In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent prot
Trang 1ribonuclease: new insights from heat and chemical
denaturation studies on variants
Concetta Giancola1, Carmine Ercole1, Iolanda Fotticchia1, Roberta Spadaccini2, Elio Pizzo3,
Giuseppe D’Alessio3and Delia Picone1
1 Department of Chemistry ‘Paolo Corradini’, University of Naples ‘Federico II’, Italy
2 Department of Biological and Environmental Sciences, Universita’ degli Studi del Sannio, Benevento, Italy
3 Department of Structural and Functional Biology, University of Naples ‘Federico II’, Italy
Keywords
calorimetric analysis; chemical denaturation;
cytotoxic ribonucleases; domain-swapping;
structure–activity relationships
Correspondence
D Picone or C Giancola, Dipartimento di
Chimica, Universita` di Napoli ‘Federico II’,
Complesso Universitario di Monte
Sant’Angelo, Via Cintia, 80126 Napoli, Italy
Fax: +39 081674409; +39 081674499
Tel: +39 081674406; +39 081674266
E-mail: delia.picone@unina.it;
concetta.giancola@unina.it
(Received 29 June 2010, revised 17
September 2010, accepted 25 October
2010)
doi:10.1111/j.1742-4658.2010.07937.x
Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer In the cytosolic reducing environment, the dimeric form
in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends The quaternary structure renders the reduced pro-tein resistant to the ribonuclease inhibitor, a propro-tein that binds most ribo-nucleases with very high affinity On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor On the basis
of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative Here, we report a comparison of the thermodynamic and chemical stability
of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity
Abbreviations
BS-RNase, bovine seminal ribonuclease; DSC, differential scanning calorimetry; GSH, glutathione; hA-BS-RNase, G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of bovine seminal ribonuclease; hA-mBS, G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; mBS, monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; MxM, dimeric form of bovine seminal ribonuclease in which the chains swap their N-termini; M=M, unswapped dimer of bovine seminal ribonuclease; NCD, noncovalent dimer; PDB, Protein Data Bank; RI, ribonuclease inhibitor; RNase A, bovine pancreatic ribonuclease; S80-BS-RNase, R80S variant of bovine seminal ribonuclease; S80-hA-BS-RNase,
R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of bovine seminal ribonuclease; S 80 -hA-mBS, R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the
monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; S 80 -mBS, R80S variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties.
Trang 2The outstanding feature of bovine seminal
ribonucle-ase (BS-RNribonucle-ase), proposed initially by Piccoli et al.,
(1992) [1] on the basis of biochemical data and
struc-turally proven a few years later [2,3], is the formation
of a dimeric form in which the chains swap their
N-termini (MxM) This phenomenon was later found
in many proteins, and is now well known under the
name of ‘3D domain swapping’ In most cases, the
swapping is associated with new biological functions,
and it has also been proposed as a possible
mecha-nism for protein aggregation in misfolding-associated
pathologies [4] To date, more than 150 structures of
swapped proteins are present in the Protein Data
Bank (PDB) Among these, BS-RNase still represents
a special case, because the native protein is isolated as
a mixture of two dimeric isoforms, MxM and an
M=M [1], with or without the exchange of N-termini
respectively, in a molar ratio of about 2 : 1 Therefore,
only for this protein, the swapping is a physiological,
equilibrium process consisting of a dimer-to-dimer
interconversion; that is, it is not associated with
varia-tion in the quaternary structure The swapping is
considered to be a prerequisite for most additional
biological properties accompanying the basal
enzy-matic activity, including a selective cytotoxic activity
towards malignant tumor cells [5] However, the X-ray
structures of the two isoforms have revealed only
minor differences [2,3], located essentially at the loop
connecting the dislocated arms to the main body of
the protein On the other hand, a so-called ‘buried
diversity’ [6] becomes evident when the protein is
con-sidered under different environments, such as cytosolic
reducing conditions
In vitro, under mild reducing conditions, the two
interchain disulfides bridging the subunits of BS-RNase
undergo selective cleavage, so that M=M is converted
into two monomers, whereas MxM maintains a
dimeric structure, stabilized by noncovalent
interac-tions of the N-termini [1] The monomeric form of
BS-RNase is readily neutralized by the ribonuclease
inhibitor (RI) [7], a protein that is abundant in
