Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analyses Muhammad S Rohman1, Takashi Tadokoro1, Clement Angkawidjaja1, Yumi Abe1, Hiroyoshi Matsumura2,3, Yuichi Koga1, Kazufumi Takano1,3 and Shigenori Kanaya1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan CREST, JST, Osaka, Japan Keywords crystal structure; destabilization mechanism; RNase HI; Shewanella oneidensis MR-1; thermostabilizing mutations Correspondence S Kanaya, Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan Fax: +81 6879 7938 Tel: +81 6879 7938 E-mail: kanaya@mls.eng.osaka-u.ac.jp (Received 26 September 2008, revised 11 November 2008, accepted 19 November 2008) doi:10.1111/j.1742-4658.2008.06811.x The Arg97 fi Gly and Asp136 fi His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 °C, respectively, in Tm, and 3.5 and 6.1 kJỈmol)1, respectively, in DG(H2O) These mutations also stabilized the So-RNase HI derivative (4·-RNase HI) with quadruple thermostabilizing mutations in an additive manner As a result, the resultant sextuple mutant protein (6·-RNase HI) was more stable than the wild-type protein by 28.8 °C in Tm and 27.0 kJỈmol)1 in DG(H2O) To analyse the effects of the mutations on the protein structure, the crystal structure of the 6·-RNase HI protein was deter˚ mined at 2.5 A resolution The main chain fold and interactions of the side-chains of the 6·-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6·-RNase HI protein We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability Psychrophiles and psychrotrophs are defined as microorganisms that can grow even at around °C [1] Enzymes from these microorganisms are usually less stable than those from mesophiles and thermophiles [2–4] It has been reported that a decreased number of ion pairs and hydrogen bonds, decreased hydrophobic interactions and packing at the core, an increased fraction of nonpolar surface area, a decreased surface hydrophilicity, decreased helix stability and a decreased number of proline residues in the loop regions are responsible for their thermolability [5–8] However, the destabilization mechanism of these enzymes remains to be fully understood One promising strategy to understand this mechanism is to Abbreviations 4·-RNase HI, So-RNase HI derivative with Asn29 fi Lys, Asp39 fi Gly, Met76 fi Val and Lys90 fi Asn mutations; 5·-RNase HI, 4·-RNase HI derivative with additional Arg97 fi Gly mutation; 6·-RNase HI, 5·-RNase HI derivative with additional Asp136 fi His mutation; D136HRNase HI, So-RNase HI derivative with Asp136 fi His mutation; Ec-RNase HI, E coli RNase HI; GdnHCl, guanidine hydrochloride; PDB, Protein Data Bank; R97G-RNase HI, So-RNase HI derivative with Arg97 fi Gly mutation; So-RNase HI, RNase HI from Shewanella oneidensis MR-1; Tt-RNase HI, RNase HI from Thermus thermophilus FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 603 Destabilization mechanism of psychrotrophic RNase HI M S Rohman et al construct the thermostabilized mutants of a given psychrophilic or psychrotrophic enzyme and analyse their stabilization mechanisms RNase H (EC 3.1.26.4) is an enzyme that specifically cleaves the RNA strand of RNA ⁄ DNA hybrids [9] The enzyme is widely present in bacteria, archaea, eukaryotes and retroviruses [10] RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is a monomeric protein with 158 amino acid residues [11] It shows amino acid sequence identity of 67% with its mesophilic counterpart Escherichia coli RNase HI (Ec-RNase HI), for which structure– stability–function relationships have been extensively studied [12] The crystal structure of So-RNase HI has been determined [11] This structure strongly resembles that of Ec-RNase HI Nevertheless, So-RNase HI is less stable than Ec-RNase HI by 22.4 °C in Tm and 12.5 kJỈmol)1 in DG(H2O) [11] We used So-RNase HI as a model protein to analyse the destabilization mechanism of a psychrotrophic protein We have recently shown that four single mutations identified by directed evolution stabilize So-RNase HI by 3.6–6.7 °C in Tm and 1.7–5.2 kJỈmol)1 in DG(H2O) [13] They include Asn29 fi Lys, Asp39 fi Gly, Met76 fi Val and Lys90 fi Asn The effects of these mutations are roughly additive, and a combination of these mutations strikingly increases the stability of So-RNase HI to a level similar to that of Ec-RNase HI These results suggest that Asn29, Asp39, Met76 and Lys90 are not optimal for the stability of So-RNase HI and their replacement with other residues increases stability However, the stabilization mechanisms of the protein with these mutations remain to be understood In addition, it remains to be determined whether the four residues mentioned above are the only ones that are not optimal for the stability of So-RNase HI It has been reported that Ec-RNase HI is stabilized by the Lys95 fi Gly [14] or Asp134 fi His [15] mutation by approximately °C in Tm at pH 5.5 Because Lys95 and Asp134 are conserved as Arg97 and Asp136, respectively, in So-RNase HI, and the structures around these residues are