Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli Yutaka Suzuki 1 , Kazufumi Takano 1,2 and Shigenori Kanaya 1 1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan 2 PRESTO, JST, Suita, Osaka, Japan When polypeptides are synthesized at ribosomes, pep- tide bonds are connected in trans form. In the case of peptide bonds N-terminal of the proline residues, how- ever, some of them form cis peptide bonds in correctly folded proteins [1]. Consequently, trans-to-cis conver- sions of these peptide bonds (prolyl isomerizations) should occur during protein folding reactions. As dem- onstrated in some refolding experiments [2,3], prolyl isomerizations are relatively slow and can be the rate limiting step in protein folding reactions. The cis-trans isomerizations of peptide bonds N-terminal of the pro- line residues are catalyzed by peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8) [4]. Three structurally unrelated families of PPIases are known. They are cyclophilins, parvulins, and FK506-binding proteins (FKBPs) [5]. We have previously shown that the cellular content of FKBP22 (SIB1 FKBP22) from a psychrotrophic Keywords domain structure; FKBP22; PPIase; psychrotrophic bacterium; thermal stability Correspondence S. Kanaya, Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan Tel ⁄ Fax: +81 6 6879 7938 E-mail: kanaya@mls.eng.osaka-u.ac.jp (Received 27 September 2004, revised 23 October 2004, accepted 29 October 2004) doi:10.1111/j.1742-4658.2004.04468.x FKBP22 from a psychrotrophic bacterium Shewanella sp. SIB1, is a dimer- ic protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. Accord- ing to homology modeling, it consists of an N-terminal domain, which is involved in dimerization of the protein, and a C-terminal catalytic domain. A long a3 helix spans these domains. An N-domain with the entire a3 helix (N-domain + ) and a C-domain with the entire a3 helix (C-domain + ) were overproduced in Escherichia coli in a His-tagged form, purified, and their biochemical properties were compared with those of the intact protein. C-domain + was shown to be a monomer and enzymatically active. Its opti- mum temperature for activity (10 °C) was identical to that of the intact protein. Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N-domain is involved in sub- strate-binding. The differential scanning calorimetry studies revealed two distinct heat absorption peaks at 32.5 °C and 46.6 °C for the intact protein, and single heat absorption peaks at 44.7 °C for N-domain + and 35.6 °C for C-domain + . These results indicate that the thermal unfolding transi- tions of the intact protein at lower and higher temperatures represent those of C- and N-domains, respectively. Because the unfolding temperature of C-domain + is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibil- ity around the active site. This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22. Abbreviations ALPF, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide; CD, circular dichroism; DSC, differential scanning calorimetry; FKBP, FK506-binding protein; MIP, macrophage infectivity potentiator; PPIase, peptidyl prolyl cis-trans isomerase. 632 FEBS Journal 272 (2005) 632–642 ª 2005 FEBS bacterium Shewanella sp. SIB1 increases at 4 °C, as compared to that at 20 °C [6]. This protein is a member of the macrophage infectivity potentiator (MIP)-like FKBP subfamily proteins and shows amino acid sequence identities of 56% to Escherichia coli FKBP22 [7], 43% to E. coli FkpA [8], and 41% to Legionella pneumophila MIP [9]. SIB1 FKBP22 exists as a homo- dimer and exhibits the PPIase activity like other MIP- like FKBP subfamily proteins. However, the optimum temperature of this protein for activity (10 °C) is much lower than that of E. coli FKBP22 (> 25 °C). We pro- pose that this activity facilitates efficient folding of pro- teins containing cis prolines in psychrotrophic bacteria at low temperatures. According to the crystal structures of L. pneumophila MIP [10] and E. coli FkpA [11], these proteins are com- posed of N- and C-domains, which are spanned by a 40 amino acid long a3 helix. The N-domain consists of a1 and a2 helices and an N-terminal region of a3 helix. The C-domain consists of six b-strands (b1–b6), a4 helix, and a C-terminal region of a3 helix. The N-domain is unique to the MIP-like FKBP subfamily proteins. This domain is involved in dimerization of the protein and the interface between two monomers is sta- bilized by the hydrophobic interactions of a1 and a2 helices. In contrast, the C-domain (except a3 helix) is conserved in all FKBP family proteins and contains the entire PPIase active-site, suggesting that all FKBP fam- ily proteins share a common catalytic mechanism. The a3 helix seems to be required to control the positions of the