mam-malian cells, whereas the quaternary structure of the
reduced dimer, henceforth called the noncovalent dimer (NCD), allows this protein to evade RI binding [8] It has been proposed that RI prevents endogenous RNA degradation by binding monomeric ribonucleases with very high affinity [9] A schematic representation
of the different forms that BS-RNase can adopt in different environments is given in Fig 1, to emphasize that, in the reducing conditions of the cytosol, BS-RNase exists both as a monomer and as swapped NCD stabilized by noncovalent interactions NCD, which is a transient species because, in solution, it dis-sociates into two monomers, is considered to be the form responsible for the cytotoxic activity of the enzyme, given its resistance to RI Furthermore, it has been reported that the structural determinants that favor the proper quaternary structure [6,10] and the stability in solution of the swapped form play a significant role in the additional biological properties
of the enzyme
In this study, we have investigated the relationship between the cytotoxic activity and the stability of the NCD form of BS-RNase in comparison with variants obtained by replacing the 16–20 hinge loop region and⁄ or Arg80 with the corresponding residues of
Structured digital abstract
l MINT-8050499 : BS-RNase (uniprotkb: P00669 ) and BS-RNase (uniprotkb: P00669 ) bind ( MI:0407 ) by biophysical ( MI:0013 )
l MINT-8050482 : BS-RNase (uniprotkb: P00669 ) and BS-RNase (uniprotkb: P00669 ) bind ( MI:0407 ) by classical fluorescence spectroscopy ( MI:0017 )
l MINT-8050435 : BS-RNase (uniprotkb: P00669 ) and BS-RNase (uniprotkb: P00669 ) bind ( MI:0407 ) by circular dichroism ( MI:0016 )
Fig 1 Crystal structures of the multiple forms of BS-RNase: mBS, PDB code 1N1X; M=M, PDB code 1R3M; MxM, PDB code 1BSR; NCD, PDB code 1TQ9.
Trang 3bovine pancreatic ribonuclease (RNase A) It is
worth noting that none of these residues is actually
implicated in the catalytic activity We have already
reported that neither substitution, i.e the changes in
the 16–20 region or the change at Arg80, significantly
affects the swapping propensity of BS-RNase [11,12]
However, when the R80S mutation is inserted into the
construct containing the 16–20 hinge loop region of
RNase A [R80S⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of
BS-RNase (S80-hA-BS-RNase)], the MxM⁄ M=M
molar ratio in the equilibrium mixture is changed from
2 : 1 to 1 : 2 [12] On the basis of the hypothesis that
ascribes the special functions of BS-RNase to the
swapped form, in this study we investigated the
biolog-ical activity of this mutant, and found that it loses
almost all of the cytotoxic activity Interestingly, the
single R80S substitution is sufficient to reduce the
anti-tumor activity almost to the same extent, leading us to
assign to this residue a pre-eminent role in the
cyto-toxic activity of BS-RNase, independently of the hinge
sequence Furthermore, evaluation of the chemical and
thermal stability of the NCD forms of the variant
pro-teins in comparison with those of the parent one
sup-ports the hypothesis that the reduced swapped dimer
represents the bioactive form of BS-RNase
Results
BS-RNase and its R80S variant (S80-BS-RNase), its
G16S⁄ N17T ⁄ P19A ⁄ S20A variant (hA-BS-RNase) and
R80S/G16S/N17T/P19A/S20A variant (S80-hA-BS-RNase)
were expressed in monomeric form, with Cys31 and
Cys32 linked covalently and reversibly to two
glutathi-one (GSH) moieties, as already reported [12,13] The
correctness of the fold of each monomeric protein was
confirmed by comparing their CD spectra and Kunitz
enzymatic activity on yeast RNA [14] with those of
parent monomeric BS-RNase (data not shown)
Fur-thermore, we compared the 2D NMR spectra of all
the monomeric variant proteins with that of the parent
monomeric N67D variant with Cys31 and Cys32
linked to GSH moieties (mBS), and found that the
res-onances of most amide signals are almost coincident,
the differences being essentially restricted to the
back-bone and side chain of the mutated residue(s) and of
those closest (in space) This is illustrated in detail for
the R80S variant of mBS (S80-mBS) in Fig 2: panel
(A) shows the overlay of the 1H-15N-HSQC spectrum
with that of mBS, and panel (B) gives a detail of the
3D structure of monomeric BS-RNase (PDB
code 1N1X), showing the local environment of Arg80
It is very evident that the shifted residues belong to
the region encompassing Arg80 (78–84), to the hinge
(15 and 16) and to regions 45–49 and 101–103, which are less than 4 A˚ from the Arg80 side chain The overlay of the 1H-15N-HSQC spectrum of mBS with those of its G16S⁄ N17T ⁄ P19A ⁄ S20A mBS and R80S⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variants, indicated as hA-mBS and S80-hA-mBS respectively, is shown
