We determined the crystal structure of the sextuple mutant protein of So-RNase HI 6·-RNase HI, in which the Arg97 fi Gly and Asp136fi His mutations were combined with the four thermostabil
Trang 1fashion as revealed by mutational and X-ray
crystallographic analyses
Muhammad S Rohman1, Takashi Tadokoro1, Clement Angkawidjaja1, Yumi Abe1,
Hiroyoshi Matsumura2,3, Yuichi Koga1, Kazufumi Takano1,3and Shigenori Kanaya1
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan
2 Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan
3 CREST, JST, Osaka, Japan
Psychrophiles and psychrotrophs are defined as
micro-organisms that can grow even at around 0C [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 promis-ing strategy to understand this mechanism is to
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 6 6879 7938
Tel: +81 6 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 Arg97fi Gly and Asp136 fi His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7C, respectively, in Tm, and 3.5 and 6.1 kJÆmol)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 28.8C in Tm and 27.0 kJÆmol)1 in DG(H2O) To analyse the effects of the mutations on the pro-tein structure, the crystal structure of the 6·-RNase HI propro-tein 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 coun-terparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability
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; D136H-RNase HI, So-D136H-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.
Trang 2construct the thermostabilized mutants of a given
psy-chrophilic 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
iden-tity 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 22.4C in Tm
and 12.5 kJÆmol)1in 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 3.6–6.7C in Tmand 1.7–5.2 kJÆmol)1 in DG(H2O)
[13] They include Asn29fi Lys, Asp39fi Gly,
Met76fi 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 Lys95fi Gly [14] or Asp134 fi His [15]
muta-tion by approximately 7C in Tm at pH 5.5 Because
Lys95 and Asp134 are conserved as Arg97 and
Asp136, respectively, in So-RNase HI, and the
struc-tures around these residues are conserved in So-RNase
HI [9], the Arg97fi 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
informa-tive to examine whether these mutations increase the
stability of So-RNase HI and its derivative (4·-RNase
HI) with quadruple thermostabilizing mutations
identi-fied by directed evolution
In this report, we show that the Arg97fi Gly and
Asp136fi His mutations increase the stability of
So-RNase HI and 4·-RNase HI We determined the
crystal structure of the sextuple mutant protein of So-RNase HI (6·-RNase HI), in which the Arg97 fi Gly and Asp136fi 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 destabili-zation mechanism of So-RNase HI
Results
Stabilization of So-RNase HI with Arg97fi Gly and Asp136fi His mutations
To examine whether the single Arg97fi Gly and Asp136fi 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 do not seriously affect the conformation of the protein The specific activities of R97G-RNase HI and D136H-RNase HI were 99% and 65%, respectively, of that of the wild-type protein (Table 1)
The stabilities of R97G-RNase HI and D136H-RNase HI against thermal denaturation were analysed
at pH 5.5 in the presence of 1 m guanidine hydrochlo-ride (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 1 The tem-perature of the midpoint of the transition, Tm, was 30.4C for the wild-type protein, 35.8 C for R97G-RNase HI and 40.1C for D136H-RNase HI Thus, R97G-RNase HI is more stable than the wild-type protein by 5.4C in Tm and 3.9 kJÆmol)1 in DDGm D136H-RNase HI is more stable than the wild-type protein by 9.7C in Tmand 7.0 kJÆmol)1in 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 characteriz-ing the urea-induced denaturation curves of the wild-type and mutant proteins are summarized in Table 2 The apparent free energy changes of unfolding in the absence of denaturant, DG(H2O), and the urea
Trang 3concen-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 kJÆmol)1 and 0.4 m,
respectively, for R97G-RNase HI, and 6.1 kJÆmol)1
and 0.7 m, respectively, for D136H-RNase HI Thus,
the stabilities of the mutant proteins against urea-induced denaturation show good agreement with those against thermal denaturation
Stabilization of 4·-RNase HI with Arg97 fi Gly and Asp136fi His mutations
To examine whether the Arg97fi 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 evo-lution 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 Arg97fi Gly and Asp136 fi His mutations, respec-tively These mutant proteins were overproduced in
E coli and purified to give a single band on SDS-PAGE 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 do not seriously affect the protein confor-mation The specific activities of the 5·-RNase HI and 6·-RNase HI proteins were 65% and 43%, respec-tively, 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 Arg97fi Gly and Asp136 fi His mutations on the enzymatic activity of the protein are not seriously
Table 1 Activities and thermostabilities of So-RNase HI and its derivatives.