two C-domains of the homodimer, such that these domains are located with an appropriate distance and orientation. Because of the high similarity in the amino acid sequence of SIB1 FKBP22 with that of L. pneumo- phila MIP and E. coli FkpA, SIB1 FKBP22 might have a similar three-dimentional structure. The stability–activity relationships of MIP-like FKBP subfamily proteins remain to be analyzed. Because the prolyl isomerization is a spontaneous reaction and the rate for this reaction increases as the reaction tempera- ture increases, the PPIase activity cannot be accurately determined at > 30 °C. Therefore, it seems difficult to analyze the stability–activity relationships of PPIases from mesophilic and thermophilic organisms. SIB1 FKBP22 seems to be an excellent model to analyze these relationships because its optimum temperature for activ- ity is 10 °C. In addition, because this protein is expected to consist of N- and C-domains, it would be informative to construct the SIB1 FKBP22 variants containing either one of these domains and compare their activities and stabilities with those of the intact protein. In this report, the N- and C-domains of SIB1 FKBP22 were overproduced in E. coli and purified in an amount sufficient for physicochemical studies. By comparing their activities and stabilities with those of the intact protein, we showed that the unfolding tem- perature of SIB1 FKBP22 is much higher than the optimum temperature for activity. Based on these results, we discuss a role of each domain of SIB1 FKBP22 and a cold-adaptation mechanism of this protein. Results Design A model for the three-dimensional structure of the His-tagged form of SIB1 FKBP22 (SIB1 FKBP22*) was constructed based on the crystal structure of L. pneumophila MIP [10] (Fig. 1). According to this model, SIB1 FKBP22 consists of N- and C-domains. The N-domain of one molecule interacts with that of another molecule to form a homodimer. The C-domain represents a catalytic domain. Based on this model, three types of the SIB1 FKBP22 variants con- taining either one of these two domains were designed. They are N-domain + , C-domain + , and C-domain – . The primary structures of these variants are schemati- cally shown in comparison with that of the intact pro- tein in Fig. 2. Because a long a3 helix spans both the N- and C-domains, and because the region containing only a1 and a2 helices seems to be too short to fold correctly, N-domain + was designed such that it contains the entire a3 helix. Likewise, C-domain + and C-domain – were designed such that the former Fig. 1. A tertiary model of SIB1 FKBP22 homodimer. The a3 helix (Val52–Arg93), which spans both the N- and C-domains, is most deeply shaded. The N-domain without a3 helix (Met1–Ala51), which is involved in dimerization, is moderately shaded, and the C-domain without a3 helix (Asp94–Ile205), which is involved in catalytic func- tion, is most lightly shaded. Y. Suzuki et al. Stability and activity of SIB1 FKBP22 domains FEBS Journal 272 (2005) 632–642 ª 2005 FEBS 633 contains the entire a3 helix and the latter does not contain it. Overproduction and purification Upon induction for overproduction at 10 °C, N-domain + and C-domain + accumulated in the cells in a soluble form, whereas C-domain – accumulated in the cells in inclusion bodies (Fig. 3). C-domain – was solubilized in the presence of 6 m urea and refolded by removing urea with a yield of nearly 100%. All pro- teins were purified to give a single band on SDS ⁄ PAGE (data not shown). The molecular masses of N-domain + , C-domain + , and C-domain – were estimated to be 15 kDa, 26 kDa, and 17 kDa, respectively, by SDS ⁄ PAGE (Fig. 3). These values are slightly larger than the calculated ones from their amino acid sequences including a His- tag (12 042 for N-domain + , 19 149 for C-domain + , and 14 085 for C-domain – ). The molecular mass of SIB1 FKBP22* estimated by SDS ⁄ PAGE (29 kDa) has been reported to be larger than that determined by EMI-MS, which is identical to the calculated one (23 947) [6]. Slow migration in the gel may be a char- acteristic common to SIB1 FKBP22* and its variants. The molecular masses of N-domain + and C-domain + were also estimated to be 39 kDa and 23 kDa, respect- ively, by gel filtration column chromatography. The former and latter values are larger than the calculated ones by 3.2 and 1.2 times, respectively, suggesting that N-domain + exists as a trimer and C-domain + exists as a monomer. However, the molecular mass of a dimeric form of SIB1 FKBP22* estimated by gel filtra- tion column chromatography has been reported to be larger than that determined by sedimentation equilib- rium analytical ultracentrifuge by 1.5 times [6]. This discrepancy is probably caused by the unusual mole- cular shape of the