How oligomerization contributes to the thermostability of an archaeon protein. Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii.

To study how oligomerization may contribute to the thermostability of archaeon proteins, we focused on a hexameric protein, protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii (StoPIMT). The crystal structure shows that StoPIMT has a distinctive hexameric structure composed of monomers consisting of two domains: an S-adenosylmethionine-dependent methyltransferase fold domain and a C-terminal alpha-helical domain. The hexameric structure includes three interfacial contact regions: major, minor, and coiled-coil. Several C-terminal deletion mutants were constructed and characterized. The hexameric structure and thermostability were retained when the C-terminal alpha-helical domain (Tyr(206)-Thr(231)) was deleted, suggesting that oligomerization via coiled-coil association using the C-terminal alpha-helical domains did not contribute critically to hexamerization or to the increased thermostability of the protein. Deletion of three additional residues located in the major contact region, Tyr(203)-Asp(204)-Asp(205), led to a significant decrease in hexamer stability and chemico/thermostability. Although replacement of Thr(146) and Asp(204), which form two hydrogen bonds in the interface in the major contact region, with Ala did not affect hexamer formation, these mutations led to a significant decrease in thermostability, suggesting that two residues in the major contact region make significant contributions to the increase in stability of the protein via hexamerization. These results suggest that cooperative hexamerization occurs via interactions of "hot spot" residues and that a couple of interfacial hot spot residues are responsible for enhancing thermostability via oligomerization.

The protein L-isoaspartyl-O-methyltransferase (PIMT 1 ; EC 2.1.1.77) functions as an enzyme in the repair of age-damaged proteins in which asparagines and aspartates have been spontaneously deamidized and isomerized into L-isoaspartyl residues; it catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of the ␣-carboxyl group of the L-isoaspartyl residues to form L-iso-Asp-␣-methyl ester (26,27). Crystal structures of PIMT have been reported for both mesophiles and thermophiles (Thermotoga maritima (28), Pyrococcus furiosus (29), humans (30,31), and Drosophila melanogaster (32)), revealing similar three-dimensional structures. The structures include an AdoMet-dependent methyltransferase fold, which is a modified Rossman fold consisting of a central seven-stranded ␤-sheet flanked by ␣-helices on both sides; this structure is common to the entire AdoMet-dependent methyltransferase family, including those having DNA, RNA, proteins, polysaccharides, lipids, and small molecules as substrates for methyltransferase reactions (33). We have succeeded in clarifying the functional and structural properties of PIMT from the thermophilic archaeon Sulfolobus tokodaii (StoPIMT), whose optimal growth temperature is 80°C, and have found a unique hexa- 1 The abbreviations used are: PIMT, protein L-isoaspartyl-O-methyltransferase; StoPIMT, PIMT from S. tokodaii; StoPIMT-dxxx, a series of deletion mutants of StoPIMT containing only the sequence from Met1 to amino acid number xxx; StoPIMT-WT-mutant, a StoPIMT double mutant in which Thr 146 and Asp 204 are both replaced with Ala; StoPIMT-d205-mutant, a StoPIMT-d205 double mutant in which Thr 146 and Asp 204 are replaced with Ala; GdnHCl, guanidine hydrochloride; SEC, size exclusion chromatography; DSC, differential scanning calorimetry; PfuPIMT, PIMT from Pyrococcus furiosus; AdoMet, Sadenosylmethionine; AdoHcy, S-adenosyl-L-homocysteine; MBP, maltose-binding protein.
meric structure that was not previously reported in the AdoMetdependent methyltransferase family.
It has been proposed that oligomerization is critically important for the stability of archaeon proteins (10, 20 -25), and recent structural studies have demonstrated that many archaeon proteins have a homo-oligomeric structure. Some reports have shown correlations between oligomerization and the hyperthermostability of archaeon proteins (20,21). Oligomerization is usually through interfacial interaction (34 -36), in which subunits cooperatively interact with each other in several ways (e.g. domain swapping and coiled-coil association).