conserved in So-RNase HI [9], the Arg97 fi Gly and Asp136 fi His mutations are also expected to increase the stability of So-RNase HI However, these mutations have not been identified by directed evolution Therefore, it would be informative to examine whether these mutations increase the stability of So-RNase HI and its derivative (4·-RNase HI) with quadruple thermostabilizing mutations identified by directed evolution In this report, we show that the Arg97 fi Gly and Asp136 fi His mutations increase the stability of So-RNase HI and 4·-RNase HI We determined the 604 crystal structure of the sextuple mutant protein of So-RNase HI (6·-RNase HI), in which the Arg97 fi Gly and Asp136 fi His mutations were combined with the four thermostabilizing mutations identified by directed evolution Based on this structure, which is basically identical to that of the wild-type protein, except for the mutation sites, we discuss the destabilization mechanism of So-RNase HI Results Stabilization of So-RNase HI with Arg97 fi Gly and Asp136 fi His mutations To examine whether the single Arg97 fi Gly and Asp136 fi His mutations stabilize So-RNase HI, two mutant proteins, R97G-RNase HI and D136H-RNase HI, were constructed These mutant proteins were overproduced in E coli in a soluble form and purified to give a single band on SDS-PAGE (data not shown) The far-UV CD spectra of these mutant proteins were similar to that of the wild-type protein (data not shown), suggesting that these mutations not seriously affect the conformation of the protein The specific activities of R97G-RNase HI and D136HRNase HI were 99% and 65%, respectively, of that of the wild-type protein (Table 1) The stabilities of R97G-RNase HI and D136HRNase HI against thermal denaturation were analysed at pH 5.5 in the presence of m guanidine hydrochloride (GdnHCl) by monitoring the change in the CD values at 220 nm Thermal denaturation of these mutant proteins was fully reversible in this condition The thermodynamic parameters characterizing the thermal denaturation curves of the wild-type and mutant proteins are summarized in Table The temperature of the midpoint of the transition, Tm, was 30.4 °C for the wild-type protein, 35.8 °C for R97GRNase HI and 40.1 °C for D136H-RNase HI Thus, R97G-RNase HI is more stable than the wild-type protein by 5.4 °C in Tm and 3.9 kJỈmol)1 in DDGm D136H-RNase HI is more stable than the wild-type protein by 9.7 °C in Tm and 7.0 kJỈmol)1 in DDGm The stabilities of the mutant proteins against urea-induced denaturation were also analysed by monitoring the change in the CD values at 220 nm Urea-induced denaturation of these proteins was fully reversible in this condition and showed a two-state transition The thermodynamic parameters characterizing the urea-induced denaturation curves of the wildtype and mutant proteins are summarized in Table The apparent free energy changes of unfolding in the absence of denaturant, DG(H2O), and the urea concen- FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS M S Rohman et al Destabilization mechanism of psychrotrophic RNase HI Table Activities and thermostabilities of So-RNase HI and its derivatives Protein Specific activitya (unitsỈmg)1) Relative activitya (%) Tm b (°C) DTmb (°C) DDGmb (kJỈmol)1) DHmb (kJỈmol)1) So-RNase HI R97G-RNase HI D136H-RNase HI 4·-RNase HI 5·-RNase HI 6·-RNase HI Ec-RNase HIc 7.8 7.7 5.1 5.5 5.1 3.4 9.1 100 99 65 70 65 43 120 30.4 35.8 40.1 49.1 52.5 59.2 52.8 – 5.4 9.7 18.7 22.1 28.8 22.4 – 3.9 7.0 13.5 15.9 20.7 – 217 264 267 359 366 433 325 a The enzymatic activity was determined at 30 °C using M13 DNA ⁄ RNA hybrid as a substrate, as described in Experimental procedures Each experiment was carried out at least twice and the average value is shown Errors are within 15% of the values reported b Parameters characterizing the thermal denaturation of So-RNase HI and its derivatives The thermal denaturation curves of these proteins were measured at pH 5.5 in the presence of M GdnHCl The thermal denaturation of these proteins was reversible in this condition The melting temperature (Tm) is the temperature of the midpoint of the thermal denaturation transition The difference in the melting temperature between the wild-type and mutant proteins (DTm) was calculated as Tm(mutant))Tm(wild-type) DHm is the enthalpy change of unfolding at Tm calculated by van’t Hoff analysis The difference between the free energy change of unfolding of the mutant protein and that of the wildtype protein at Tm (DDGm) was estimated by the equation, DDGm = DTmDSm(wild-type), where DSm(wild-type) is the entropy change of the wild-type protein at Tm [44] The DSm(wild-type) value of 0.72 kJỈmol)1, which has been determined previously [11], was used to calculate the DDGm values Each experiment was carried out at least twice and the average value is shown Errors are within ± 0.3 °C for Tm, ± 26 kJỈmol)1 for DHm, ± 0.12 kJỈmol)1ỈK)1 for DSm and ± 0.3 kJỈmol)1 for DDGm c Data from Tadokoro et al [11] Table Parameters