protein, which is cylindrical rather ABC Fig. 3. Estimation of the amount of the proteins in soluble and insoluble forms by SDS ⁄ PAGE. N-domain + (A), C-domain – (B), and C-domain + (C) were overproduced in E. coli as described for SIB1 FKBP22* [6]. The soluble (lane S) and insoluble (lane P) fractions after sonication lysis were analyzed by 15% (for C-domain + ) and 17% (for N-domain + and C-domain – ) SDS ⁄ PAGE. The gel was stained with Coomassie Brilliant Blue. Arrows indicate the recombinant proteins overproduced in the cells. The positions of the standard proteins contained in a low mole- cular mass marker kit (Pharmacia Biotech, Piscataway, NJ, USA) are shown alongside the gels, together with their molecular masses. Fig. 2. Schematic representations of the pri- mary structures of SIB1 FKBP22* and its variants. A His-tag attached to the N-termini of the proteins is represented by shaded box. The a-helices and b-strands are repre- sented by cylinders and arrows, respect- ively. These secondary structures are arranged based on a tertiary model of SIB1 FKBP22. Numbers indicate the positions of the residues relative to the initiator methio- nine residue. The ranges of the N- and C-domains are also shown. Stability and activity of SIB1 FKBP22 domains Y. Suzuki et al. 634 FEBS Journal 272 (2005) 632–642 ª 2005 FEBS than globular. Because N-domain + is expected to assume a similar cylindrical structure, sedimentation equilibrium analytical ultracentrifugation was per- formed to determine its molecular mass in solution. The data fitted well to a single-species model with no evidence of aggregation, and the molecular mass was determined to be 23 431 Da. This value is 1.9 times larger than that calculated from the amino acid sequence, indicating that N-domain + exists as a dimer. CD spectra The far-UV CD spectra of N-domain + , C-domain + , C-domain – , and SIB1 FKBP22* were measured at 10 °C (Fig. 4A). The spectrum of N-domain + , which gave a broad trough with a double minimum at 208 and 222 nm, was similar to that of SIB1 FKBP22*, although the depth of the trough in this spectrum is larger than that in the SIB1 FKBP22* spectrum. The helical content was calculated to be 51% for N-domain + and 38% for SIB1 FKBP22* from these spectra using the method of Wu et al. [12]. These val- ues were comparable to those calculated from a ter- tiary model of SIB1 FKBP22* (60% for N-domain + and 34% for SIB1 FKBP22*), suggesting that N-domain + assumes a similar helical structure to that of the N-domain in the intact molecule. On the other hand, the CD spectra of C-domain + and C-domain – gave a broad trough with a single minimum at 207 nm and one without any clear minimum, respectively. The depths of these troughs were considerably smaller than that in the SIB1 FKBP22* spectrum. The near-UV CD spectra of these proteins were also measured at 10 °C (Fig. 4B). These spectra reveal the three-dimensional environments of aromatic residues such as Trp and Tyr. SIB1 FKBP22* contains one tryptophan residue (Trp157), which is conserved in the FKBP family proteins and required for PPIase activity, and seven tyrosine residues. Because most of these residues (one tryptophan and six tyrosine residues) are located in its C-domain, the near-UV CD spectrum of SIB1 FKBP22* may reflect the conformation of the C-domain. The spectrum of C-domain + was similar to that of SIB1 FKBP22*, suggesting that C-domain + assumes a similar structure to that of the C-domain in the intact molecule. In contrast, the spectrum of C-domain – was quite different from those of C-domain + and SIB1 FKBP22*, suggesting that the structure of C-domain – is considerably different from that of the C-domain in the intact molecule. PPIase activity When the PPIase activity was determined at 10 °C by the protease coupling assay using N-succinyl-Ala- Leu-Pro-Phe-p-nitroanilide (ALPF) as a substrate, C-domain + exhibited PPIase activity, whereas C-domain – did not. The catalytic efficiency (k cat ⁄ K m ) of C-domain + was estimated to be 1.43 lm )1 Æs )1 , which was 1.6 times higher than that of SIB1 FKBP22*. The temperature dependence of the PPIase activity of C-domain + was nearly identical to that of SIB1 FKBP22* (Fig. 5A). In contrast, when the PPI- ase activity was determined by the RNase T 1 refolding assay, C-domain + exhibited much less activity as com- pared to that of SIB1 FKBP22*. The acceleration effect of C-domain + on the RNase T 1 refolding reac- tion was not detected at 21 nm, but detected at 210 nm (Fig. 5B). The acceleration effect similar to that detec- ted in the presence of 210 nm C-domain + was detected in the presence of 19 nm SIB1 FKBP22*. The k cat ⁄ K m values were estimated to be 0.5 lm )1 Æs )1 for SIB1 FKBP22* and 0.015 lm )1 Æs )1 for C-domain + . Thermal