Crystal structure of alanyl-tRNA synthetase editing-domain homolog (PH0574) from a hyperthermophile, Pyrococcus horikoshii OT3 at 1.45 A resolution.
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Alanine-tRNA Ligase↗
Biomedical subjects
Katsuhide Yutani
Publications and source records attributed to Katsuhide Yutani.
Conformational Changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex.
The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.
Amino Acid Sequence↗
Conformational changes in the alpha-subunit coupled to binding of the beta 2-subunit of tryptophan synthase from Escherichia coli: crystal structure of the tryptophan synthase alpha-subunit alone.
When the tryptophan synthase alpha- and beta(2)-subunits combine to form the alpha(2)beta(2)-complex, the enzymatic activity of each subunit is stimulated by 1-2 orders of magnitude. To elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the alpha- and beta-subunits alone and together with the alpha(2)beta(2)-complex. The crystal structures of the tryptophan synthase alpha(2)beta(2)-complex from Salmonella typhimurium (Stalpha(2)beta(2)-complex) have already been reported. However, the structures of the subunit alone from mesophiles have not yet been determined. The structure of the tryptophan synthase alpha-subunit alone from Escherichia coli (Ecalpha-subunit) was determined by an X-ray crystallographic analysis at 2.3 A, which is the first report on the subunits alone from the mesophiles. The biggest difference between the structures of the Ecalpha-subunit alone and the alpha-subunit in the Stalpha(2)beta(2)-complex (Stalpha-subunit) was as follows. Helix 2' in the Stalpha-subunit, including an active site residue (Asp60), was changed to a flexible loop in the Ecalpha-subunit alone. The conversion of the helix to a loop resulted in the collapse of the correct active site conformation. This region is also an important part for the mutual activation in the Stalpha(2)beta(2)-complex and interaction with the beta-subunit. These results suggest that the formation of helix 2'that is essential for the stimulation of the enzymatic activity of the alpha-subunit is constructed by the induced-fit mode involved in conformational changes upon interaction between the alpha- and beta-subunits. This also confirms the prediction of the conformational changes based on the thermodynamic analysis for the association between the alpha- and beta-subunits.
Amino Acid Sequence↗
Stabilization due to dimer formation of phosphoribosyl anthranilate isomerase from Thermus thermophilus HB8: X-ray Analysis and DSC experiments.
The crystal structure of phosphoribosyl anthranilate isomerase (PRAI) from Thermus thermophilus HB8 (TtPRAI) was solved at 2.0 A resolution. The overall structure of TtPRAI with a dimeric structure was quite similar to that of PRAI from Thermotoga maritima (TmPRAI). In order to elucidate the stabilization mechanism of TtPRAI, its physicochemical properties were examined using DSC, CD, and analytical centrifugation at various pHs in relation to the association-dissociation of the subunits. Based on the experimental results for TtPRAI and the structural information on TtPRAI and TmPRAI, we found that: (i) the denaturation of TtPRAI at acidic pH is correlated with the dissociation of its dimeric form; (ii) the hydrophobic interaction of TtPRAI in the monomer structure is slightly greater than that of TmPRAI, but dimer interface of the TmPRAI is remarkably greater; (iii) the contributions of hydrogen bonds and ion bonds to the stability are similar to each other; and (iv) destabilization due to the presence of cavities in TtPRAI is greater than that of TmPRAI in both the monomer and dimer structures.
Aldose-Ketose Isomerases↗
Stabilization mechanism of the tryptophan synthase alpha-subunit from Thermus thermophilus HB8: X-ray crystallographic analysis and calorimetry.
In order to elucidate the thermo-stabilization mechanism of the tryptophan synthase alpha-subunit from the extreme thermophile Thermus thermophilus HB8 (Tt-alpha-subunit), its crystal structure was determined and its stability was examined using DSC. The results were compared to those of other orthologs from mesophilic and hyperthermophilic organisms. The denaturation temperature of the Tt-alpha-subunit was higher than that of the alpha-subunit from S. typhimurium (St-alpha-subunit) but lower than that of the alpha-subunit from P. furiosus (Pf-alpha-subunit). Specific denaturation enthalpy and specific denaturation heat capacity values of the Tt-alpha-subunit were the lowest among the three proteins, suggesting that entropy effects are responsible for the stabilization of the Tt-alpha-subunit. Based on a structural comparison with the St-alpha-subunit, two deletions in loop regions, an increase in the number of ion pairs and a decrease in cavity volume seem to be responsible for the stabilization of the Tt-alpha-subunit. The results of structural comparison suggest that the native structure of the Tt-alpha-subunit is better adapted to an ideally stable structure than that of the St-alpha-subunit, but worse than that of the Pf-alpha-subunit. The results of calorimetry suggest that the residual structure of the Tt-alpha-subunit in the denatured state contributes to the stabilization.
Amino Acid Sequence↗
Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies.
The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.
Archaeal Proteins↗
The crystal structure of the tryptophan synthase beta subunit from the hyperthermophile Pyrococcus furiosus. Investigation of stabilization factors.