characterizing the urea-induced denaturation of So-RNase HI and its derivativesa Protein Cm a (M) Ma (kJỈmol)1ỈM)1) DG (H2O)a (kJỈmol)1) DDG (H2O)a (kJỈmol)1) So-RNase HI R97G-RNase HI D136H-RNase HI 4·-RNase HI 5·-RNase HI 6·-RNase HI Ec-RNase HIb 2.6 3.0 3.3 4.0 4.9 5.7 4.3 8.5 8.9 9.0 9.3 8.5 8.1 8.2 22.3 26.3 30.1 37.3 41.3 45.9 34.8 – 3.5 6.1 12.2 20.0 27.0 12.5 a The urea-induced denaturation curves of these proteins were measured at pH 5.5 and 20 °C Urea-induced denaturation of these proteins was reversible in this condition The urea concentration of the midpoint of the urea-induced denaturation curve (Cm), the measurement of the dependence of DG on the urea concentration (m), and the free energy change of unfolding in H2O [DG(H2O)] were calculated from the urea-induced denaturation curves The difference in DG(H2O) [DDG(H2O)] between the wild-type and mutant proteins was calculated using the equation: DDG(H2O) = mavDCm, where mav represents the average m value (8.7 kJỈmol)1ỈM)1) and DCm = Cm(mutant))Cm(wild-type) Each experiment was carried out at least twice and the average value is shown Errors are within ± 0.1 M for Cm, ± 0.8 kJỈmol)1ỈM)1 for m and ± 1.0 kJỈmol)1 for DG(H2O) bData from Tadokoro et al [11] trations of the midpoints of the denaturation curves, Cm, of the mutant proteins were higher than those of the wild-type protein by 3.5 kJỈmol)1 and 0.4 m, respectively, for R97G-RNase HI, and 6.1 kJỈmol)1 and 0.7 m, respectively, for D136H-RNase HI Thus, the stabilities of the mutant proteins against ureainduced denaturation show good agreement with those against thermal denaturation Stabilization of 4·-RNase HI with Arg97 fi Gly and Asp136 fi His mutations To examine whether the Arg97 fi Gly and Asp136 fi His mutations stabilize the quadruple mutant protein of So-RNase HI (4·-RNase HI), in which the four thermostabilizing mutations identified by directed evolution are combined, the quintuple (5·-RNase HI) and sextuple (6·-RNase HI) mutant proteins of So-RNase HI were constructed The 5·-RNase HI and 6·-RNase HI proteins represent the 4·-RNase HI derivatives with additional Arg97 fi Gly mutation and additional Arg97 fi Gly and Asp136 fi His mutations, respectively These mutant proteins were overproduced in E coli and purified to give a single band on SDSPAGE like the wild-type protein (data not shown) The far-UV CD spectra of these mutant proteins were similar to that of the wild-type protein (data not shown), suggesting that the quintuple and sextuple mutations not seriously affect the protein conformation The specific activities of the 5·-RNase HI and 6·-RNase HI proteins were 65% and 43%, respectively, of that of the wild-type protein, and 93% and 66%, respectively, of that of the 4·-RNase HI protein (Table 1) These results suggest that the effects of the Arg97 fi Gly and Asp136 fi His mutations on the enzymatic activity of the protein are not seriously FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 605 Destabilization mechanism of psychrotrophic RNase HI M S Rohman et al changed regardless of whether they are introduced into So-RNase HI or 4·-RNase HI The stabilities of the 4·-RNase HI, 5·-RNase HI and 6·-RNase HI proteins against thermal denaturation were analysed as described for R97G-RNase HI and D136H-RNase HI Thermal denaturation of these proteins was fully reversible in this condition The thermodynamic parameters characterizing the thermal denaturation curves of these proteins are summarized in Table The temperature of the midpoint of the transition, Tm, was 49.1 °C for 4·-RNase HI, 52.5 °C for 5·-RNase HI and 59.2 °C for 6·-RNase HI Thus, the 5·-RNase HI protein is more stable than the wildtype and 4·-RNase HI proteins by 22.1 and 3.4 °C, respectively, in Tm, and 15.9 and 2.4 kJỈmol)1, respectively, in DDGm The 6·-RNase HI protein is more stable than the wild-type, 4·-RNase HI and 5·-RNase HI proteins by 28.8, 10.1 and 6.7 °C, respectively, in Tm, and 20.7, 7.2 and 4.8 kJỈmol)1, respectively, in DDGm The stabilities of the 4·-RNase HI, 5·-RNase HI and 6·-RNase HI proteins against urea-induced denaturation were also analysed by monitoring the change in the CD values at 220 nm Urea-induced denaturation of these proteins was fully reversible and showed a two-state transition The thermodynamic parameters characterizing the urea-induced denaturation curves of the wild-type and mutant proteins are summarized in Table The DG(H2O) and Cm values of the 5·-RNase HI protein were higher than those of the wild-type and 4·-RNase HI proteins by 20.0 and 7.8 kJỈmol)1 and 2.3 and 0.9 m, respectively The DG(H2O) and Cm values of the 6·-RNase HI protein were higher than those of the wild-type, 4·-RNase HI and 5·-RNase HI proteins by 27.0, 14.8 and 7.0 kJỈmol)1 and 3.1, 1.7 and 0.8 m, respectively Thus, the stabilities of the 5·-RNase HI and 6·-RNase HI proteins against ureainduced denaturation show good agreement with those against thermal denaturation, although the DDG(H2O) and DDGm values are significantly different from