ABSTRACT
Heat shock factor 1 (HSF1) and 2 (HSF2) play distinct but overlapping regulatory roles in maintaining cellular proteostasis or mediating cell differentiation and development. Upon activation, both HSFs trimerize and bind to heat shock elements (HSEs) present in the promoter region of target genes. Despite structural insights gained from recent studies, structures reflecting the physiological architecture of this transcriptional machinery remains to be determined. Here, we present co-crystal structures of human HSF1 and HSF2 trimers bound to DNA, which reveal a triangular arrangement of the three DNA-binding domains (DBDs) with protein-protein interactions largely mediated by the wing domain. Two structural properties, different flexibility of the wing domain and local DNA conformational changes induced by HSF binding, seem likely to contribute to the subtle differential specificity between HSF1 and HSF2. Besides, two more structures showing DBDs bound to "two-site" head-to-head HSEs were determined as additions to the published tail-to-tail dimer-binding structures.
ABSTRACT
Naturally occurring and computationally ab initio designed protein cages can now be considered as extremely suitable materials for new developments in nanotechnology. Via self-assembly from single identical or non-identical protomers large oligomeric particles can be formed. Virus-like particles have today found a number of quite successful applications in the development of new vaccines. Complex chimeric nanoparticles can serve as suitable platforms for the presentation of natural or designed antigens to the immune system of the host. The scaffolds can be cage forming highly symmetric biological macromolecules like lumazine synthase or symmetric self-assembling virus-like particles generated by computational ab initio design. Symmetric nanoparticle carriers display a structurally ordered array of immunogens. This feature can lead to a more favorable interaction with B-cell receptors, in comparison to the administration of single recombinant immunogens. Several pre-clinical animal studies and clinical studies have recently pointed out the efficiency of nanoparticle antigens produced recombinantly in creating strong immune responses against infectious diseases like HIV, Malaria, Borrelia, Influenza.
ABSTRACT
The role of fluctuations in protein thermostability has recently received considerable attention. In the current literature a dualistic picture can be found: thermostability seems to be associated with enhanced rigidity of the protein scaffold in parallel with the reduction of flexible parts of the structure. In contradiction to such arguments it has been shown by experimental studies and computer simulation that thermal tolerance of a protein is not necessarily correlated with the suppression of internal fluctuations and mobility. Both concepts, rigidity and flexibility, are derived from mechanical engineering and represent temporally insensitive features describing static properties, neglecting that relative motion at certain time scales is possible in structurally stable regions of a protein. This suggests that a strict separation of rigid and flexible parts of a protein molecule does not describe the reality correctly. In this work the concepts of mobility/flexibility versus rigidity will be critically reconsidered by taking into account molecular dynamics calculations of heat capacity and conformational entropy, salt bridge networks, electrostatic interactions in folded and unfolded states, and the emerging picture of protein thermostability in view of recently developed network theories. Last, but not least, the influence of high temperature on the active site and activity of enzymes will be considered.
Subject(s)
Enzyme Stability , Models, Molecular , Protein Conformation , Protein Stability , Animals , Biocatalysis , Catalytic Domain , Cold Temperature/adverse effects , Entropy , Hot Temperature/adverse effects , Humans , Protein Folding , Protein Unfolding , Static ElectricityABSTRACT
The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral ß subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.
Subject(s)
Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Riboflavin Synthase/chemistry , Riboflavin Synthase/metabolism , Archaea/enzymology , Bacteria/enzymology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Fungi/enzymology , Plants/enzymology , Pteridines/metabolism , Riboflavin/biosynthesis , Schizosaccharomyces/chemistry , Schizosaccharomyces/metabolismABSTRACT
All drosophilid alcohol dehydrogenases contain an eight-member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short-chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X-ray crystallography of the SlADH(T114V) mutant revealed a broken water chain, the overall 3D structure of the binary enzyme-NAD(+) complex was almost identical to the wild-type enzyme (SlADH(wt) ). As for the SlADH(wt) , steady-state kinetic studies revealed that catalysis by the SlADH(T114V) mutant was consistent with a compulsory ordered reaction mechanism where the co-enzyme binds to the free enzyme. The mutation caused a reduction of the k(on) velocity for NAD(+) and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme-NAD(+) -alcohol complex. Furthermore, it increased the pK(a) value of the group in the binary enzyme-NAD(+) complex that regulates the k(on) velocity of alcohol and alcohol-competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis.