The structure of the tryptophan synthase beta2 subunit (Pfbeta2) from the hyperthermophile, Pyrococcus furiosus, was determined by X-ray crystallographic analysis at 2.2 A resolution, and its stability was examined by DSC. This is the first report of the X-ray structure of the tryptophan synthase beta2 subunit alone, although the structure of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium has already been reported. The structure of Pfbeta2 was essentially similar to that of the beta2 subunit (Stbeta2) in the alpha2beta2 complex from S. typhimurium. The sequence alignment with secondary structures of Pfbeta and Stbeta in monomeric form showed that six residues in the N-terminal region and three residues in the C-terminal region were deleted in Pfbeta, and one residue at Pro366 of Stbeta and at Ile63 of Pfbeta was inserted. The denaturation temperature of Pfbeta2 was higher by 35 degrees C than the reported values from mesophiles at approximately pH 8. On the basis of structural information on both proteins, the analyses of the contributions of each stabilization factor indicate that: (a) the higher stability of Pfbeta2 is not caused by either a hydrophobic interaction or an increase in ion pairs; (b) the number of hydrogen bonds involved in the main chains of Pfbeta is greater by about 10% than that of Stbeta, indicating that the secondary structures of Pfbeta are more stabilized than those of Stbeta and (c) the sequence of Pfbeta seems to be better fitted to an ideally stable structure than that of Stbeta, as assessed from X-ray structure data.
Amino Acid Sequence↗
[Circular dichroism].
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Circular Dichroism↗
Structure and implications for the thermal stability of phosphopantetheine adenylyltransferase from Thermus thermophilus.
Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyzes the rate-limiting step in coenzyme A (CoA) biosynthesis by transferring an adenylyl group from ATP to 4'-phosphopantetheine (Ppant), yielding 3'-dephospho-CoA (dPCoA). The crystal structure of PPAT from Thermus thermophilus HB8 (Tt PPAT) complexed with Ppant has been determined by the molecular-replacement method at 1.5 A resolution. The overall fold of the enzyme is almost the same as that of Escherichia coli PPAT, a hexamer having point group 32. The asymmetric unit of Tt PPAT contains a monomer and the crystallographic triad and dyad coincide with the threefold and twofold axes of the hexamer, respectively. Most of the important atoms surrounding the active site in E. coli PPAT are conserved in Tt PPAT, indicating similarities in their substrate binding and enzymatic reaction. The notable difference between E. coli PPAT and Tt PPAT is the simultaneous substrate recognition by all six subunits of Tt PPAT compared with substrate recognition by only three subunits in E. coli PPAT. Comparative analysis also revealed that the higher stability of Tt PPAT arises from stabilization of each subunit by hydrophobic effects, hydrogen bonds and entropic effects.
Crystallography, X-Ray↗
Stimulated interaction between and subunits of tryptophan synthase from hyperthermophile enhances its thermal stability.
Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (22) composed of and 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the and subunits in the hyperthermophile protein were of the order of 108 M1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the hyperthermophile protein than in the mesophile one, indicating that the conformational change of the hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the subunit from the hyperthermophile was enhanced by 17 degrees C due to the formation of the 22 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.
Amino Acid Sequence↗
Buried water molecules contribute to the conformational stability of a protein.
This study sought to attain a better understanding of the contribution of buried water molecules to protein stability. The 3SS human lysozyme lacks one disulfide bond between Cys77 and Cys95 and is significantly destabilized compared with the wild-type human lysozyme (4SS). We examined the structure and stability of the I59A-3SS mutant human lysozyme, in which a cavity is created at the mutation site. The crystal structure of I59A-3SS indicated that there were ordered new water molecules in the cavity created. The stability of I59A-3SS is 5.5 kJ/mol less than that of 3SS. The decreased stability of I59A-3SS (5.5 kJ/mol) is similar to that of Ile to Ala mutants with newly introduced water molecules in other globular proteins (6.3 +/- 2.1 kJ/mol), but is less than that of Ile/Leu to Ala mutants with empty cavities (13.7 +/- 3.1 kJ/mol). This indicates that water molecules partially compensate for the destabilization by decreasing hydrophobic and van der Waals interactions. These results provide further evidence that buried water molecules contribute to protein stability.
Amino Acid Substitution↗
How can free energy component analysis explain the difference in protein stability caused by amino acid substitutions? Effect of three hydrophobic mutations at the 56th residue on the stability of human lysozyme.