each other for these proteins Overall structure of 6·-RNase HI The crystal structure of the 6·-RNase HI protein with the sextuple thermostabilizing mutations was deter˚ mined at 2.5 A resolution The asymmetric unit of the crystal structure consists of four protein molecules (A–D) The structures of these four protein molecules are virtually identical with one another with rmsd val˚ ˚ ues of 0.73 A between molecules D and A, 0.59 A ˚ between molebetween molecules D and B, and 0.61 A cules D and C for 148 Ca atoms In the structures of these protein molecules, however, three N-terminal (Met1–Glu3) and four C-terminal (Gln155–Ser158) residues are disordered In the structures of molecules A and B, a part of the loop between the bE strand and aV helix (Ala127–His129) is also disordered We used the structure of molecule D in this study The overall structure of 6·-RNase HI is essentially the same as that of the wild-type protein (Fig 1A) The rmsd value between the wild-type and 6·-RNase ˚ HI proteins is 0.85 A for 148 Ca atoms The shifts of the Ca coordinates of 6·-RNase HI relative to those of the wild-type protein are shown in Fig The differences between the Ca coordinates of molecules C and D are also shown in this figure as a reference Relatively large shifts were observed around Gly17 and Asn18 in a turn between the bA and bC strands, around Ser95 in a loop between the aIII and aIV Fig Stereoview of the three-dimensional structure of 6·-RNase HI The structure of molecule D of 6·-RNase HI (gold) is superimposed on the structure of the wild-type protein (green) The entire structure (A), and the structures around residue 29 (B), residue 39 (C), residue 76 (D), residues 90 and 97 (E) and residue 136 (F) are shown The side-chains of the amino acid residues are shown as stick models, in which the oxygen, nitrogen and sulfur atoms are coloured red, blue and yellow, respectively The PDB code for the wild-type protein is 2E4L For the entire structure (A), N and C represent the N- and C-termini of the protein, and a and b represent the a helix and b strand, respectively The side-chains of the mutated and parent amino acid residues at the six mutation sites are shown D39 ⁄ G39 and M76 ⁄ V76 are simply labelled as 39 and 76, respectively The side-chains of the five active site residues, D12, E50, D72, H126 and D136, are also shown For the structure around residue 29 (B), the side-chains of residues 29, T34 and E131 are shown The hydrogen bonds between N29 and Oc and T34 and Oc, and between N29 and Nd and E131 and Oe2, in the wild-type protein are shown as green broken lines, and the ion pair between the e-amino group of K29 and the carboxyl group of E131 in 6·-RNase HI is shown as a gold broken line, together with the distances For the structure around residue 39 (C), the side-chains of Y24, residue 39, F41 and Q149 are shown The hydrogen bonds between D39 and Od1 and Q149 and Ne, and between D39 and Od2 and Q149 and Ne, in the wild-type protein are shown as green broken lines together with the distances For the structure around residue 76 (D), the side-chains of L51, P54, residue 76, W106, L109, W120 and W122 are shown For the structure around residues 90 and 97 (E), the side-chains of K89, residue 90 and residue 97 are shown The distances between the e-amino groups of K89 and K90, and between the e-amino group of K90 and the guanidino group of R97, in the wildtype protein are shown For the structure around residue 136 (F), the side-chains of D12, E50, D72, H126, E133 and residue 136 are shown A p-stacking interaction between His126 and His136 in 6·-RNase HI is shown as a gold broken line, together with the distance 606 FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS M S Rohman et al Destabilization mechanism of psychrotrophic RNase HI A B C D E F FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 607 Destabilization mechanism of psychrotrophic RNase HI M S Rohman et al conformational changes predominantly around the respective mutation sites, and has only a slight effect on the backbone conformation of the protein This result indicates that the mutations affect the protein structure independently, but not cooperatively, and is consistent with the fact that the thermostabilizing effects of the mutations are roughly additive The possible stabilization mechanism of the protein by each mutation is described below, based on a local conformational change caused by each mutation Asn29 fi Lys Fig Displacement of the Ca coordinates between the 6·-RNase HI and wild-type proteins (full line) and between molecules C and D (broken line) a Helices and b strands are indicated by bars helices, and around Gly128 in a loop between the aV helix and bE strand The shifts around Gly17 and Asn18 are probably a result of fluctuations rather than perturbations caused by the mutations, because any mutation site is located close to this region The shifts around Ser95 are probably a result of the Lys90 fi Asn and ⁄ or Arg97 fi Gly mutations, and those