in Fig S1
Biological activity The monomeric proteins were converted into dimers
by removal of the protecting GSH moieties and
15 N (p
1 H (p.p.m.)
A
B
Fig 2 (A) Overlay of the 1 H– 15 N-HSQC spectra of the monomeric derivatives of BS-RNase (black) and S 80 -BS-RNase (red) at 300 K Residues whose resonances are shifted are labeled (B) Details of the 3D structure of monomeric BS-RNase (PDB code 1N1X), show-ing the local environment of Arg80 Residues that are less than 4 A˚ from the Arg80 side chain are indicated.
Trang 4reoxidation of the intersubunit disulfides, followed by
incubation at 37C to allow the interconversion of
M=M and MxM to reach equilibrium The cytotoxic
activity of the proteins towards tumor cells was
mea-sured by adding increasing concentrations (ranging
from 12.5 to 100 lgÆmL)1) of each variant to
malig-nant SVT2 cells, using BS-RNase as a positive control
For a negative control, the proteins were also assayed
on nontumor 3T3 cell cultures at a final concentration
of 100 lgÆmL)1, and found to be nontoxic (Fig S2)
The percentage of SVT2 cells surviving, illustrated in
Fig 3, show that the replacement of Arg80 by Ser
induced a significant drop in cytotoxic activity,
inde-pendently of the hinge sequence In contrast, we found
that changes in the 16–20 hinge region had only small
effects, as the cytotoxic activity of BS-RNase was only
slightly higher than that of hA-BS-RNase, and that of
S80-BS-RNase was very close to that of S80
-hA-BS-RNase
Stability of NCD versus monomeric forms
In the search for the molecular basis for the induction
of the loss of cytotoxic activity of BS-RNase variants
reported in Fig 3, we followed by CD the thermal
denaturation process of NCD derivatives, measuring
the molar ellipticity at 222 nm as a function of
temper-ature (Fig 4A) As a comparison, the melting curves
of the corresponding monomers are reported in
Fig 4B The melting temperatures (Tm values) of
NCD derivatives, which represent the midpoint of
the denaturation curve (Fig 4A), were 59.4C for
BS-RNase and 59.0C for hA-BS-RNase, i.e very
close to each other In turn, significantly lower, and
comparable, Tm values of 54.3C and 53.6 C were
found for S80-hA-BS-RNase and S80-BS-RNase,
respec-tively A similar trend was observed for the CD melting
temperatures of monomeric derivatives (Fig 4B) and
[12], which can be separated into two groups, corre-sponding to Tmvalues around 58.0C and 54.0 C for the proteins with Arg80 or Ser80, respectively
The CD melting curves of the monomers were used
to calculate the denaturation enthalpy changes by using the van’t Hoff equation (Eqn 3 in Experimental procedures), which describes two-state NMD transi-tions The data obtained, reported in Table 1, indicate that DH0
v:H: values of the monomers follow the same trend of Tm values, with those of mBS and hA-mBS close to each other and higher than the DH0
v:H: values
of S80-mBS and S80-hA-mBS, which, in turn, are close
to each other
As a further step, we performed a calorimetric analysis of all the proteins by standard differential
0
25
50
75
100
Fig 3 SVT2 cell survival after 48 h of incubation with different
amount of BS-RNase ( ), S80-BS-RNase (h), hA-BS-RNase ( ) and
S80-hA-BS-RNase ( ).
0
1 A
B
Temperature (°C)
Temperature (°C)
0
1
Fig 4 Thermal unfolding curves obtained following the change of
CD signal at 222 nm of NCDs (A) and monomeric derivatives (B) of BS-RNase ( ), hA-BS-RNase (x), S80-BS-RNase (•), and S 80 -hA-BS-RNase (h) The unfolded fraction of protein was calculated as (Q –
Q min ) ⁄ (Q max – Q min ); Q is the ellipticity at 222 nm at a given temper-ature, and Q max and Q min are the maximum and minimum values of ellipticity corresponding to the denaturated state and native state of proteins, respectively.