In this report, we first describe the crystal structure of StoPIMT at 2.8-Å resolution. The monomeric structure of StoPIMT consists of two domains, an AdoMet-dependent methyltransferase fold domain and a distinctive C-terminal ␣-helical domain. Six monomers associate into a hexamer, in which there are three contact regions per monomer, referred to as the major, minor, and C-terminal ␣-helical contact regions. We investigated whether and how oligomerization is correlated with enhancement of thermostability by constructing several deletion mutants; our results suggest the existence of "hot spot" residues in the subunit interfaces that function in cooperative oligomerization and in enhancing thermostability via oligomerization. On the basis of these observations, we discuss how oligomerization contributes to the thermostability of this protein.

EXPERIMENTAL PROCEDURES
Materials-All enzymes used in genetic engineering were obtained from Takara Shuzo (Kyoto, Japan), Toyobo (Osaka, Japan), and New England Biolabs (Beverly, MA). Isopropyl-1-thio-␤-D-galactopyranoside was obtained from Wako Fine Chemicals Inc. (Osaka, Japan). All other reagents were of biochemical research grade.
Expression and Purification of Wild-type StoPIMT Protein and Var-ious Deletion and/or Point Mutants of StoPIMTs-A transformed Escherichia coli strain, BL21 (DE3) (39), harboring expression vector encoding the protein gene, was grown at 37°C in LB supplemented with 100 g ml Ϫ1 ampicillin until the early stationary phase. To induce expression of the desired protein, Isopropyl-1-thio-␤-D-galactopyranoside was added to a final concentration of 1 mM, and the culture was grown for 3 h at 37°C. The selenomethionine derivative of StoPIMT was expressed in a methionine auxotroph, E. coli strain B834(DE3) (40), grown in M9 medium supplied with 0.1 mM selenomethionine. Cells were harvested by centrifugation at 5000 ϫ g for 10 min at 4°C; washed with 50 mM Tris-HCl (pH 8.0), 50 mM NaCl; and then resuspended in the same buffer. The suspension was sonicated, followed by centrifugation at 8000 ϫ g for 30 min at 4°C. The soluble fraction was incubated at 70°C for 30 min and then centrifuged at 8000 ϫ g for 30 min at 20°C. Crystallization of StoPIMT Proteins-Procedures for crystallization were in principle those followed by Tanaka et al. (38). StoPIMT was dialyzed against 10 mM Tris-HCl, pH 8.0, and concentrated to 20 mg ml Ϫ1 . Initial crystallization conditions were screened by the sparse matrix method at 20°C, using the Crystal Screen kit and Crystal Screen 2 kit (Hampton Research). Crystals of StoPIMT most suitable for further analyses could be grown by the hanging drop vapor diffusion method from 100 mM citrate buffer, pH 5.6, 0.8 M lithium sulfate, 0.5 M ammonium sulfate.
X-ray Diffractions-X-ray diffraction of selenomethionine-substituted StoPIMT was performed on the beamline BL41XU at SPring-8 (Harima, Japan), under cryogenic conditions (100 K). For multiwavelength anomalous diffraction phasing, three wavelengths were chosen on the basis of the fluorescence spectrum of the selenium K absorption edge, corresponding to the maximum fЉ (peak, 0.9793 Å), minimum fЈ (edge, 0.9795 Å), and the reference point (remote, 0.9700 Å). The threewavelength diffraction data set was collected to a resolution of 2.8 Å with a MAR Research CCD detector. The crystal of selenomethioninesubstituted StoPIMT belongs to space group F23 with unit cell dimensions of a ϭ b ϭ c ϭ 277.80 Å. The data were indexed and integrated with the program MOSFLM (41) and were further scaled and merged with the program SCALA in the CCP4 package (42).