around Gly128 are probably caused by the Asp136 fi His mutation The details of these shifts are described in the Discussion section The solvent accessibilities of the amino acid residues that are located around the mutation sites, including the parent and mutated residues at these sites, were calculated on the basis of their accessible surface areas in a native and extended structure Comparison of these values for the wild-type and 6·-RNase HI proteins indicated that the solvent accessibilities of all residues, except for residues 29, 39, 126 and 133, were not seriously changed by the sextuple mutations The solvent accessibilities of residues 29 and 39 were signifi˚ cantly increased from 20 to 39 A2 and decreased from ˚ 44 to 17 A2 by the Asn29 fi Lys and Asp39 fi Gly mutations, respectively The solvent accessibilities of His126 and Glu133 were significantly decreased from ˚ 68 to 35 and 60 to 44 A2, respectively, as a result in the shift of a loop between the aV helix and bE strand Discussion In this study, we have shown that the simultaneous introduction of six thermostabilizing mutations causes 608 The 6·-RNase HI structure around residue 29 is compared with that of the wild-type protein in Fig 1B Asn29 and Lys29 are located in the bB strand and are partially exposed to the solvent by 20 and 39%, respectively In the wild-type protein, Asn29 forms hydrogen bonds with Thr34 and Glu131, which are located in the bC strand and aV helix, respectively In 6·-RNase HI, Lys29 forms an ion pair with Glu131 The distances between the Nf atom of Lys29 and the Oe2 atom of ˚ Glu131 are 2.7, 4.1, 3.3 and 3.3 A for molecules A, B, C and D, respectively Thus, by the Asn29 fi Lys mutation, one ion pair is introduced and two hydrogen bonds are eliminated at the mutation site Both the hydrogen bond and ion pair have been reported to contribute to protein stabilization [16,17] However, the finding that So-RNase HI is stabilized by the Asn29 fi Lys mutation by 3.6 °C in Tm and 3.5 kJỈmol)1 in DG(H2O) [13] suggests that the stabilization effect caused by the introduction of an ion pair at the mutation site is stronger than the destabilization effect caused by the elimination of two hydrogen bonds at the same site Several proteins have also been reported to be stabilized by the introduction of ion pairs [18–21] Asp39 fi Gly So-RNase HI is stabilized by the Asp39 fi Gly mutation by 5.8 °C in Tm and 3.5 kJỈmol)1 in DG(H2O) [13] The 6·-RNase HI structure around residue 39 is compared with that of the wild-type protein in Fig 1C The deviation in the shifts of this residue in ˚ molecules A–D is less than 0.4 A Asp39 and Gly39 are located in the bC strand and exposed to the solvent by 44 and 17%, respectively In the vicinity of residue 39, Tyr24, Phe41 and Gln149 are located Tyr24 and Phe41 are almost fully buried inside the protein molecule, whereas Gln149 is relatively well exposed to the solvent We have shown previously that the Asp39 fi Ala mutation also stabilizes the protein to a similar level as that of D39G-RNase HI [13] FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS M S Rohman et al Asp39 is changed to Ala (Ala37) in Ec-RNase HI, which is buried inside the protein molecule by 83% Therefore, the Asp39 fi Gly mutation stabilizes the protein, probably because hydrophobic interactions around the mutation site increase In the wild-type protein, Asp39 forms hydrogen bonds with Gln149 However, these hydrogen bonds may not seriously contribute to the stabilization of the protein, because the hydrogen bond partner, Gln149, can form hydrogen bonds with water molecules Met76 fi Val So-RNase HI is stabilized by the Met76 fi Val mutation by 6.7 °C in Tm and 5.2 kJỈmol)1 in DG(H2O) [13] The 6·-RNase HI structure around residue 76 is compared with that of the wild-type protein in Fig 1D Met76 and Val76 are located in the aII helix within a hydrophobic core and almost fully buried inside the protein molecule by more than 98% The structures of the 6·-RNase HI and wild-type proteins have a cavity around residue 76 within a hydrophobic ˚ core The volume of this cavity is 92 A3 for the wild˚ type protein, and 110, 113, 112 and 111 A3 for molecules A, B, C and D, respectively, of 6·-RNase HI, indicating that the cavity volume increases with the ˚ Met76 fi Val mutation by roughly 20 A3, which is comparable with the volume of a methylene group The side-chain of Met is larger than that of Val, and the difference between them is equivalent to one methylene group in size Therefore, the decrease in the size of the side-chain of residue 76 accounts for the increase in the cavity volume by the mutation We have shown previously that Ec-RNase HI is stabilized by filling a cavity with methyl or methylene groups [22,23] However, one of the mutant proteins of Ec-RNase HI, in which a cavity is filled by the Ala52 fi Met mutation, is less stable than another, in which a cavity is filled by the Ala52 fi Val mutation, by 3.9 °C in Tm [23] These results suggest that the filling