Trang 5scanning calorimetry (DSC) measurements The
calori-metric profiles of all proteins are reported in Figs S3
and S4 for monomers and NCDs respectively The
denaturation enthalpies of the monomeric derivatives
obtained from the DSC curves, reported as DH0
cal in Table 1, were in good agreement with the van’t Hoff
enthalpies derived from DSC and CD curves, thus
confirming that the thermal denaturation process for
these proteins is a two-state transition process An
inspection of the whole set of thermodynamic
parame-ters of monomeric forms, collected in Table 1, shows
that hA-mBS and mBS have comparable stabilities,
indicating that the substitution of four residues in the
hinge region does not significantly perturb the global
stability of the monomeric form of BS-RNase On the
other hand, the single mutation R80S leads to
decreases of about 4C in the melting temperature
and of about 100 kJÆmol)1 in the value of DH0
cal This destabilization is well reflected in DG0 values, showing
that Arg80 is crucial for the stability of monomeric
form of BS-RNase
For the NCD forms, thermal denaturation was
found to be an irreversible process, because there was
no refolding upon cooling of the protein solutions
The irreversibility of the denaturation process does not
allow Gibbs energy calculations, but only a
compari-son of the melting temperatures and unfolding
enthalpy changes DH0
caland DH0
v:H: values, both calcu-lated from calorimetric profiles, are very similar and
the DH0
cal=DH0
v:H: ratio is in the range 0.98–1.07,
sug-gesting that the unfolding of the dimers is close to
being a one-step process (Table 1) The DH0
v:H: from
CD and DSC curves, relative to the unfolding of the secondary and tertiary structure respectively, are for each dimer very close, indicating simultaneous collapse
of both structures As also indicated in Table 1, the enthalpy changes for the NCD forms are very similar
to each other For a comparison of the DH0
cal values of the dimers with those of the corresponding monomers, the enthalpy changes of NCD forms were calculated
at the melting temperatures of the corresponding monomers, using the Kirchhoff equation Values of
607 kJÆmol)1, 642 kJÆmol)1, 581 kJÆmol)1 and 605 kJÆmol)1 were obtained for NCD forms of BS-RNase, hA-BS-RNase, S80-BS-RNase and S80-hA-BS-RNase, respectively All values are less than twice those of the respective monomers This indicates a loss of interac-tions of the monomers in the dimeric structures In conclusion, all of the reported data indicate that the R80S mutation is crucial for the loss in the enthalpic content of NCDs of BS-RNase, engendering a lower melting temperature of the R80S variants
Urea denaturation of NCD forms The conformational stability of the NCD forms against the denaturing action of urea in comparison with the corresponding monomers was investigated by means of steady-state fluorescence and CD measure-ments at pH 7.0
Monomeric proteins showed sigmoidal transition curves when the change in fluorescence intensity was
Table 1 Thermodynamic melting parameters of the unfolding process of monomers and NCDs of BS-RNase mutants T m , denaturation temperature; DH0(T m ), calorimetric enthalpy change; DH 0
v:H: , van’t Hoff enthalpy change; DC 0 ðT m Þ, excess heat capacity change; DS 0
(T m ), entropy change; DG 0
298 , denaturation Gibbs energy change at 298 K.
Tm(C)
DH 0 (Tm) (kJÆmol)1)
DH 0 v:H:
(kJÆmol)1)
DC 0 ðT m Þ (kJÆmol)1ÆK)1)
DS 0 (Tm) (kJÆmol)1ÆK)1)
DG 0 298 (kJÆmol)1)
408 ± 16 a
420 ± 17a
328 ± 13 a
316 ± 13 a
528 ± 21a
588 ± 17 a
553 ± 22 a
547 ± 21 a
a DH 0
v:H: from CD measurements.