stability Heat induced unfolding of N-domain + , C-domain + , and SIB1 FKBP22* were analyzed by differential Fig. 4. CD spectra of SIB1 FKBP22* and its variants. The far-UV (A) and near-UV (B) CD spectra of SIB1 FKBP22* (dashed line), N-domain + (heavy thick line), C-domain – (thin line), and C-domain + (moderately thick line) are shown. All spectra were measured at 10 °C as described under Experimental procedures. Y. Suzuki et al. Stability and activity of SIB1 FKBP22 domains FEBS Journal 272 (2005) 632–642 ª 2005 FEBS 635 scanning calorimetry (DSC) (Fig. 6, Table 1). All DSC curves were reproduced by repeating thermal scans, indicating that thermal unfoldings of these proteins are highly reversible. The denaturation curve of SIB1 FKBP22* clearly showed two well separated transi- tions. Deconvolution of the thermogram according to a non-two-state denaturation model gives melting tem- perature (T m ) values of 32.5 °C and 46.6 °C for these transitions. These T m values are nearly equal to those of C-domain + (35.6 °C) and N-domain + (44.7 °C), suggesting that the thermal unfolding transitions of SIB1 FKBP22* at lower and higher temperatures rep- resent those of its C-domain and N-domain, respect- ively. For unfolding of N-domain + , the van’t Hoff enthalpy (DH vH ) was roughly two times larger than the calorimetric enthalpy (DH cal ). Because N-domain + exists as a dimer, this result possibly reflects a coupling of the unfolding of N-domain + to dissociation of the homodimer. Similarly, the unfolding reaction of C-domain + seems to contain complex processes, as indicated by the DH cal ⁄DH vH ratio far from unity. Comparison of thermal stability of SIB1 FKBP22* and E. coli FKBP22* To examine whether SIB1 FKBP22* is less stable than its mesophilic counterpart, heat induced unfolding of E. coli FKBP22* was analyzed by DSC. However, thermodynamic parameters including T m could not be obtained because of the poor reversibility of this pro- tein in thermal unfolding. Therefore, thermal stabilities of SIB1 FKBP22* and E. coli FKBP22* were analyzed by circular dichroism (CD). The far-UV CD spectra of SIB1 FKBP22* and E. coli FKBP22* were measured at various temperatures and the spectra of SIB1 FKBP22* at 10 and 50 °C are shown in comparison with those of E. coli FKBP22* at 20 and 80 °Cin Fig. 7. The spectrum of SIB1 FKBP22* at 10 °Cis identical to that shown in Fig. 4A. The spectra of Table 1. Thermodynamic parameters for heat induced unfolding of SIB1 FKBP22*, C-domain + and N-domain + recorded by microcalori- metry. The melting temperature (T m ), calorimetric enthalpy (DH cal ), and van’t Hoff enthalpy (DH vH ) were obtained from the DSC curves shown in Fig. 6, using ORIGIN software (MicroCal, Inc.). Protein T m (°C) DH cal (kJÆmol )1 ) DH vH (kJÆmol )1 ) SIB1 FKBP22* 32.5 82.8 404.2 46.4 194.8 303.9 C-domain+ 35.6 171.8 232.4 N-domain + 44.7 140.9 259.2 Fig. 6. DSC curves of N-domain + , C-domain + , and SIB1 FKBP22*. The DSC curves of N-domain + (thick line), C-domain + (thin line), and SIB1 FKBP22* (dashed line), which were measured at a scan rate of 1 °CÆmin )1 , are shown. These proteins were dissolved in 20 mM sodium phosphate (pH 8.0) at 0.6 mgÆmL )1 . Fig. 5. PPIase activities of C-domain + . (A) The temperature dependence of the PPIase activity of C-domain + (–d–), which was determined by protease coupling assay using ALPF as a substrate, is shown in comparison with that of SIB1 FKBP22* (–s–). The catalytic efficiency, k cat ⁄ K m , was calculated according to Harrison & Stein [34]. The experiment was carried out in duplicate. Each plot represents the average value and errors from the average values are shown. (B) The increase in tryptophan fluorescence at 323 nm during refolding of RNase T 1 (0.2 lM) is shown as a function of the refolding time. Refolding reaction was carried out at 10 °C in the absence (dotted line), or presence of 21 n M of C-domain + (thick solid line), 210 nM of C-domain + (thin solid line) or 19 nM of SIB1 FKBP22* (dashed line). Stability and activity of SIB1 FKBP22 domains Y. Suzuki et al. 636 FEBS Journal 272 (2005) 632–642 ª 2005 FEBS SIB1 FKBP22* at 10 °C and E. coli FKBP22* at 20 °C, which represent the spectra of these proteins in a native form, were similar to each other, suggesting that the tertiary structures of these proteins are similar to each other. With a temperature shift from 10 to 50 °C, the spectrum of SIB1 FKBP22*, which gave a broad trough with double minimum [h] values of )11 200 at 209 nm and )12 100 at 222 nm, was greatly changed so that it exhibits a trough with a minimum [h] value of )7800 at 207 nm, which is accompanied by a shoulder with a [h] value of )5700 at 220 nm. A similar spectral change was observed for E. coli FKBP22* when the temperature was shifted