Subject(s)
Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Water/chemistry , Alcohol Dehydrogenase/antagonists & inhibitors , Alcohol Dehydrogenase/genetics , Alcohols/metabolism , Animals , Catalytic Domain , Crystallography, X-Ray , Drosophilidae , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Conformation , Threonine/chemistryABSTRACT
The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R(cryst) = 23.7% (R(free) = 28.4%) at a resolution of 3.5 A. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.
Subject(s)
Bacillus anthracis/enzymology , Enzyme Inhibitors/chemistry , Multienzyme Complexes/chemistry , Thermodynamics , Amino Acid Sequence , Enzyme Inhibitors/metabolism , Models, Molecular , Molecular Sequence Data , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Substrate SpecificityABSTRACT
Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K(i) of 70microM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K(i) 38microM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve pi-pi stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.
Subject(s)
Antitubercular Agents/pharmacology , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Antitubercular Agents/chemistry , Binding Sites , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/chemistry , Indoles/chemistry , Molecular Structure , Mycobacterium tuberculosis/enzymologyABSTRACT
The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.
Subject(s)
Aeropyrum/enzymology , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Isocitrate Dehydrogenase/chemistry , Isocitrate Dehydrogenase/metabolism , Aeropyrum/genetics , Amino Acid Substitution , Archaeal Proteins/genetics , Enzyme Stability , Hot Temperature , Hydrogen-Ion Concentration , Isocitrate Dehydrogenase/genetics , Kinetics , Mutagenesis, Site-Directed , Protein Folding , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermodynamics , Unfolded Protein Response , UreaABSTRACT
The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 A resolution to a final R factor of 23.7% (R(free) = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.
Subject(s)
Crystallography, X-Ray , Haloferax volcanii/chemistry , Proliferating Cell Nuclear Antigen/chemistry , Amino Acid Sequence , Cloning, Molecular , Escherichia coli/genetics , Haloferax volcanii/metabolism , Models, Molecular , Molecular Sequence Data , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Salts/metabolism , Sequence AlignmentABSTRACT
We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuclease for ss DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two-domain dumbbell-like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non-catalytic ssDNA-binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double-stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3' and 5' extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure-specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.
Subject(s)
DNA, Archaeal/metabolism , DNA, Single-Stranded/metabolism , Endonucleases/chemistry , Endonucleases/metabolism , Pyrococcus abyssi/enzymology , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , DNA, Archaeal/chemistry , DNA, Single-Stranded/chemistry , Endonucleases/genetics , Endonucleases/isolation & purification , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mycobacterium tuberculosis/enzymology , Protein Conformation , Protein Multimerization , Protein Structure, Tertiary , Sequence Alignment , Substrate SpecificityABSTRACT
The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 A crystal structure of the N-terminal domain (residues 1-268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed.
Subject(s)
Bacterial Proteins/chemistry , DNA Helicases/chemistry , Sulfolobus solfataricus/enzymology , Amino Acid Sequence , Archaeal Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , DNA Helicases/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Methanobacteriaceae/enzymology , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Sequence Deletion , Structural Homology, Protein , Zinc/chemistryABSTRACT
The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.