Trang 6recorded at the wavelength maximum, Imax, as a
func-tion of urea concentrafunc-tion (insets in Fig 5), whereas
the curves of NCDs displayed two transitions (Fig 5)
The values of urea concentration at half-completion of
transition, C½, are shown in Table 2, which also
reports the values found by monitoring the molar
ellip-ticity at 222 nm with CD measurements; these reflect
conformational changes of the secondary structures for
monomers (insets in Fig 6) and NCDs (Fig 6) Also
in this case, two distinct C½ values, the first value in
the 2–3 m range and the second close to the C½ value
of the corresponding monomeric form (Table 2), were
observed for the dimers To investigate in more detail
the mechanism of urea denaturation, we followed this
process at different protein concentrations, but
focus-ing on the parent BS-RNase and on the sfocus-ingle-point
R80S variant The results are reported inFig 7, where
the folded fraction is reported as a function of the urea
concentration at four different protein concentrations,
in the range 0.1–25 lm According to Rumfeldt et al
[15], the variation in the curve shape from sigmoidal to
biphasic observed when the protein concentration
increases confirms the presence of a dimeric
intermedi-ate in the dissociation process of both NCD variants
The biphasic curves for the NCD forms of BS-RNase
and S80-BS-RNase at the highest concentration, where
the intermediate is present in significant amounts, were
analyzed according to the three-state equilibrium
model (N2MI2M2U) [16] The following values for the
Gibbs energy changes and m-values were obtained:
DG1= 14 kJÆmol)1, m1= 5 kJÆmol)1Æm)1, DG2 = 80
kJÆmol)1, m2= 19 kJÆmol)1Æm)1 for NCD BS-RNase;
and DG1= 12 kJÆmol)1, m1= 6 kJÆmol)1Æm)1, DG2=
43 kJÆmol)1, m2= 15 kJÆmol)1Æm)1 for S80-BS-RNase The Gibbs energy values indicate that the perturbative action of the urea is greater for the second transition,
I2M2U, than for the first transition, N2MI2, for both dimers The m-values also indicate that the surface area exposed to solvent in the first transition is smaller than that in the second transition A comparison between the two NCD forms shows that the R80S mutation decreases the stability mainly in the step
I2M2U, and, if we assume that the final state is the same for both NCD forms, this suggests that the R80S mutation decreases the stability of the intermediate
Structural models of the NCD forms
In the search for the possible origin of the reduced activity of S80 variants in both aggregation states, i.e
in the monomeric and noncovalent swapped dimeric forms, we examined the corresponding 3D structures All attempts to obtain crystals suitable for X-ray anal-ysis of any form of the two S80-BS-RNase variants had hitherto been unsuccessful Supported by the similarity of NMR spectra (Figs 2 and S1) of the monomers, suggesting that the global architecture of all the variant proteins is very similar to that of the parent BS-RNase, and by the close similarity among the X-ray structures of swapped isoforms of hA-BS-RNase and BS-RNase [11], models of the 3D structures of all proteins were obtained starting from the X-ray structure of the corresponding form of the parent BS-RNase, i.e the monomeric derivative (PDB
0.0 0.2 0.4 0.6 0.8 1.0
[Urea] M
0.0
0.2
0.4
0.6
0.8
1.0
[Urea] M
0.0
0.2
0.4
0.6
0.8
1.0
[Urea] M
0.0 0.2 0.4 0.6 0.8 1.0
[Urea] M
Fig 5 Urea-induced transition curves for NCDs of BS-RNase variants and for the corresponding monomers (inset), followed
by fluorescence spectroscopy.
(A) BS-RNase (B) hA-BS-RNase.
(C) S80-BS-RNase (D) S80-hA-BS-RNase The unfolded fraction represents the fraction
of denaturated protein, calculated as (I ) I min ) ⁄ (I max ) I min ); I is the fluorescence intensity at a given temperature, and I max and Iminare the maximum and minimum values of fluorescence intensity corresponding to the denaturated state and native state of proteins, respectively.
Trang 7code 1N1X) and the noncovalent swapped dimer (PDB
code 1TQ9) A representation of the structural models
built for S80-hA-BS-RNase, which, among the variants
examined in this study, is the one hosting the highest
number of substitutions, is reported inFig 8
A careful inspection of these structures reveals that
all of the variant proteins examined, and notably both
of the S80 variants, are characterized by the presence
of a decreased number of hydrogen bonds with respect
to the parent protein This trend is even more evident
in the NCD derivatives: in these forms, all of the
mutants have fewer intersubunit hydrogen bonds
than the native protein In particular, focusing on
residue 80, a contact between the side chains of Arg80 and Ser18 is detectable only in parent BS-RNase Fur-thermore, the same protein and hA-BS-RNase are sta-bilized by a contact between a core residue of one subunit (Gln101) and the hinge residues of the other subunit (Ser20 in the case of BS-RNase and Ser18 in the case of hA-BS-RNase)
Discussion
The antitumor activity of dimeric ribonucleases relies
on their quaternary structure, which enables the pro-teins to avoid inhibition by RI and provides good sta-bility in solution We have already shown that Pro19, Leu28 and, possibly, Gly16 play a relevant role in the cytotoxicity, because they ensure the correct quater-nary assembly of the NCD derivative of BS-RNase [17] However, the hinge residues and Leu28 have a synergistic effect, because to observe a drastic reduc-tion of the cytotoxic activity they have to be replaced simultaneously [6,18]
In contrast, the data reported in this article show that the substitution of the whole hinge region induces only a small reduction in the basal cytotoxic activity
of BS-RNase, as in the case of the single mutants P19A and L28Q [6] Surprisingly, the replacement of Arg80 by Ser significantly reduces the cytotoxic activ-ity, as both S80variants are less active than the parent BS-RNase On the one hand, this shows the impor-tance of Arg80, although it is irrelevant for the cata-lytic activity or the swapping extent of BS-RNase [12];
on the other hand, it indirectly confirms that the
Table 2 Urea-induced denaturation parameters of monomers and
dimers of BS-RNase mutants, monitored by fluorescence and CD
spectroscopy [Urea]1⁄ 2values were the denaturant concentrations
at half-completion of the transition.