from 20 to 80 °C. The spectra of SIB1 FKBP22* at 50 °C and E. coli FKBP22* at 80 °C were not seri- ously changed at higher temperatures, indicating that these spectra represent the spectra of these proteins in a denatured form. In these conditions, SIB1 FKBP22* was fully reversible in thermal denaturation, whereas E. coli FKBP22* was not. The reversibility of E. coli FKBP22* was roughly 70%. The thermal denaturation curves of SIB1 FKBP22* and E. coli FKBP22* were measured by monitoring a change in the CD values at 222 nm (Fig. 8). SIB1 FKBP22* apparently unfolded through an intermedi- ate state. The T m values for the first and second transi- tions were roughly estimated to be 32 and 44 °C, respectively, which were comparable with those determined by DSC. As compared to SIB1 FKBP22*, E. coli FKBP22* unfolded at higher temperatures, indicating that it is more stable than SIB1 FKBP22*. However, it is unclear whether this protein unfolds through an intermediate state as well, because this intermediate state was not clearly detected. The ther- mal unfolding curve of this protein did not fit the the- oretical curve, which was drawn on the assumption that the protein unfolds in a single cooperative fashion (data not shown). Discussion Unfolding of SIB1 FKBP22* In this study, SIB1 FKBP22* was shown to unfold in a complex non-two-state mechanism with two peaks apparent in the DSC curve. Construction of the N-domain + and C-domain + , which lack the C- and N-domains, respectively, followed by DSC analyses, clearly showed that two peaks of heat capacity observed in thermal unfolding of SIB1 FKBP22* rep- resent unfoldings of its N- and C-domains. In this thermal unfolding process, the C- and N-domains unfold at lower and higher temperatures, respectively. It has been reported that a phosphoglycerate kinase [13] and a chitobiase [14] from psychrophilic bacteria consist of a heat labile domain and a heat stable domain. Bentahir et al. [13] have proposed that a heat labile domain provides a sufficient flexibility around the active site, and a heat stable domain provides a sufficient rigidity to the substrate-binding site, so that Fig. 8. Thermal denaturation curves of SIB1 FKBP22* and E. coli FKBP22*. The [h] values of SIB1 FKBP22* (trace 1) and E. coli FKBP22* (trace 2) at 222 nm are shown as a function of tempera- ture. The proteins were dissolved in 20 m M sodium phosphate (pH 8.0) at 0.30 mgÆmL )1 for SIB1 FKBP22* and 0.29 mgÆmL )1 for E. coli FKBP22*. A cell with an optical path length of 2 mm was used. Temperature was linearly raised at 1 °CÆmin )1 . Fig. 7. Far-UV CD spectra of SIB1 FKBP22* and E. coli FKBP22*. The CD spectra of SIB1 FKBP22* measured at 10 °C (thick line) and 50 °C (thick dashed line), and those of E. coli FKBP22* measured at 20 °C (thin line) and 80 °C (thin dashed line) are shown. The spectra were measured as described under Experimental procedures. Y. Suzuki et al. Stability and activity of SIB1 FKBP22 domains FEBS Journal 272 (2005) 632–642 ª 2005 FEBS 637 the enzymatic reaction is efficiently achieved at low temperatures. Because the C-terminal catalytic domain of SIB1 FKBP22 represents a heat labile domain, the instability of this domain may be required to increase the flexibility of the active-site at low temperatures. Stability and activity of SIB1 FKBP22* SIB1 FKBP22* was shown to be much less stable than E. coli FKBP22*. Its optimal temperature for activity has been reported to be greatly shifted downward as compared to that of E. coli FKBP22* [6]. Cold-adap- tation has been specified by the increase in the cata- lytic efficiency at low temperatures, the downward shift in the optimum temperatures for activity, and the reduction in the conformational stability [15]. There- fore, SIB1 FKBP22 can be defined as a cold-adapted enzyme, although it is less active than E. coli FKBP22* even at low temperatures [6]. Several cold- adapted enzymes have also been reported to be less active than their mesophilic counterparts [16–19]. Analyses of the thermal stability of SIB1 FKBP22* by DSC (Fig. 3) and CD (Fig. 8) indicate that unfold- ing of this protein is initiated at > 25 °C. In fact, the CD spectrum of SIB1 FKBP22* at 20 °C was nearly identical to that at 10 °C (data not shown), suggesting that the conformation of this protein is not seriously changed upon temperature shift from 10 to 20 °C. Thermal unfolding of C-domain + is also initiated at > 25 °C. Nevertheless, SIB1 FKBP22* and C-domain + both exhibit the maximal PPIase activity at 10 °C and their activities are greatly reduced at 20 °C. These results suggest that a subtle conforma- tional change around the active-site causes a great reduction of the enzymatic activity. The large differ- ence in the temperatures for enzymatic inactivation and structural