Structure Solution and Refinement-The initial phasing and subsequent phase improvement were done with the program SOLVE/RE-SOLVE (43)(44)(45). First we performed multiwavelength anomalous diffraction phasing and phase improvement using the entire threewavelength data set, which resulted in an uninterpretable electron density map. Therefore, the single-wavelength anomalous dispersion strategy in the program SOLVE was applied for structure solution using only the diffraction data collected at the peak wavelength. Positional and individual B factor refinement was carried out with the program CNS (47), using reflections ranging from 15 to 2.8 Å. A random 10% of all observed reflections were set aside for cross-validation analysis and were used to monitor throughout the refinement by calculating the free R value (R free ). Noncrystallographic symmetry restraints were used, and the restraint weight was gradually decreased as model refinement progressed. The water molecules were automatically located by peak searching on the SIGMAA-weighted mF o Ϫ DF c map, and some water molecules, which occupied irrelevant positions, were deleted on the basis of their real space correlation coefficient and/or maximum density level using the procedure in the program CNS (47). Finally, the crystallographic R and R free values converged to 19.8 and 24.5%, respectively. The stereochemical quality of the final refined model was analyzed with the program PROCHECK (48). The crystallographic parameters and refinement statistics are summarized in Table I.

Hot Spots for Oligomerization and Thermostability of Protein
Size Exclusion Chromatographic Analyses of StoPIMT and Its Mutants-Size exclusion chromatography using a HiLoad Superdex 200-pg column (16 ϫ 300 mm; Amersham Biosciences) was carried out for analysis of the extent of multimerization of StoPIMT and its mutants. The column was equilibrated with 50 mM Tris-HCl, pH 8.0, containing 200 mM NaCl, and then 2 ml of purified protein was applied to the column at a flow rate of 2.5 ml min Ϫ1 .
Differential Scanning Calorimetry (DSC)-All DSC measurements were carried out with a nanoDSC II calorimeter (Calorimetry Science Corp.). Proteins were dialyzed against phosphate-buffered saline buffer (125 mM phosphate buffer, pH 7.0, containing 150 mM NaCl). The dialysis buffer was used as a reference solution for the DSC scan. Protein samples of 1.2-1.7 mg ml Ϫ1 were heated from 0 to 125°C at a scanning rate of 1 K min Ϫ1 .
CD Spectrometry-To compare the tolerance to GdnHCl of StoPIMT and its mutants, purified and concentrated proteins were diluted in 50 mM Tris-HCl (pH 8.0) buffer containing various concentrations of GdnHCl (0 -8 M). After 24 h, CD spectra were measured in an AVIV circular dichroism spectrometer (Proteiron Co.): path length, 1.0 mm; resolution, 0.2 nm; average time, 4 s. The results are expressed as the mean residue ellipticity ([]), and [] values at 222 nm were plotted as a function of GdnHCl concentration.

RESULTS
Crystal Structure of StoPIMT-The crystal structure of StoPIMT was determined at 2.8-Å resolution by the singlewavelength anomalous dispersion method (Table I). The asymmetric unit contains four StoPIMT monomers with a Matthews' coefficient (V M ) value of 4.21 Å 3 Da Ϫ1 and a solvent content of 70.6%. The monomeric structure was composed of seven ␤ strands (␤ 1 -␤ 7 ) and eight ␣-helices (␣ 1 -␣ 8 ), forming two domains: the AdoMet-dependent methyltransferase fold domain (blue to gold; Fig. 1A), which is common to all of the enzymes in this family (33), and a characteristic C-terminal ␣-helical domain (red; Fig. 1A).
The former is described as a modified Rossman fold consisting of a central seven-stranded ␤-sheet (␤ 1 -␤ 7 ) flanked by ␣-helices (␣ 1 -␣ 7 ) on both sides. In contrast to the other AdoMetdependent methyltransferases from various species whose crystal structures have been published (28 -32), in all PIMT proteins the two strands ␤ 6 and ␤ 7 are exchanged relative to the arrangement of the Rossman fold (28 -32). The structure of StoPIMT reported here also included this rearrangement of two ␤ strands (Fig. 1B), and therefore the StoPIMT structure can be superimposed well on the other PIMT structures from T. maritima, P. furiosus, humans, and Drosophila; the respective root mean square differences for superposition are 1.52 Å for 175 C␣ atoms, 1.23 Å for 190 C␣ atoms, 1.39 Å for 170 C␣ atoms, and 1.42 Å for 163 C␣ atoms.
