifying an individual cysteine residue as bonded or free, which is around 2% accuracy improvement. The average accuracy for disulfide bonding connectivity prediction is also improved, which yields overall sensitivity of 73.42% and specificity of 91.61%.
Our computational results have shown that the context-based scores are effective features to enhance the prediction accuracies of both disulfide bonding state prediction and connectivity prediction. Our disulfide prediction algorithm is implemented on a web server named "Dinosolve" available at: http://hpcr.cs.odu.edu/dinosolve.