Protein
Specific activity a
(unitsÆmg)1)
Relative activitya(%)
T mb
(C)
DT mb
(C)
DDG mb
(kJÆmol)1)
DH mb
(kJÆmol)1)
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 mea-sured at pH 5.5 in the presence of 1 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 (DT m ) was calculated as T m (mutant) )T m (wild-type) DH m is the enthalpy change of unfolding at
Tmcalculated by van’t Hoff analysis The difference between the free energy change of unfolding of the mutant protein and that of the wild-type 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 T m [44] The DS m (wild-type) value of 0.72 kJÆmol)1, which has been determined previously [11], was used to calculate the DDG m values Each experiment was carried out at least twice and the average value is shown Errors are within ± 0.3 C for T m , ± 26 kJÆmol)1 for DHm, ± 0.12 kJÆmol)1ÆK)1for DSmand ± 0.3 kJÆmol)1for DDGm c Data from Tadokoro et al [11].
Table 2 Parameters characterizing the urea-induced denaturation
of So-RNase HI and its derivatives a
Protein
C ma
( M )
Ma (kJÆmol)1Æ M )1)
DG (H 2 O)a (kJÆmol)1)
DDG (H 2 O)a (kJÆmol)1)
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
mea-surement 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
differ-ence in DG(H 2 O) [DDG(H 2 O)] between the wild-type and mutant
proteins was calculated using the equation: DDG(H 2 O) = m av DC m ,
where mav represents the average m value (8.7 kJÆmol)1Æ M )1) and
DCm= Cm(mutant) )C m (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 kJÆmol)1Æ M )1 for m and ± 1.0 kJÆmol)1 for
DG(H2O) b Data from Tadokoro et al [11].
Trang 4changed 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
denatur-ation 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 1 The temperature of the midpoint of the
transition, Tm, was 49.1C 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
wild-type and 4·-RNase HI proteins by 22.1 and 3.4 C,
respectively, in Tm, and 15.9 and 2.4 kJÆmol)1,
respec-tively, 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 6.7C, respectively, in
Tm, and 20.7, 7.2 and 4.8 kJÆmol)1, respectively, in
DDGm
The stabilities of the 4·-RNase HI, 5·-RNase HI
and 6·-RNase HI proteins against urea-induced
dena-turation were also analysed by monitoring the change
in the CD values at 220 nm Urea-induced
denatur-ation 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 2 The DG(H2O) and Cmvalues 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 kJÆmol)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 kJÆmol)1and 3.1, 1.7
and 0.8 m, respectively Thus, the stabilities of the 5·-RNase HI and 6·-RNase HI proteins against urea-induced 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 molecules D and B, and 0.61 A˚ between mole-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) res-idues 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 2 The dif-ferences 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 1 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 dis-tances 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 wild-type 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.
Trang 5D
F
E
Trang 6helices, 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
Lys90fi Asn and ⁄ or Arg97 fi Gly mutations, and
those around Gly128 are probably caused by the
Asp136fi 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 A˚2 and decreased from
44 to 17 A˚2 by the Asn29fi 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 A˚2, 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
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 pos-sible stabilization mechanism of the protein by each mutation is described below, based on a local confor-mational change caused by each mutation
Asn29fi Lys The 6·-RNase HI structure around residue 29 is com-pared 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%, respec-tively 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 Asn29fi 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 kJÆmol)1in 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]
Asp39fi Gly So-RNase HI is stabilized by the Asp39 fi Gly muta-tion by 5.8C in Tm and 3.5 kJÆmol)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 sol-vent 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 Asp39fi Ala mutation also stabilizes the protein
to a similar level as that of D39G-RNase HI [13]
Fig 2 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.
Trang 7Asp39 is changed to Ala (Ala37) in Ec-RNase HI,
which is buried inside the protein molecule by 83%
Therefore, the Asp39fi 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
hydr-ogen bonds with water molecules
Met76fi Val
So-RNase HI is stabilized by the Met76fi Val
muta-tion by 6.7C in Tm and 5.2 kJÆmol)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 A˚3 for the
wild-type protein, and 110, 113, 112 and 111 A˚3 for
mole-cules A, B, C and D, respectively, of 6·-RNase HI,
indicating that the cavity volume increases with the
Met76fi Val mutation by roughly 20 A˚3, 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
meth-ylene 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
Ala52fi Met mutation, is less stable than another, in
which a cavity is filled by the Ala52fi Val mutation,
by 3.9C in Tm[23] These results suggest that the
fill-ing of a cavity with Met is not as effective as the fillfill-ing
of a cavity with Val with respect to protein
stabiliza-tion 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]
Lys90fi 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