The C-terminal ␣-helical domain, which consists of a long loop and one ␣-helix (␣ 8 ), protrudes from the methyltransferase fold, and the three ␣ 8 -helices in three separate monomers related by the crystallographic 3-fold symmetry axis form a unique coiled-coil assembly ( Fig. 1D; see "Hexameric Assembly of StoPIMT"). Such a coiled-coil assembly has not been identified in any other solved AdoMet-dependent methyltransferase. We should also note that the C-terminal 30 residues corresponding to this domain show no significant sequence similarity to any sequence in the Swiss-Prot/TrEMBL data base (available on the World Wide Web at ca.expasy.org/).
In the structure of P. furiosus PIMT, very clear electron density corresponding to the cofactor S-adenosyl-L-homocysteine (AdoHcy) was recognized (29). In the present structure of StoPIMT, the F o Ϫ F c electron density map indicated that an AdoHcy molecule was bound to two of four molecules in the asymmetric unit (chains A and C in Fig. 1C). AdoHcy was associated with Thr 54 O␥, Gly 80 O, Glu 99 O⑀, Gly 197 O, and Gly 125 N, suggesting that the recognition manner seems to be identical to that of other PIMTs. As with P. furiosus PIMT (PfuPIMT), this was an unexpected result, because no cofactor was added in the purification and/or crystallization steps for either protein. The bound AdoHcy is presumably derived from PIMT activity during the growth of the E. coli cells, and the enzyme must have retained the cofactor through all of the experimental procedures.
Hexameric Assembly of StoPIMT-The PIMTs reported so far function as monomers, and there has been no report of their oligomerization. In contrast, the crystal structure of StoPIMT reveals that it forms a hexamer (Figs. 1, C and D), which is consistent with the results of size exclusion chromatography (Fig. 1E).
We now describe the elaborate hexameric assembly of this enzyme. The four monomers in the asymmetric unit create two disulfide-bridged dimers. Two disulfide linkages are formed between chains A and B and between chains C and D (Fig. 1C), using the identical cysteine residue Cys 149 . Interestingly, this Cys 149 residue appears only in StoPIMT (Fig. 2). The two monomers forming a disulfide-bridged dimer are related by a noncrystallographic 2-fold axis of symmetry. Three of these disulfide-bridged dimers assemble into a hexamer having crystallographic 3-fold symmetry. Therefore, the StoPIMT hexamer is a trimer of disulfide-bridged dimers with a point group symmetry 32, and two independent hexamers can be generated from the four molecules in the asymmetric unit (Fig. 1C).
In the hexameric structure, three distinct intermolecular interfaces are observed. One is the "major contact," which corresponds to the interface between two disulfide-linked monomers (red arrows in Fig. 1D). The major contact contains three hydrogen bonds (Thr 149 O␥-Gly 201Ј O, Glu 153 O⑀-Glu 153Ј O⑀, and Val 167 N-Asp 204Ј O␦) and one salt bridge (Lys 150 N-Glu 153Ј O⑀), and the area of interaction is 947 Å 2 (Fig. 1D). Another interface is the "minor contact," a smaller intermolecular interface between adjacent methyltransferase fold domains, which are related by crystallographic 3-fold symmetry (blue arrows in Fig. 1D). The minor contact has an area of 749 Å 2 and contains only one hydrogen bond (Asn 32 N␦-Ile 47Ј O). The third contact is formed among three identical C-terminal ␣-helices (␣ 8 ) related by crystallographic 3-fold symmetry.  1. Crystal structure of StoPIMT and the hexameric structure in solution. A, stereo ribbon diagram of a monomer of StoPIMT, which consists of an AdoMet-dependent methyltransferase fold domain and a C-terminal ␣-helical domain. The ribbon model is colored according to the sequence by a rainbow color ramp going from blue at the N terminus to red at the C terminus. Bound AdoHcy is also shown as a ball-and-stick model. B, topology diagrams of a monomer of StoPIMT (top) and typical AdoMet-dependent methyltransferase fold domain of rRNA methyltrans-These three ␣-helices form a distinctive coiled-coil assembly and form a hydrophobic core composed of Ile 209 , Val 213 , Leu 216 , Ile 220 , and Ile 223 . The coiled-coil structure seems to be additionally stabilized by one salt bridge between Arg 212 and Glu 217 . The StoPIMT hexamer contains two coiled-coil structures, which protrude from the methyltransferase fold domain and extend in opposite directions along the crystallographic 3-fold axis (Fig. 1D).