[Urea]1⁄ 2 (M) CD
[Urea]1⁄ 2 (M) Fluorescence
5.60 ± 0.04
3.00 ± 0.06 5.70 ± 0.03
NCD S 80 -hA-BS-RNase 1.60 ± 0.05
4.50 ± 0.15
1.60 ± 0.20 4.60 ± 0.20
5.60 ± 0.01
2.62 ± 0.03 5.64 ± 0.01
NCD S80-BS-RNase 2.07 ± 0.02
5.12 ± 0.02
2.17 ± 0.03 5.09 ± 0.02
0.0 0.2 0.4 0.6 0.8 1.0
[Urea] M
0.0 0.2 0.4 0.6 0.8 1.0
[Urea] M
0.0 0.2 0.4 0.6 0.8 1.0
[Urea] M
0.0 0.2 0.4 0.6 0.8 1.0
[Urea] M
Fig 6 Urea-induced transition curves for
NCDs of BS-RNase variants and for the
corresponding monomers (inset), followed
by CD spectroscopy (A) BS-RNase (B)
hA-BS-RNase (C) S80-BS-RNase (D)
S 80 -hA-BS-RNase The unfolded fraction of
protein was calculated as (Q ) Q min ) ⁄
(Q max ) Q min ); Q is the ellipticity at 222 nm
at a given temperature, and Q max and Q min
are the maximum and minimum values of
ellipticity corresponding to the denaturated
state and native state of proteins,
respectively.
Trang 8exchange of the N-terminal arms in BS-RNase-like
proteins is not sufficient to elicit the antitumor activity
[10] We are aware that, in principle, the substitution
of a basic residue on the protein surface might reduce
the cytotoxic activity, by affecting the electrostatic
interaction with the cell membrane and perhaps the
internalization process [19], but, as shown by
Notomi-sta [20], the side of BS-RNase with the strongest
posi-tive potential is the one hosting the N-termini, which
is located opposite to Arg80 (Fig 8)
The observation of models built with the X-ray
structure of the reduced dimer of BS-RNase (PDB
code 1TQ9) as template indicate that all of the variants
maintain a quaternary structure very close to that of
the parent protein, but are characterized by weaker
interactions between subunits This result is also in
agreement with thermodynamic data and thermal and
chemical dissociation, which indicate a lower stability
of the variant proteins with respect to BS-RNase,
prin-cipally for both Ser80 variants
We also investigated the dissociation mechanism, and found that it is not affected by the mutations investigated here The experimental data related to thermal denaturation processes of both Ser80 variant proteins can be interpreted by surmising a simple two-step process, from native dimer to denatured monomers, whereas the presence of biphasic chemical denaturation profiles, exhibited by both fluorescence and CD curves, suggests the existence of a thermody-namically stable intermediate induced by urea This discrepancy is not unexpected, because the two dena-turation processes are induced by different perturbing agents, proceed through different mechanisms (in the case of the thermal denaturation process, the interme-diate state is not present in significant amounts) and end with completely distinct denatured states [21] It is possible that the chaotropic effect of urea initially causes a small perturbation of the secondary and ter-tiary structures of the proteins (DG1 for the N2MI2 transition is smaller than DG2 of the I2M2U transi-tion), and urea then stabilizes the intermediate through its hydrogen-bonding ability Furthermore, the com-parison of the values obtained for the first step indi-cates that, in all variants examined, the C½ value of the first step of the denaturation process is decreased with respect to that of the parent protein, indicating
0.0
0.2
0.4
0.6
0.8
1.0
[Urea] M A
0.0
0.2
0.4
0.6
0.8
1.0
[Urea] M B
Fig 7 Urea-induced transition curves of NCDs of BS-RNase (A)
and S80-BS-RNase (B), followed by fluorescence spectroscopy at
different protein concentrations: (•), 0.1 lM; ( ), 1 lM; (h), 7 lM;
( ), 25 lM.