To elucidate the molecular mechanism of thermal stability, it is essential to determine what are the major free energy components that contribute significantly to the total free energy difference caused by amino acid mutations. In this work, we carried out free energy calculations based on all-atom molecular dynamics simulations to investigate the effect of three hydrophobic mutations at the same position, I56A, I56V and I56F of human lysozyme. The calculated free energy differences are in good agreement with the experimental values in all cases. From free energy component analysis, we found that small changes in stability in the I56A and I56V mutants originate from the short-range Lennard-Jones interactions, whereas the I56F mutant is largely destabilized owing to the changes in the long-range electrostatic interactions. The calculated results are also compared with the free energy components determined by an empirical relationship based on the native-state structure and thermodynamic data. Although this relationship has been shown to be very successful in reproducing the stability changes caused by various amino acid substitutions in several proteins, the changes of stability in I56V and I56F mutants are not reproduced very well. By comparing the free energy components calculated by these two approaches, we showed that the effect of the long-range interaction on the stability changes may be underestimated in the empirical relationships when the structural change caused by mutation is relatively small, as in I56F. It is also suggested that estimation of the change in accessible surface area, deltadeltaASA, may be overestimated if the structure around the mutation site in the denatured state is native-like, which would cause overestimation of the free energy change as in the case of I56V. Our results clearly show that the combined approach of the free energy calculation based on the all-atom molecular dynamics simulation and the empirical relationships is very useful for understanding the detailed mechanism of protein stability.
Amino Acid Substitution↗
Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus.
Expression of AT-rich genes from microorganisms such as archaea is often inefficient in Escherichia coli. The trpA and trpB genes encoding the tryptophan synthase subunits were cloned from the hyperthermophilic archaeon Pyrococcus furiosus. No apparent difference in codon bias was found between the genes. However, using a conventional cloning vector having the lac promoter, the trpB gene was expressed poorly in E. coli, whereas the trpA gene was overexpressed. The expression of the trpB gene was remarkably enhanced (>12-fold) by the introduction of an overlapping leader open reading frame. The expression of the trpA gene was also improved ( approximately 1.5-fold). This approach may be useful for overexpressing various kinds of AT-rich genes.
AT Rich Sequence↗
An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study.
It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.
Amyloid↗
Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability.
Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.
Arginine↗
The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus.
In order to understand the thermodynamic and kinetic basis of the intrinsic stability of proteins from hyperthermophiles, the folding-unfolding reactions of cysteine-free pyrrolidone carboxyl peptidase (Cys142/188Ser) (PCP-0SH) from Pyrococcus furiosus were examined using circular dichroism (CD) and differential scanning calorimetry (DSC) at pH 2.3, where PCP-0SH exists in monomeric form. DSC showed a strong dependence of the shape and position of the unfolding profiles on the scan rate, suggesting the stability of PCP-0SH under kinetic control. On DSC timescales, even at a scan rate of 1 deg. C/hour, heat denaturation of PCP-0SH was non-equilibrium. However, over a long period of incubation of the heat-denatured PCP-0SH at pre-transition temperatures, it refolded completely, indicating reversibility with very slow relaxation kinetics. The rates of refolding of the heat-denatured PCP-0SH determined from the time-resolved DSC and CD spectroscopic progress curves were found to be similar within experimental error, confirming the mechanism of refolding to be a two-state process. The equilibrium established with a relaxation time of 5080 seconds (at t(m)=46.5 degrees C), which is unusually higher than the relaxation times observed for mesophilic and hyperthermophilic proteins. The long relaxation time may lead to the apparent irreversibility of an unfolding process occurring on the DSC experiment timescale. The refolding rate (9.8 x 10(-5) s(-1)) peaked near the t(m) (=46.5 degrees C), whereas the stability profile reached maxima (11.8 kJ mol(-1)) at 17 degrees C. The results clearly indicate the unusual mode of protein destabilization via a drastic decrease in the rate of folding at low pH and still maintaining a high activation energy barrier (284 kJ mol(-1)) for unfolding, which provides an effective kinetic advantage to unusually stable proteins from hyperthermophiles.
Calorimetry, Differential Scanning↗
Elongation in a beta-structure promotes amyloid-like fibril formation of human lysozyme.
To understand the mechanism of amyloid fibril formation of a protein, we examined wild-type and three mutant human lysozymes containing both amyloidogenic and non-amyloidogenic proteins: I56T (amyloidogenic); EAEA, which has four additional residues (Glu-Ala-Glu-Ala-) at the N-terminus located on a beta-structure; and EAEA-I56T, which is an I56T mutant of EAEA. All formed amyloid-like fibrils through an in the increase contents of alpha-helix with increasing concentration of ethanol. The order of propensity for amyloid-like fibril formation in highly concentrated ethanol solution is EAEA-I56T > EAEA > I56T > wild-type. This order is almost the reverse of the order of conformational stability of these proteins, wild-type > EAEA > I56T > EAEA-I56T. The important views in this work are as follows. (i) Artificially modified proteins formed amyloid fibrils in vitro. This means that amyloid formation is a generic property of polypeptide chains. (ii) The amyloidogenic mutation Ile56 to Thr caused the destabilization and promoted fibril formation in the wild-type and EAEA human lysozymes, indicating that instability facilitates amyloid formation. (iii) The mutant protein EAEA human lysozyme had higher propensity for fibril formation than the amyloidogenic mutant protein, indicating that amyloid formation is controlled not only by stability but also by other factors. In this case, appending polypeptide chains to a beta-structure accelerated amyloid formation.
Amino Acid Substitution↗