The Effects of the C-terminal Loop Region and Coiled-coil Domain on Multimerization of StoPIMT-Comparison of the amino acid sequence homology of several PIMTs (Fig. 2) shows that StoPIMT is the only one with a long C-terminal loop and helix (i.e. from Asp 205 to Thr 231 ). To investigate the effects of the C-terminal loop and helical domain on oligomerization, first, we fused the C-terminal ␣-helical domain of StoPIMT (residues 205-231) at its C terminus with maltose-binding protein (MBP) or glutathione S-transferase as models. We next prepared several deletion mutants of StoPIMT and investigated the oligomerization states of wild-type and mutant proteins by chromatography (Table II and Fig. 4). StoPIMT-d205 had a hexameric structure, indicating that deletion of the helical domain did not hinder association of hexameric StoPIMT. The hexameric structure was also retained in StoPIMT-d204 and StoPIMT-d203, but the SEC results for StoPIMT-d202 showed three fractions corresponding to monomers, dimers, and hexamers (Fig. 4). Interestingly, rechromatography of the peak corresponding to the hexamer of StoPIMT-d202 yielded dimer and monomer peaks (data not shown), indicating that the hexamer of the truncated mutant is unstable. These results reveal that Tyr 203 makes a significant contribution to hexamerization. Additional deletions from StoPIMT-d202 resulted in proteins (StoPIMT-d199 and StoPIMT-d197) having only monomers and dimers, without hexamers. The addition of a slight molar excess of 2-mercaptoethanol to the fractions corresponding to dimers of StoPIMT-d202, StoPIMT-d199, and StoPIMT-d197 resulted in dissociation of the dimeric structures into monomers (data not shown). This indicates that two monomers of the deletion mutant proteins associate with each other by a disulfide bond without appropriate contact between the major contact regions. Considferase from Bacillus subtilis (Protein Data Bank code 2ERC (58)) (bottom). C, schematic diagram of four molecules (chains A, B, C, and D) in the asymmetric unit (dashed box) and two adjacent hexamers generated by crystallographic symmetry. The hexamer belongs to point group 32 (crystallographic 3-fold ϩ pseudo-2-fold). The asterisks represent the positions of Tyr 203 that significantly contribute to hexamerization. The intermolecular disulfide linkage between Cys 149 residues and the positions of the major and minor contacts are also shown. A stereo diagram of disulfide-bridged dimer (chains A and B) is shown in Fig. 7A. D, ribbon diagram of hexameric structure of StoPIMT; the 3-fold axis of symmetry is perpendicular to the plane of the paper. The major and minor contact regions are indicated by red and blue triangles, respectively. E, results of size exclusion chromatography of StoPIMT on HiLoad Superdex200. The elution points of standard marker proteins are shown along the top. ering that the deleted residues from 198 to 205 and the cysteine residue that forms the intermolecular disulfide link are located in the interface of the major contact region, deletion of Tyr 203 apparently affects not only the major but also the minor contact interfaces.