Subject(s)
Enzyme Inhibitors/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Oxamic Acid/pharmacology , Riboflavin Synthase/antagonists & inhibitors , Bacillus subtilis/enzymology , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Kinetics , Models, Molecular , Molecular Structure , Mycobacterium tuberculosis/enzymology , Oxamic Acid/analogs & derivatives , Oxamic Acid/chemical synthesis , Oxamic Acid/chemistry , Schizosaccharomyces/enzymology , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Stability and function of a large number of proteins are crucially dependent on the presence of disulfide bonds. Recent genome analysis has pointed out an important role of disulfide bonds for the structural stabilization of intracellular proteins from hyperthermophilic archaea and bacteria. These findings contradict the conventional view that disulfide bonds are rare in those proteins. A specific protein, known as protein disulfide oxidoreductase (PDO) is recognized as a potential key enzyme in intracellular disulfide-shuffling in hyperthermophiles. The structure of this protein consists of two combined thioredoxin-related units which together, in tandem-like manner, form a closed protein domain. Each of these units contains a distinct CXXC active site motif. Both sites seem to have different redox properties. A relation to eukaryotic protein disulfide isomerase is suggested by the observed structural and functional characteristics of the protein. Enzymological studies have revealed that both, the archaeal and bacterial forms of this protein show oxidative and reductive activity and are able to isomerize protein disulfides. The variety of active site disulfides found in PDO's from hyperthermophiles is puzzling. It is assumed, that PDO enzymes in hyperthermophilic archaea and bacteria may be part of a complex system involved in the maintenance of protein disulfide bonds.
Subject(s)
Archaea/enzymology , Archaeal Proteins/metabolism , Disulfides/metabolism , Protein Disulfide-Isomerases/metabolism , Amino Acid Motifs/physiology , Archaea/genetics , Archaeal Proteins/genetics , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Oxidation-Reduction , Protein Disulfide-Isomerases/genetics , Protein Structure, Tertiary/physiology , Thioredoxins/genetics , Thioredoxins/metabolismABSTRACT
The effect of D-H...S(gamma)-Fe hydrogen bonding on the reduction potential of rubredoxin was investigated by the introduction of an O-H...S(gamma)-Fe hydrogen bond on the surface of Pyrococcus abyssi rubredoxin. The formation of a weak hydrogen bond between Ser44-O(gamma) and Cys42-S(gamma) in mutant W4L/R5S/A44S increased the reduction potential by 56 mV. When side effects of the mutation were taken into account, the contribution of the additional cluster hydrogen bond to the reduction potential was estimated to be +65 mV. The structural analysis was based on ultrahigh-resolution structures of oxidized P. abyssi rubredoxin W4L/R5S and W4L/R5S/A44S refined to 0.69 and 0.86 A, respectively.
Subject(s)
Iron/chemistry , Pyrococcus abyssi/metabolism , Rubredoxins/chemistry , Crystallization , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/genetics , Hydrogen Bonding , Mutation , Protein Conformation , Rubredoxins/genetics , Serine/chemistry , Serine/genetics , SolutionsABSTRACT
The dynamics of a hyperthermophilic protein fragment in a water environment, as studied by performing molecular dynamics (MD) simulations at various temperatures, is compared to the dynamical behavior of a homologous mesophilic protein simulated under identical conditions. The effects on the stability of the spatial arrangement and mobility of the charged residues in solution were quantified by calculating free energy changes upon salt bridge formation in these proteins. Electrostatic free energy terms derived from a thermodynamic cycle were obtained by solving the linearized Poisson-Boltzmann equation for a series of protein conformations generated by MD simulations and placed subsequently in a continuum solvent medium. Our results show that the ion pairs are electrostatically stabilizing in most of the cases, but their individual contributions vary significantly. The greater contribution of the charged residues to the stability of the hyperthermophilic protein as compared with the mesophilic counterpart was evidenced only by the calculations that included conformations sampled at 343 and 373 K. The "dynamic" structure of the hyperthermophilic protein fragment simulated at elevated temperatures reveals an optimum placement of the ionizable residues within the protein structure as well as the role of their cooperative interactions in promoting thermal stability. The thermodynamic properties such as electrostatic free energy differences, configurational entropies, and specific heat capacities calculated in the dynamic context of the protein structure provided new insight into the mechanism of protein thermostabilization.
Subject(s)
Glutamate Dehydrogenase/chemistry , Temperature , Thermotoga maritima/enzymology , Amino Acid Sequence , Binding Sites , Enzyme Stability , Glutamate Dehydrogenase/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Sequence Alignment , Thermodynamics , Thermotoga maritima/chemistryABSTRACT
Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 A. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 A and a complete native data set was collected to 2.65 A resolution.