unfolding has been observed for cold-adapted a-amylase and family 8 xylanase from an Antarctic bacterium [20,21]. The apparent optimal temperatures of these proteins for enzymatic activities are much lower than the temperatures at which any significant conformational event occurs. In contrast, the optimal temperatures for the activities of their mesophilic and thermophilic counterparts closely cor- relate with the temperatures for their structural transi- tions. Thus, the large difference in the temperatures for enzymatic inactivation and structural unfolding seems to be a characteristic feature of cold-adapted enzymes. It has been proposed that this difference is caused by a cold-adaptation strategy termed ‘localized flexibility’ [20]. Although an increase in flexibility around the active site increases k cat by reducing the energy cost of conformational change during the cata- lytic reaction, it should increase K m concomitantly. By restricting the increase of flexibility within small areas, cold-adapted enzymes prevent unfavorable increases in K m [22]. SIB1 FKBP22 probably adopts a similar strategy for cold-adaptation. Structural importance of a3 helix Two types of the SIB1 FKBP22* variants, which con- tain the C-domain, were designed based on its tertiary model. C-domain + contains an entire a3 helix, whereas C-domain – does not contain it. These two proteins differ greatly in their biochemical properties. C-domain + was overproduced in E. coli in a soluble form and exhibited the PPIase activity. Its near-UV CD spectrum was similar to that of SIB1 FKBP22*. In contrast, C-domain – was overproduced in E. coli in inclusion bodies and exhibited little PPIase activity. Its near-UV CD spectrum was quite different from that of SIB1 FKBP22*. These results strongly suggest that a3 helix is required to facilitate folding of the C-domain, or to stabilize it, so that the C-domain assumes a native conformation. It has previously been reported that limited proteolysis of L. pneumophila MIP allows the separation of their N- and C-domains such that the C-domain contains the C-terminal half of the a3 helix [23,24]. In addition, the C-domain of E. coli FkpA shows a high tendency to form inclusion bodies when it is overproduced in E. coli in a form without a3 helix [25]. These results are consistent with our results. According to the crystal structure of L. pneu- mophila MIP, there are three distinct contacts between the C-terminal region of a3 helix and the C-domain [10]. These contacts may also be conserved in the structure of SIB1 FKBP22. Role of N- and C-domains Most organisms contain multiple PPIases within a sin- gle cell. They are usually composed of several domains; one is common to the members of each family and specifies the family to which that PPIase belongs, and the others are unique to the particular PPIase and thought to be related to the protein’s distinct function. The C- and N-domains of MIP-like FKBP subfamily proteins represent the former and latter domains, respectively. Therefore, biochemical charac- terizations of N-domain + and C-domain + will facili- tate understanding of the roles of these domains in the intact molecule. The observation that N-domain + exists as a dimer, whereas C-domain + exists as a monomer supports a tertiary model of SIB1 FKBP22, in which the a1 and a2 Stability and activity of SIB1 FKBP22 domains Y. Suzuki et al. 638 FEBS Journal 272 (2005) 632–642 ª 2005 FEBS helices form the dimerization core of the protein. In addition, we showed that the PPIase activity of C-domain + determined by the RNase T 1 refolding assay was greatly reduced as compared to that of the intact protein. These results suggest that a dimeric structure of SIB1 FKBP22 is responsible for its high PPIase activity for protein substrates. Alternatively, N-domain contains a binding site for protein substrates. Similar results have been reported for other MIP-like FKBP subfamily pro- teins. For example, The C-domain of L. pneumophila MIP produced upon limited proteolysis has been repor- ted to exist as a monomer and exhibit weak PPIase activity for protein substrate [23]. Likewise, the C-domain of E. coli FkpA is devoid of chaperone-like function, although it shows PPIase activity [11,25]. Fur- thermore, it has been reported that human FKBP12 which intrinsically consists of a single domain, exhibited lower activity for RNase T 1 substrate and higher activity for tetrapeptide substrates than E. coli FkpA [25]. How- ever, the reason why C-domain alone exhibits a weak activity for protein substrates remains to be clarified. Further structural and functional studies of these pro- teins will be required to clarify this reason. Experimental procedures Cells and plasmids Psychrotrophic bacterium Shewanella sp. SIB1 was