Comparison of Thermostability and Tolerance to GdnHCl of Wild-type and Mutant StoPIMTs-The thermostability of each protein was measured by differential scanning calorimetry (DSC). Fig. 5A shows DSC results for wild-type StoPIMT and its deletion mutants that have hexameric structures, and Fig.  5B shows the results for deletion mutants that form only mo-nomeric and dimeric structures. Table III summarizes the denaturation enthalpy values and peak temperatures of the profiles.
All of the samples showed irreversible transition and precipitated after the DSC run, so we analyzed the profile mainly in terms of the peak temperatures. The thermogram of wild-type StoPIMT shows a peak at 98.7°C with a slight shoulder, and those of StoPIMT-d205 and StoPIMT-d204 show peaks at ϳ101°C without a shoulder. The peak for StoPIMT-d203 occurs at a lower temperature with a shoulder, and the hexameric fraction of StoPIMT-d202 shows three peaks; in contrast,    the monomer fraction of StoPIMT-d202 showed two peaks at ϳ85 and 90°C. The monomeric fraction of StoPIMT-d199 and StoPIMT-d197 showed one peak at ϳ85°C.

Hot Spots for Oligomerization and Thermostability of Protein
To evaluate the stability of StoPIMT and its mutants to a chemical denaturant, the effects of GdnHCl on their denaturation were investigated by CD spectroscopy. Fig. 6 shows the CD intensities at 222 nm for each protein at various GdnHCl concentrations. The transition midpoints for StoPIMT, StoPIMT-d205, -d204, -d203, and -d202, were 4.2, 4.5, 3.4, 3.4, and 3.2 M GdnHCl, respectively. The monomer and dimer fractions of StoPIMT-d202, -d199, and -d197 showed transition midpoints of ϳ3.0 M GdnHCl.
Effects of Mutation of Two Residues in the Dimeric Interface on the Thermo/Chemicostability of StoPIMT-Thus far, our results show that the hexamerization of StoPIMT seems to generally correlate with its thermostability and tolerance to GdnHCl and that some residues in the major contact region (i.e. Tyr 203 -Asp 204 -Asp 205 ) make critical contributions to the protein's hexamerization and thermo/chemicostability. The crystal structure of StoPIMT shows that two hydrogen bonds, Thr 146 -Glu 201 and Asp 204 -Val 167 , and one disulfide bond, Cys 149 -Cys 149Ј are located in the major contact region (Fig. 7A). To address the role of interfacial residues in the major contact region, we first constructed several mutants in which one residue in the major or minor contact region was replaced with Ala or Ser; however, little effect on hexamerization or thermostability of the protein was observed (data not shown). We then constructed two double mutants: the StoPIMT mutant, in which Thr 146 and Asp 204 were both replaced with Ala (StoPIMT-WT-mutant), and the deletion mutant StoPIMT-d205, in which these residues were also replaced with Ala (StoPIMT-d205-mutant). Size exclusion chromatography showed that the StoPIMT-WT-mutant and the StoPIMT-d205mutant proteins existed as hexamers in solution (Fig. 7B). Appreciably lower tolerance to GdnHCl, however, was observed for both double mutants; the transition midpoints were almost the same as for the hexamer of StoPIMT-d202, which did not form stable hexamers but readily dissociated into dimers and monomers (Fig. 7C). DSC measurements also showed two peaks for the double mutants; one peak is identical to that for the unstable hexameric structure of StoPIMT-d202 and has a maximum at 90.7°C, and the other is from the same as that for hexamers of wild-type and mutants that have maximums at ϳ98°C (Fig. 7D). DISCUSSION Oligomerization has been suggested to make a critical contribution to the stability of proteins (10, 20 -25), and recent structural studies have demonstrated that many archaeon proteins have homo-oligomeric structures. Some reports have shown correlation between oligomerization and the hyperthermostability of archaeon proteins (20,21). Oligomerization is usually through interfacial interactions (34 -36), in which subunits cooperatively interact with each other in several ways (e.g. domain-swapping and coiled-coil association) (49,50). The crystal structure of StoPIMT includes three areas of intermolecular contact (i.e. major, minor, and C-terminal ␣-helical coiled-coil contact regions); therefore, the hexameric structure of StoPIMT has interfacial interactions and coiled-coil associations. To address how oligomerization of archaeon proteins contributes to their hyperthermostability, here we focused on StoPIMT.