Subject(s)
Exonucleases/chemistry , Pyrococcus abyssi/enzymology , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Protein ConformationABSTRACT
Lumazine synthase is an enzyme involved in riboflavin biosynthesis in many plants and microorganisms, including numerous human pathogens. The fact that the enzymes of the riboflavin biosynthesis pathway are not present in the human or animal host makes them potential targets for anti-infective agents. The crystal structure of lumazine synthase from Candida albicans was solved by molecular replacement and refined at 2.5-Angstrom resolution. The results of crystallographic investigations and sedimentation equilibrium experiments clearly indicated the presence of pentameric assemblies of the enzyme either in crystals or in solution. Isothermal titration calorimetry measurements of the binding reactions of four different inhibitors revealed high affinity for all four compounds with binding constants in the micromolar range. Structural comparison with previously determined structures of the enzyme.ligand complexes of other orthologue allowed modeling of the binding of four different inhibitors into the active site of lumazine synthase from Candida albicans.
Subject(s)
Antifungal Agents/pharmacology , Candida albicans/enzymology , Multienzyme Complexes/metabolism , Amino Acid Sequence , Antifungal Agents/chemistry , Cloning, Molecular , Drug Design , Enzyme Inhibitors/metabolism , Models, Molecular , Molecular Sequence Data , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/chemistry , Protein Conformation , Sequence Homology, Amino Acid , UltracentrifugationABSTRACT
We report here the crystal structure of a protein from Pyrococcus furiosus (Phr) that represents the first characterized heat shock transcription factor in archaea. Phr specifically represses the expression of heat shock genes at physiological temperature in vitro and in vivo but is released from the promoters upon heat shock response. Structure analysis revealed a stable homodimer, each subunit consisting of an N-terminal winged helix DNA-binding domain (wH-DBD) and a C-terminal antiparallel coiled coil helical domain. The overall structure shows as a molecular chimera with significant folding similarity of its DBD to the bacterial SmtB/ArsR family, while its C-terminal part was found to be a remote homologue of the eukaryotic BAG domain. The dimeric protein recognizes a palindromic DNA sequence. Molecular docking and mutational analyses suggested a novel binding mode in which the major specific contacts occur at the minor groove interacting with the strongly basic wing containing a cluster of three arginine residues.
Subject(s)
Archaeal Proteins/chemistry , Heat-Shock Proteins/chemistry , Protein Structure, Quaternary , Protein Structure, Secondary , Pyrococcus furiosus/chemistry , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Base Sequence , Crystallography, X-Ray , Dimerization , Evolution, Molecular , Gene Expression Regulation, Archaeal , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Point Mutation , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Pyrococcus furiosus/genetics , Sequence Alignment , Static ElectricityABSTRACT
Isocitrate dehydrogenase from Archaeoglobus fulgidus (AfIDH) has an apparent melting temperature (T(m)) of 98.5 degrees C. To identify the structural features involved in thermal stabilization of AfIDH, the structure was solved to 2.5 A resolution. AfIDH was strikingly similar to mesophilic IDH from Escherichia coli (EcIDH) and displayed almost the same number of ion pairs and ionic networks. However, two unique inter-domain networks were present in AfIDH; one three-membered ionic network between the large and the small domain and one four-membered ionic network between the clasp and the small domain. The latter ionic network was presumably reduced in size when the clasp domain of AfIDH was swapped with that of EcIDH and the T (m) decreased by 18 degrees C. Contrarily, EcIDH was only stabilized by 4 degrees C by the clasp domain of AfIDH, a result probably due to the introduction of a unique inter-subunit aromatic cluster in AfIDH that may strengthen the dimeric interface in this enzyme. A unique aromatic cluster was identified close to the N-terminus of AfIDH that could provide additional stabilization of this region. Common and unique heat adaptive traits of AfIDH with those recently observed for hyperthermophilic IDH from Aeropyrum pernix (ApIDH) and Thermotoga maritima (TmIDH) are discussed herein.