of a cavity with Met is not as effective as the filling of a cavity with Val with respect to protein stabilization The Met residue at the hydrophobic core is less preferable than Val for protein stability, probably because its solvation free energy is higher than that of Val [24], and its linear side-chain is rotated more freely than the branched one of Val [25] Destabilization mechanism of psychrotrophic RNase HI region of the aIII helix and a long loop between the aIII and aIV helices, respectively In the vicinity of Lys90, Lys89 is located Lys89, Lys90 and Arg97 are well exposed to the solvent by 80%, 69% and 95%, respectively So-RNase HI is stabilized by the Lys90 fi Asn mutation by 4.1 °C in Tm and 1.7 kJỈmol)1 in DG(H2O) [13] It has been reported that the avoidance of unfavourable electrostatic repulsions is more effective in increasing protein stability than is the creation of stabilizing surface ion pairs [26] Therefore, the Lys90 fi Asn mutation stabilizes the protein, probably because positive charge repulsions between Lys90 and Lys89 and ⁄ or between Lys90 and Arg97 are eliminated The Arg97 fi Gly mutation stabilizes the wild-type and 4·-RNase HI proteins by 5.4 and 3.4 °C, respectively, in Tm, and 3.5 and 8.0 kJỈmol)1, respectively, in DG(H2O) It has been reported that the Lys95 fi Gly mutation stabilizes Ec-RNase HI by 6.8 °C in Tm, because the strain caused by the left-handed backbone structure in the typical : 5-type loop is eliminated [14,27] Non-Gly residues are energetically unfavourable for the left-handed helical conformation because of the steric hindrance between the backbone oxygen atom and side-chain Cb atom The Arg97 fi Gly mutation probably stabilizes the protein with a similar mechanism In fact, Arg97 in the structure of the wild-type protein assumes a left-handed helical conformation with the (/, w) values of (68.4°, 35.2°) This conformation is not seriously changed by the Arg97 fi Gly mutation, because the (/, w) values of Gly97 in the 6·-RNase HI structure are (54.0°, 66.3°) The reason why the effects of this mutation on the thermal stabilities of the wildtype and 4·-RNase HI proteins are not consistent with those on the conformational stabilities (stabilities against urea denaturation) remains to be clarified It should be noted that a loop region (residues 94–97) is shifted towards the aIII helix at most by 0.5, ˚ 3.4, 1.5 and 3.0 A in the structures of molecules A, B, C and D, respectively, of 6·-RNase HI when compared with that in the structure of the wild-type protein As shown in Fig 2, the largest shift is observed for the Ca atom of Ser95 Elimination of the positive charge repulsions among Lys89, Lys90 and Arg97 may be responsible for this shift However, the mutation sites at residues 90 and 97 are close to the protein–protein contacts in the crystal packing, which may account for the large deviation in the loop shift among the molecules A–D Lys90 fi Asn and Arg97 fi Gly The 6·-RNase HI structure around residues 90 and 97 is compared with that of the wild-type protein in Fig 1E Lys90 and Arg97 are located in the C-terminal Asp136 fi His The Asp136 fi His mutation stabilizes the wild-type and 5·-RNase HI by 9.7 and 6.7 °C, respectively in FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 609 Destabilization mechanism of psychrotrophic RNase HI M S Rohman et al Tm, and 6.1 and 7.0 kJỈmol)1, respectively in DG(H2O), indicating that the stabilizing effect of this mutation is independent of those of the other five mutations The 6·-RNase HI structure around residue 136 is compared with that of the wild-type protein in Fig 1F Asp136 and His136 are located in the aV helix and exposed to the solvent by 42% and 39%, respectively In the structure of the wild-type protein, many acidic residues, such as Asp12, Glu50, Asp72 and Glu133, are clustered in the vicinity of Asp136 It has been reported that the corresponding mutation (Asp134 fi His) stabilizes Ec-RNase HI by 7.0 °C in Tm as a result of elimination of negative charge repulsions [15] The Asp136 fi His mutation probably stabilizes the protein with a similar mechanism A loop containing His126 is greatly shifted by, at ˚ most, 4.0 and 4.1 A in the structures of molecules C and D, respectively, of 6·-RNase HI when compared with that in the structure of the wild-type protein This shift is largest amongst those observed in the 6·-RNase HI structure (Fig 2) With this shift, two His residues, His126 and His136, make a p-stacking interaction (Fig 1F) The distances of this interaction are ˚ 3.9 and 3.7 A for molecules C and D, respectively A p-stacking interaction has been reported to contribute to protein stabilization [28] However, this interaction may not be a major stabilization factor of the mutant protein with the Asp136 fi His mutation, because this interaction is not observed in the structures of the Ec-RNase HI variants with the corresponding mutation [31,32] According to the crystal structures of these Ec-RNase HI variants, the position of