isolated from water deposits in a Japanese oil reservoir [26]. E. coli JM109 [recA1 , supE44, endA1, hsdR17, gyrA96, relA1, thi, D(lac-proAB) ⁄ F¢, traD36, proAB + , lacI q lacZDM15] was obtained from Toyobo Co., Ltd. (Kyoto, Japan). E. coli BL21(DE3) [F – , ompT, hsdS B (r B – ,m B – ), gal, dcm (DE3)] and plasmid pET-28a were obtained from Novagen (Madi- son, WI, USA). Plasmid pUC18 was obtained from Takara Shuzo Co., Ltd. (Kyoto, Japan). The E. coli transformants were grown in Luria–Bertani medium containing 50 mgÆL )1 ampicillin or 35 mgÆL )1 kanamycin. Plasmid construction Plasmid pSIB1-Nd, pSIB1-Cd, and pSIB1-a3+Cd for over- production of a His-tagged form of the N-domain of SIB1 FKBP22 with entire a3 helix (N-domain + ), C-domain with- out a3 helix (C-domain – ), and C-domain with entire a3 helix (C-domain + ), respectively, were constructed by ligating a part of the SIB1 FKBP22 gene amplified by PCR into pET- 28a as follows. Genomic DNA was prepared from a Sarkosyl lysate of the Shewanella sp. SIB1 cells [27] and used as a tem- plate. The gene encoding Met1–Asp94 of SIB1 FKBP22 was amplified by PCR and ligated into the NdeI–SacI sites of pET-28a to produce plasmid pSIB1-Nd. Likewise, the genes encoding Gly95–Ile205 and Gly47–Ile205 of SIB1 FKBP22 were amplified by PCR and ligated into pET-28a to produce plasmids pSIB1-Cd and pSIB1- a3+Cd, respectively. The sequences of the 5¢ PCR primers were 5¢-AGAGAGAA TT CATATGTCAGATTTGTTCAG-3¢ for N-domain + ,5¢- CTGAAAACGCTAAG CATATGGGTATTACGA-3¢ for C-domain – , and 5¢-CTTGCTGATGCACATATGGGGAA AGAAAGC-3¢ for C-domain + , where underlined bases show the position of the NdeI site. The sequences of the 3¢ PCR primers were 5¢-GACTCT GAGCTCGTAATCTAGT CACGCTTA-3¢ for N-domain + , where underlined bases show the position of the SacI site, and 5¢- GGCCACT GGATCCAACTACAGCAATTCTCA-3¢ for C-domain – and C-domain + , where underlined bases show the position of the BamHI site. PCR was performed with GeneAmp PCR system 2400 (PerkinElmer, Tokyo, Japan) using KOD polymerase (Toyobo Co., Ltd) according to the procedures recommended by the supplier. Overproduction and purification His-tagged forms of SIB1 FKBP22 (SIB1 FKBP22*) and E. coli FKBP22 (E. coli FKBP22*) were overproduced and purified as described previously [6]. N-domain + , C-domain – , and C-domain + were overproduced in the E. coli BL21(DE3) cells transformed with plasmids pSIB1- Nd, pSIB1-Cd, and pSIB1-a3+Cd, respectively, and puri- fied, as described for SIB1 FKBP22* [6], except for the purification of C-domain – . For purification of C-domain – , which was overproduced in inclusion bodies, the cells were disrupted by sonication and centrifuged at 15 000 g for 30 min at 4 °C. The pellet was dissolved in 20 mm sodium phosphate (pH 8.0) containing 6 m urea and 0.5% (w ⁄ v) Triton X-100, and incubated overnight at 4 °C. After cen- trifugation at 15 000 g for 30 min at 4 °C to remove insol- uble materials, the protein was refolded by dialysis against 20 mm sodium phosphate (pH 8.0), and purified as des- cribed for SIB1 FKBP22* using metal chelating affinity chromatography and gel filtration chromatography [6]. Production of the recombinant proteins in the E. coli cells, as well as their purities, were analyzed by SDS ⁄ PAGE [28] on a 15 or 17% polyacrylamide gel, followed by stain- ing with Coomassie Brilliant Blue. Protein concentration Protein concentrations were determined from the UV absorption on the basis that the absorbance at 280 nm of a 0.1% solution is 0.68 for SIB1 FKBP22*, 0.12 for N-domain + , 1.01 for C-domain – , 0.75 for C-domain + and 0.69 for E. coli FKBP22*. These values were calculated by using  ¼ 1576 m )1 Æcm )1 for Tyr and 5225 m )1 Æcm )1 for Trp at 280 nm [29]. For N-domain + , which contains only one tyrosine residue and no tryptophan residues, a method Y. Suzuki et al. Stability and activity of SIB1 FKBP22 domains FEBS Journal 272 (2005) 632–642 ª 2005 FEBS 639 of Scopes [30] was used to confirm the accuracy of its concentration. In this method, the protein concentration (mgÆmL )1 ) is calculated from A 205nm ⁄ (31 · b), where A 205nm represents absorbance at 205 nm and b represents an optical path length (cm). Molecular mass The molecular masses of purified proteins were estimated by gel filtration column chromatography using a Superdex 200 16 ⁄ 60 gel filtration column (Amersham Biosciences, Piscat- away, NJ, USA) equilibrated with 50 mm Tris ⁄ HCl (pH 8.0) containing 50 mm NaCl. Elution was performed at a flow rate of 0.5 mLÆmin )1 . Bovine serum albumin (67 kDa), ovalbumin (44 kDa), chymotrypsinogen A (25 kDa), and RNase A (14 kDa) were used as standard proteins. The molecular mass of N-domain + in solution was deter- mined by sedimentation equilibrium analytical ultracentri- fugation. Sedimentation