A
B
Fig 8 (A) Crystal structure of the NCD form of BS-RNase (PDB code 1TQ9) (B) Homology model of S 80 -hA-BS-RNase The Arg80– Ser18 and Ser20–Gln101 hydrogen bonds, which are detectable only in BS-RNase, are indicated.
Trang 9that the interactions between the hinge region and
resi-due 80 are involved in the early stages of the chemical
denaturation process As a consequence, S80
-hA-BS-RNase is the most prone to unfolding
Our data suggest a correlation between the cytotoxic
activity of BS-RNase and its derivatives and the
stability of the corresponding reduced swapped forms
This is also in agreement with the finding that
cyto-toxic RNases are, in general, very stable enzymes, and
with the relationship between resistance to unfolding
and cytotoxic activity observed for different variants
of RNase A [22,23] In conclusion, the enhancement of
the conformational stability of the NCD derivative
represents a good approach to increase the toxicity of
BS-RNase towards cancer cells In addition, the
find-ing that the structure and stability of the dimeric
inter-mediate, shown by urea denaturation studies, play a
key role in the dissociation process suggests further
investigations of the dissociation mechanism that may
help in the design of new cancer chemotherapeutic
agents based on BS-RNase
Experimental procedures
Protein samples
All of the experimental procedures for obtaining significant
amounts of BS-RNase and its variants starting from the
cor-responding pET-22b(+) plasmid cDNA have already been
described in detail elsewhere [12,13] As in the previous
stud-ies, all of the constructs were coding for an Asp residue at
position 67, instead of an Asn as in the wild-type protein, to
avoid side effects caused by the spontaneous deamidation of
Asn67 [24,25] All of the proteins were expressed in
Escheri-chia colicells and purified in monomeric form, with Cys31
and Cys32 linked to two GSH molecules Monomers with
Cys31 and Cys32 in the reduced form, prepared as described
previously [12], were either carboxyamidomethylated with
iodoacetamide [26], to obtain the monomeric proteins used
directly for analysis, or dialyzed against 0.1 m Tris⁄ acetate
(pH 8.4) for 20 h at 4C, to obtain dimers In both cases,
the last step of the purification procedure was gel filtration
chromatography on a G-75 column (75· 3 cm)
Freshly prepared dimeric proteins were essentially made
least 72 h, to allow the mixture to reach equilibrium
The protein concentration was measured by UV
spectro-photometry, assuming e = 0.5 (0.1%, 278 nm, 1 cm) for
monomers and e = 0.465 for dimers
NCDs
The NCDs were prepared according to the protocol
previ-ously described by Piccoli et al [1], from the exchanged
form of the corresponding dimer The reduction of the
under nonreducing conditions The NCD forms were kept
at 4C until used for kinetic or thermodynamic analyses
Cytotoxicity studies
Cytotoxicity was evaluated by performing the 3-(4,5-dim-ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduc-tion inhibireduc-tion assay as described previously [27] Simian virus-40-transformed mouse fibroblasts (SVT2 cells) and the parental nontransformed Balb⁄ C 3T3 line (3T3 cells) were obtained from the ATCC (Manassas, VA, USA) The cells were plated on 96-well plates at a density of 2.5· 103
cells per well in 100 lL of medium containing BS-RNase or one
of its variants (12.5, 25, 50 or 100 lgÆmL)1), and incubated for 24 and 48 h at 37C Cell survival is expressed as the absorbance of blue formazan measured at 570 nm [27] with
an automatic plate reader (Victor3 Multilabel Counter; Per-kin Elmer, Shelton, CT, USA) Each curve reports the aver-age of three independent assays Standard deviations in all assays were in the range of 5–10%
DSC
DSC measurements were performed on a third-generation
were analyzed with a previously described program [28] The excess heat capacity of the protein in solution in the sample cell was measured against a reference cell filled with the buf-fer solution in the temperature range of 4–80C The excess heat functionhDC0
Pi was obtained after baseline subtraction, assuming that the baseline is given by the linear temperature dependence of the native state heat capacity The denatur-ation enthalpies, DH0(Tm), were obtained by integrating the area under the heat capacity versus temperature curves Tm