His124, which corresponds to His126 of So-RNase HI, varies for different proteins, because of the intrinsic flexibility of the loop containing His124 and the crystal packing effect Destabilization mechanism of So-RNase HI A combination of the six thermostabilizing mutations increases the stability of So-RNase HI by 28.8 °C in Tm and 27.0 kJỈmol)1 in DG(H2O) Five of the six substituted residues in the resultant sextuple mutant protein (6·-RNase HI) are found in the corresponding positions of at least one of the amino acid sequences of its mesophilic and thermophilic counterparts Lys29 is conserved as Arg27 in Ec-RNase HI and Arg31 in Thermus thermophilus RNase HI (Tt-RNase HI) Gly39 and Gly97 are conserved as Gly41 and Gly100 in Tt-RNase HI, respectively Val76 is conserved as Val74 in Ec-RNase HI Asn90 is conserved as Gln96 in RNase HI from a cyanobacterium Arg and Gln are similar to Lys and Asn, respectively, in size and charge ⁄ polarity Another substituted residue His136 is 610 not found in other RNase H sequences, because the original residue (Asp136) is one of the active site residues However, a possibility that RNase H with His at this position exists in nature cannot be ruled out, because the mutation of this Asp residue to His greatly stabilizes both So-RNase HI and Ec-RNase HI without seriously affecting the activity These results suggest that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts by increasing the number of amino acid residues unfavourable for protein stability in a localized fashion, in which these residues independently contribute to the destabilization of the protein Experimental procedures Cells and plasmids E coli MIC2067 [F),k), IN(rrnD, rrnE)1, rnhA339::cat, rnhB716::kam] was kindly donated by M Itaya [31] kDE3 lysogen of this strain, E coli MIC2067(DE3), was constructed previously in our laboratory [32] Plasmids pET500M [11] and pET500M4x [13] for the overproduction of So-RNase HI and 4·-RNase HI, respectively, were also previously constructed in our laboratory Mutagenesis The genes encoding R97G-RNase HI, D136H-RNase HI, 5·-RNase HI and 6·-RNase HI were constructed by sitedirected mutagenesis using PCR as described previously [33] Plasmid pET500M or pET500M4x was used as template The mutagenic primers were designed such that the codons for Arg97 (CGT) and Asp136 (GAT) were changed to those for Gly (GGT) and His (CAT), respectively The nucleotide sequences of the genes encoding the mutant proteins were confirmed using a Prism 310 DNA sequencer (Applied Biosystems, Tokyo, Japan) Overproduction and purification of the wild-type and mutant proteins were carried out as described previously [11] The protein concentration was determined from the UV absorption at 280 nm, assuming that the absorption coefficient at this wavelength (2.1 for 0.1% solution) was not changed by the mutation Enzymatic activity The RNase H activity was determined at 30 °C and pH 8.0 by measuring the radioactivity of the acid-soluble digestion product from 3H-labelled M13 DNA ⁄ RNA hybrid, as described previously [34] The reaction mixture contained 10 pmol of the substrate and an appropriate amount of enzyme in 20 lL of 10 mm Tris ⁄ HCl (pH 8.0) containing 10 mm MgCl2, 50 mm NaCl, mm 2-mercaptoethanol and 50 lgỈmL)1 BSA One unit is defined as the amount of FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS M S Rohman et al Destabilization mechanism of psychrotrophic RNase HI The far-UV CD spectra were measured on a J-725 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) at °C The protein was dissolved in 10 mm sodium acetate (pH 5.5) The protein concentration and optical path length were 0.1– 0.2 mgỈmL)1 and mm, respectively The mean residue ellipticity h, which has units of degỈcm2Ỉdmol)1, was calculated using an average amino acid molecular weight of 110 Hampton Research (Alise Viejo, CA, USA) (Crystal Screens I and II and Crystal Screen Cryo I) and Emerald Biostructures (Bainbridge Island, WA, USA) (Wizard I and II) The conditions were surveyed using a sitting-drops vapour diffusion method at °C Drops were prepared by mixing lL each of the protein and reservoir solutions, and were vapour equilibrated against 100 lL of reservoir solution Native crystals suitable for X-ray diffraction analysis appeared after weeks using Crystal Screen II solution No 26 [30% poly(ethylene glycol), MME 5000, 0.1 m Mes, pH 6.5, 0.2 m ammonium sulfate] The crystal was cryoprotected in mother liquor containing 20% sucrose prior to mounting for X-ray diffraction Thermal denaturation Structure determination and refinement Thermal denaturation curves of So-RNase HI and its derivatives were measured as described previously [11] The proteins were dissolved in 10 mm sodium acetate (pH 5.5) containing m GdnHCl The protein concentration and optical path length were 0.1–0.2 mgỈmL)1 and mm, respectively The temperature of the protein solution was increased linearly by approximately 1.0 °CỈmin)1 Thermal denaturation of these proteins was reversible