equilibrium experiments were performed at 10 °C for 20 h with a Beckman Optima XL-A Analytical Ultracentrifugate using an An-60 Ti rotor at a speed of 28 000 r.p.m. Before measurements, the protein solutions were dialyzed overnight against 20 mm sodium phosphate (pH 8.0) at 4 °C. The initial loading concentra- tion of the protein was 1.8 mgÆmL )1 . The protein concen- tration distribution within the cell was monitored by the absorbance at 280 nm. Analysis of the sedimentation equili- bria was performed using the program xlavel (Beckman, Tokyo, Japan, version 2). Enzymatic activity The PPIase activity was determined by protease-coupling assay [31,32] and RNase T 1 refolding assay [33]. For the protease-coupling assay, chymotrypsin was used as the protease and N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (ALPF; Wako Chemicals, Osaka, Japan) was used as the substrate. The reaction mixture (2.1 mL) contained 35 mm Hepes buffer (pH 7.8), 25 lm tetrapeptide substrate, and the appropriate amount of the enzyme. The reaction mix- ture was incubated at reaction temperature (4, 10, 15 or 25 °C) for 3 min prior to the addition of chymotrypsin. The reaction was initiated by the addition of 30 lLof 0.76 mm chymotrypsin. The isomerization reaction cata- lyzed by PPIases was measured by monitoring the change in the concentration of p-nitroaniline, because p-nitroaniline is not released from the substrate when the peptide bond N-terminal of the proline residue is in the cis conformation. The concentration of p-nitroaniline was determined from the absorption at 390 nm with the molar absorption coeffi- cient value of 8900 m )1 Æcm )1 using a Hitachi U-2010 UV ⁄ VIS spectrophotometer (Hitachi Instruments, Tokyo, Japan). The catalytic efficiency (k cat ⁄ K m ) was calculated from the relationship k cat ⁄ K m ¼ (k p – k n ) ⁄ E, where E repre- sents the concentration of the enzyme, and k p and k n represent the first-order rate constants for the release of p-nitroaniline from the substrate in the presence and absence of the enzyme, respectively [34]. For the RNase T 1 refolding assay, RNase T 1 was first unfolded by incubating the solution containing 50 mm Tris ⁄ HCl (pH 8.0), 1 mm EDTA, 5.6 m guanidine hydro- chloride, and 16 lm RNase T 1 (Funakoshi, Tokyo, Japan) at 10 °C overnight. Refolding was then initiated by diluting this solution 80-fold with 50 mm Tris ⁄ HCl (pH 8.0) containing SIB1 FKBP22* or C-domian + . The final concentrations of RNase T 1 , SIB1 FKBP22*, and C-domian + were 0.2 lm, 19 nm, and 21 or 210 nm, respectively. The refolding reaction was monitored by measuring the increase in tryptophan fluorescence with an F-2000 spectrofluorometer (Hitachi Instruments). The excitation and emission wavelengths were 295 and 323 nm, respectively, and the band width was 10 nm. The refolding curves were analyzed with double expo- nential fit [35]. The k cat ⁄ K m values were calculated from the relationship described above, where k p and k n represent the first-order rate constants for the faster refolding phase of RNase T 1 in the presence and absence of the enzyme, respectively. Circular dichroism The CD spectra were recorded on a J-725 automatic spec- tropolarimeter from Japan Spectroscopic Co., Ltd. (Tokyo, Japan). The proteins were dissolved in 20 mm sodium phos- phate (pH 8.0) and incubated for 30 min at the temperatures indicated prior to the CD measurement. For measurement of the far-UV CD spectra (200–260 nm), the protein concen- tration was approximately 0.2 mgÆmL )1 and a cell with an optical path length of 2 mm was used. For measurement of the near-UV CD spectra (240–320 nm), the protein concen- tration was 0.4–1.0 mgÆmL )1 and a cell with an optical path length of 10 mm was used. The mean residue ellipticity, h, which has units of degÆcm 2 Ædmol )1 , was calculated by using an average amino acid molecular mass of 110. Differential scanning calorimetry DSC measurements were carried out on a high-sensitivity VP-DSC controlled by the vpviewer TM software package (Microcal, Inc., Northampton, MA, USA) at a scan rate of 1 °CÆmin )1 . Prior to the measurements, samples were fil- tered through 0.22 lm pore size membranes and then de- gassed in a vacuum. The protein concentrations during the measurements were  0.5 mgÆmL )1 . The reversibility of thermal denaturation was verified by reheating the samples. Homology modeling A model for dimeric structure of SIB1 FKBP22 was built by SWISS-MODEL (Swiss Institute of Bioinfomatics) Stability and activity of SIB1 FKBP22 domains Y. 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The sequences of the 5¢ PCR primers were 5¢-AGAGAGAA TT CATATGTCAGATTTGTTCAG-3¢ for N-domain + ,5¢- CTGAAAACGCTAAG CATATGGGTATTACGA-3¢ for C-domain – , and 5¢-CTTGCTGATGCACATATGGGGAA AGAAAGC-3¢