Hexamerization of StoPIMT: Correlation with Thermostability and Existence of Hot Spots-Analyses of the oligomerization states of wild-type and mutant StoPIMT proteins indicates that the normal hexameric structure is retained even after deletion of the C-terminal ␣-helical domain; dissociation of hexamers into monomers and dimers occurred only after removal of the next three residues (Tyr 203 -Asp 204 -Asp 205 ). SEC analyses indicated that the presence of Tyr 203 is critical for oligomerization of the protein, and DSC measurements indicated the critical contribution of Asp 204 to the thermostability of the protein. Deletion of Asp 205 resulted in decreased tolerance to GdnHCl. In the crystal structure of StoPIMT, the loop region including these residues is located in the major contact region (Figs. 1D and 7A). Replacement of some residues in the minor contact region with Ala affected neither the thermostability nor the tolerance to GdnHCl (data not shown). Although dimer formation via disulfide bonds was observed in each deletion mutant, replacement of the sole Cys residue with Ser had no effect on either oligomerization or thermostability of the protein (data not shown), suggesting that the disulfide bond between these Cys residues also makes little contribution to the thermostability of the protein. Taken together, these results suggest that adequate association of monomers involving the major contact region is critical for hexamerization and hyperthermostability and also suggest that the three residues, Tyr 203 -Asp 204 -Asp 205 , are critical for hexamerization and hyperthermostability. As proposed from studies of the effects of mutations on several types of interactions, including receptor- ligand interactions (51,52), antigen-antibody interactions (53,54), and homodimer formation (55,56), we propose that these residues also be defined as hot spot residues (57) for hexamerization and/or hyperthermostability. Formation of oligomeric structures was shown to be correlated with enhanced stability of StoPIMT. The electrostatic potential on the surface of the minor contact region of the StoPIMT monomer is relatively neutral, although that of the major contact region is relatively negatively charged (Fig. 8). Accordingly, association between the neutral surfaces of the minor contacts of two monomers leads to a decrease in neutral surface area and exposure of relatively more charged surfaces of the protein to the solvent. It has been proposed that an increased number of polar residues at the molecular surface enhances protein thermostability (3)(4)(5)(6)(7)(8)(9)(10). Enhanced thermostability in StoPIMT may thus be partly due to the decrease in neutral surface area and the consequent increase in charged surface area that it can achieve by hexamerization. We should note that the minor contact region of one monomer cannot interact with its counterpart on another monomer without appropriate contact between the major contact regions, especially including Tyr 203 -Asp 204 -Asp 205 .
The stabilities of StoPIMT and StoPIMT-d205 in the pres-ence of GdnHCl were decreased by replacing Thr 146 and Asp 204 with Ala; they became almost as easily denatured as the deletion mutant StoPIMT-d202. StoPIMT-WT-mutant and StoPIMT-d205-mutant had a peak at ϳ90°C in the DSC analyses, which was not observed in the wild-type and mutant proteins having stable hexameric structures, suggesting that deletion of some interactions in the major contact caused some structural changes. The significant decreases in the tolerance to GdnHCl of StoPIMT-WT-mutant and StoPIMT-d205-mutant might be correlated with the appearance of this peak in the DSC analyses of the mutants. The two replaced residues are involved in intermolecular contacts in the major contact region (Fig. 7A). The significant decrease in thermostability and tolerance to GdnHCl resulting from these mutations indicates the critical contribution of the side chains of Thr 146 and Asp 204 to stability via hydrogen bond formation. These mutants, however, do have hexameric structures, suggesting that the thermostability and tolerance to GdnHCl of StoPIMT may be triggered by favorable interactions in the major contact region, including hydrogen bond formation by these two residues. We should note that Asp 204 is located in the C-terminal loop, which was shown to be essential for hexamerization. Therefore, we could conclude that the protein is hexamerized via the C- terminal loop including Tyr 203 -Asp 204 -Asp 205 and that thermostability can be achieved by rearrangement of the contacts of Thr 146 and Asp 204 in the major contact region. These results again suggest the existence of hot spot residues for the formation of hexamers and acquisition of thermostability. We should also note that cooperative association of monomers into a hexamer via the hot spot residues is important for the thermostable architecture of the protein.