in the presence of m GdnHCl The temperature of the midpoint of the transition, Tm, was calculated from curve fitting of the resultant CD values versus temperature data on the basis of a least-squares analysis The enthalpy (DHm) and entropy (DSm) changes for thermal denaturation at Tm were calculated by van’t Hoff analysis X-Ray diffraction data sets of the 6·-RNase HI crystal were collected at 100 K using synchrotron radiation at the BL44XU station in SPring-8, using a DIP6040 multiple enzyme producing lmol of acid-soluble material per minute The specific activity was defined as the enzymatic activity per milligram of protein CD spectra Urea-induced denaturation Urea-induced denaturation curves of So-RNase HI and its derivatives were measured at 20 °C as described previously [11] The proteins (0.1–0.2 mgỈmL)1) were dissolved in 10 mm sodium acetate (pH 5.5) containing 100 mm NaCl and the appropriate concentrations of urea The protein solution was incubated for at least h at 20 °C before the measurement The urea-induced denaturation of these proteins was fully reversible On the assumption that the unfolding equilibria of these proteins follow a two-state mechanism, the pre- and post-transition baselines were extrapolated linearly, and the difference in free energy between the folded and unfolded states, DG, and the free energy change of unfolding in H2O, DG(H2O), were calculated by the equations given by Pace [35] Crystallization and data collection Table Data collection and refinement statistics for 6·-RNase HI Beamline BL44XU ˚ Wavelength (A) ˚ Resolution (A) Observations Unique reflections Completeness (%) Rmerge (%)a Average I ⁄ r(I) Refinement ˚ Resolution limit (A) 1.0 50.0–2.49 (2.59)2.49) 329 997 23 782 100 (100) 14.6 (52.5) 28.4 (6.39) Space group ˚ Cell unit (A) No of molecules No of protein atoms No of water molecules R-factor (%) Rfree (%)b rmsd ˚ Bond length (A) Bond angles (deg) ˚ Mean B factors (A2) Main chain Side-chain Ramachandran plot statistics (%) Most favoured regions Additionally allowed regions Generously allowed regions 47.52–2.49 P41212 a = b = 68.24, c = 272.82 a = b = c = 90° 4721 255 19.3 24.7 0.025 2.316 26.99 29.30 88.3 10.9 0.8 P P Rmerge = Ihkl ) ⁄ Ihkl, where Ihkl is the intensity measurement for reflections with indices hkl and is the mean intensity for multiply recorded reflections b Rfree was calculated using 5% of the total reflections chosen randomly and omitted from refinement a The 6·-RNase HI protein was concentrated using a Centricon ultrafiltration system (Millipore, Billerica, MA, USA) to approximately 10 mgỈmL)1 The crystallization conditions were initially screened using crystallization kits from FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 611 Destabilization mechanism of psychrotrophic RNase HI M S Rohman et al imaging plate diffractometer (Bruker AXS Inc., Madison, WI, USA) These data sets were indexed, integrated and scaled using the hkl2000 program [36] The crystal structure was solved by the molecular replacement method using molrep [37] in the ccp4 program suite [38] There were four molecules per asymmetric unit, with a solvent content of ˚ 47% and Matthews coefficient of 2.34 [39] The refined A structure of So-RNase HI [Protein Data Bank (PDB) code 2E4L] was used as a starting model Refinement of the structure was performed using the programs cns [40] and refmac [41] The final model was built using coot [42], with R-factor and Rfree values of 19.3% and 24.7%, respectively The Ramachandran plot produced by procheck [43] shows that 100% of the residues in the structure fall in the most favoured and allowed regions The statistics for data collection and refinement are summarized in Table The figures were prepared using pymol (http://www.pymol.org) The accessible surface areas of the protein in native and extended structures were calculated using ACCESS_Surf of MSI InsightII Ver 2000 module (Molecular Simulation Inc ⁄ Accelrys Inc., San Diego, CA, USA) The extended structure was built using Biopolymer of the same module The volume of the cavity within the hydrophobic core was calculated using voidoo software [44] PDB accession number The coordinates and structure factors of 6·-RNase HI have been deposited in PDB under accession code 2ZQB Acknowledgements The synchrotron radiation experiments were performed at the beam line BL44XU in SPring-8 with the approval of the Institute for Protein Research, Osaka University, Osaka, Japan (2008A6909) This work was supported in part by a Grant-in-Aid for Scientific Research on 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Ec -RNase HI and Arg31 in Thermus thermophilus RNase HI (Tt -RNase HI) Gly39 and Gly97 are conserved as Gly41 and Gly100 in Tt -RNase HI, respectively Val76 is conserved as Val74 in Ec -RNase HI Asn90... with Arg97 fi Gly and Asp136 fi His mutations To examine whether the single Arg97 fi Gly and Asp136 fi His mutations stabilize So -RNase HI, two mutant proteins, R97G -RNase HI and D136H -RNase HI, were