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%, respec-tively So-RNase HI is stabilized by the Lys90fi Asn mutation by 4.1C in Tmand 1.7 kJÆmol)1in DG(H2O) [13] It has been reported that the avoidance of unfa-vourable electrostatic repulsions is more effective in increasing protein stability than is the creation of stabilizing surface ion pairs [26] Therefore, the Lys90fi Asn mutation stabilizes the protein, probably because positive charge repulsions between Lys90 and Lys89 and⁄ or between Lys90 and Arg97 are eliminated The Arg97fi Gly mutation stabilizes the wild-type and 4·-RNase HI proteins by 5.4 and 3.4 C, respec-tively, in Tm, and 3.5 and 8.0 kJÆmol)1, respectively, in DG(H2O) It has been reported that the Lys95fi Gly mutation stabilizes Ec-RNase HI by 6.8C in Tm, because the strain caused by the left-handed backbone structure in the typical 3 : 5-type loop is eliminated [14,27] Non-Gly residues are energetically unfavour-able for the left-handed helical conformation because of the steric hindrance between the backbone oxygen atom and side-chain Cb atom The Arg97fi Gly mutation probably stabilizes the protein with a similar mecha-nism 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 wild-type 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
Asp136fi His The Asp136fi His mutation stabilizes the wild-type and 5·-RNase HI by 9.7 and 6.7 C, respectively in
Trang 8Tm, and 6.1 and 7.0 kJÆmol)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
com-pared 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
(Asp134fi His) stabilizes Ec-RNase HI by 7.0 C in
Tm as a result of elimination of negative charge
repul-sions [15] The Asp136fi 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
inter-action (Fig 1F) The distances of this interinter-action 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 Asp136fi 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
dif-ferent 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 28.8C in
Tm and 27.0 kJÆmol)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
not found in other RNase H sequences, because the original residue (Asp136) is one of the active site resi-dues 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 with-out seriously affecting the activity These results sug-gest that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts by increasing the number of amino acid residues unfa-vourable 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 con-structed 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 site-directed mutagenesis using PCR as described previously [33] Plasmid pET500M or pET500M4x was used as tem-plate 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 pro-teins 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 concen-tration 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 30C 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, 1 mm 2-mercaptoethanol and
50 lgÆmL)1 BSA One unit is defined as the amount of
Trang 9enzyme producing 1 lmol of acid-soluble material per
minute The specific activity was defined as the enzymatic
activity per milligram of protein
CD spectra
The far-UV CD spectra were measured on a J-725
spectropo-larimeter (Japan Spectroscopic, Tokyo, Japan) at 4C The
protein was dissolved in 10 mm sodium acetate (pH 5.5)
The protein concentration and optical path length were 0.1–
0.2 mgÆmL)1and 2 mm, respectively The mean residue
ellip-ticity h, which has units of degÆcm2Ædmol)1, was calculated
using an average amino acid molecular weight of 110
Thermal denaturation
Thermal denaturation curves of So-RNase HI and its
deriv-atives were measured as described previously [11] The
pro-teins were dissolved in 10 mm sodium acetate (pH 5.5)
containing 1 m GdnHCl The protein concentration and
optical path length were 0.1–0.2 mgÆmL)1 and 2 mm,
respectively The temperature of the protein solution was
increased linearly by approximately 1.0CÆmin)1 Thermal
denaturation of these proteins was reversible in the presence
of 1 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
calcu-lated by van’t Hoff analysis
Urea-induced denaturation
Urea-induced denaturation curves of So-RNase HI and its
derivatives were measured at 20C as described previously
[11] The proteins (0.1–0.2 mgÆmL)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 2 h at 20C before the
measurement The urea-induced denaturation of these
pro-teins 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
calcu-lated by the equations given by Pace [35]
Crystallization and data collection
The 6·-RNase HI protein was concentrated using a
Centr-icon ultrafiltration system (Millipore, Billerica, MA, USA)
to approximately 10 mgÆmL)1 The crystallization
condi-tions were initially screened using crystallization kits from
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 4C Drops were prepared by mixing 1 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 anal-ysis appeared after 2 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 cryopro-tected in mother liquor containing 20% sucrose prior to mounting for X-ray diffraction
Structure determination and refinement 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
Table 3 Data collection and refinement statistics for 6·-RNase HI.
R merge (%)a 14.6 (52.5)
Refinement Resolution limit (A ˚ ) 47.52–2.49
a = b = c = 90
rmsd
Mean B factors (A˚2 )
Ramachandran plot statistics (%)
Additionally allowed regions 10.9 Generously allowed regions 0.8
a Rmerge= P
Ihkl) <I hkl > ⁄ P
Ihkl, where Ihklis the intensity measure-ment for reflections with indices hkl and <I hkl > is the mean inten-sity for multiply recorded reflections b Rfreewas calculated using 5% of the total reflections chosen randomly and omitted from refinement.
Trang 10imaging plate diffractometer (Bruker AXS Inc., Madison,
WI, USA) These data sets were indexed, integrated and
scaled using the hkl2000 program [36] The crystal
struc-ture 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 2 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 Rfreevalues of 19.3% and 24.7%,
respec-tively 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 3 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 Priority Areas ‘Systems Genomics’ from
the Ministry of Education, Culture, Sports, Science,
and Technology of Japan, and by an Industrial
Tech-nology Research Grant Program from the New Energy
and Industrial Technology Development Organization
(NEDO) of Japan
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