Contribution of C-terminal Coiled-coil Contact Region to Hexamerization and Thermostability of StoPIMT-The thermostability and tolerance to GdnHCl of StoPIMT-d205 and StoPIMT-d204 were greater than that of StoPIMT, despite deletion of the ␣ 8 -helix, which participates in the trimerized coiled-coil structure. In the crystal structure, the asymmetric unit contains four molecules of StoPIMT. Their superimposition on the AdoMet-dependent methyltransferase fold domain of each protein revealed that the C-terminal ␣-helical domains, especially the N and C termini of ␣ 8 , did not overlap well on each other. The C-terminal ␣-helical domain of StoPIMT is flexible, and therefore packing within a hexamer via the coiledcoil structure is relatively loose in comparison with the major and minor contact regions. Deletion of this flexible domain in StoPIMT leads to some enhancement of hyperthermostability; for example, Russell et al. (11) reported that shortening a loop region is one of the factors that increase thermostability in citrate synthase from thermophiles. Thus, one may conclude that oligomerization of proteins via association of coiled-coil regions does not enhance their stability but may lead to slight destabilization.
Structural Comparison of StoPIMT with PfuPIMT and Human PIMT and Its Relation to Thermostability-The crystal structure of PfuPIMT has been reported (29), revealing that PfuPIMT has a monomeric structure. Although the root mean square difference of StoPIMT superimposed on PfuPIMT is only 1.23 Å, and their amino acid sequence homology is greater than 35%, PfuPIMT has higher thermostability than StoPIMT. The crystal structure of PfuPIMT may be characterized as including intramolecular hydrogen bonds, ion pairs on the protein surface, and less hydrophobic surface with many charged residues than StoPIMT. The numbers of intramolecular hydrogen bonds whose lengths were within 3.0 Å and of ion pairs whose lengths were within 3.3 Å when StoPIMT was superposed on homologous sequences in PfuPIMT and human PIMT were thus calculated and compared. There were 120 hydrogen bonds in StoPIMT, but 147 and 145 in PfuPIMT and human PIMT, respectively. Nine and five ion pairs were observed in (monomeric) PfuPIMT and human PIMT, respectively, but the numbers of ion pairs in four StoPIMT monomers within an asymmetric unit in the crystal structure were four (three molecules) and six (one molecule). These results indicate that thermostability of StoPIMT does not originate from increases in numbers of intramolecular hydrogen bonds and ion pairs. We can therefore consider that oligomerization may be an alternative means for the development of thermal stability in StoPIMT. It is interesting to note that different mechanisms make PIMT hyperthermostable in the two archaeons.
Conclusions-X-ray crystallography of StoPIMT has revealed its hexameric structure. The hexameric associations resulted from three interfacial contacts: major, minor, and coiled-coil contact regions. Although the C-terminal ␣-helical domain promotes multimerization of StoPIMT by interacting to form coiled coils, this domain is not needed for hexamerization of StoPIMT and does not contribute importantly to increasing its thermostability. Mutation in and/or deletions that include the major contact regions affect the oligomerization and thermostability of StoPIMT, suggesting that local structures within the major contact region contribute importantly to oligomerization and to increasing the protein's stability via hexamerization. The existence of interfacial hot spot residues for promoting thermostability via oligomerization and cooperative hexamerization via interactions of two hot spot residues are suggested.