is the melting temperature, and corresponds to the maximum
of each DSC peak The entropy changes corresponding to the thermal denaturation of the monomers, DS0(Tm), were determined by integrating the curve obtained by dividing the heat capacity curve by the absolute temperature
Thermodynamic analysis
The van’t Hoff enthalpies were obtained by DSC profiles, utilizing the equation [29]:
DH0 v:H:¼ aRT2
mDC0
PðTmÞ=DH0ðTmÞ ð1Þ where a originates from the stoichiometry of the reaction (a = 4 or a = 6 for monomer or dimer denaturation,
DC0
PðTmÞ is the value of the excess heat capacity function at
Tm, and DH0(Tm) is the calorimetric enthalpy calculated by direct integration of the area under the DSC peak For the
Trang 10monomers, the entropy changes, DS0(Tm), were determined
by integrating the curve obtained by dividing the heat
capacity curve by the absolute temperature, and the
dena-turation Gibbs energies at 298 K were calculated by
com-bining the classical Kirchoff equations:
DG0ð298Þ ¼ DH0ðTmÞ Tm 298
Tm
DC0
pðTmÞ
ðTm 298Þ þ 298 DCp0ðTmÞ lnTm
298
ð2Þ
NMR
Two-dimensional NMR spectra were acquired at 300 K on
on Bruker DRX600 spectrometer by using standard pulse
sequence libraries For the natural abundance 1H–15
N-HSQC spectra, the protein concentration was 2.5 mm in
95% H2O⁄ 5% D2O (pH 5.65) 1H chemical shifts are
rela-tive to the water signal at 4.70 p.p.m at 300 K, and 15N
chemical shifts were indirectly referenced to the 1H
chemi-cal shifts according to gyromagnetic ratios [30]
CD spectroscopy
CD measurements were performed on a JASCO 715 CD
spectrophotometer equipped with a thermoelectrically
con-trolled cell holder (JASCO PTC-348) that allows
measure-ments at a controlled temperature Quartz cuvettes with
0.1 cm optical pathlength were used Unless otherwise
reported, the protein concentration was 0.2 mgÆmL)1 The
CD spectra were recorded from 250 to 200 nm at 4C, and
normalized by subtraction of the buffer spectrum Molar
ellipticity per mean residue [h] in deg cm2Ædmol)1was
calcu-lated from the equation [h] = 100[h]obs⁄ lC, where [h]obs is
the ellipticity measured in degrees, l is the pathlength of the
cell (cm) and C is the protein molar concentration CD
spectra were recorded with a response of 4 s, a 1.0 nm
denatur-ation curves were recorded in temperature mode at 222 nm,
and a scan rate of 1.0CÆmin)1 The enthalpy changes were
calculated using origin 7.5 to fit CD melting curves by the
van’t Hoff equation:
@ln K
@ð1=TÞ
P
0
where K is the equilibrium constant and R is the gas
con-stant
The urea-induced transition curves at 4C were obtained
by recording the CD signal at 222 nm for each independent
sample All of the measurements were performed after
over-night incubation of the samples at 4C The values of urea
concentration at half-completion of transition, [urea]1 ⁄ 2,
were calculated with the Boltzmann equation of origin 7.0
Fluorescence analyses
Intrinsic protein fluorescence was recorded with a Perkin Elmer LS50B spectrofluorimeter equipped with a circulating water bath Unless otherwise stated, the protein
monomers and 7 lm for the dimers The excitation wave-length was set at 274 nm, and the emission was measured between 250 and 400 nm The spectra were recorded at 4C with a 1 cm cell and a 10 nm emission slit width, and cor-rected for background signal The urea-induced transition curves were obtained by recording the change in fluorescence intensity at maximum wavelength as a function of denatur-ant concentration The maximum emission wavelength of proteins was recorded at 303 nm Measurements were per-formed after overnight incubation of samples at 4C The values of urea concentration at half-completion of transi-tion, [urea]1 ⁄ 2, were calculated as for the CD measurements The biphasic curves at 25 lm were analyzed according to the three-state equilibrium model with a dimeric intermedi-ate, N2MI2M2U, where N2 is the native dimeric stintermedi-ate, I2 the dimeric intermediate and U the unfolded monomer, respectively [16] Fitting of the data was performed with the
changes, DG1and DG2, and the values of m1 and m2 for the N2MI2 and I2M2U transitions, respectively The m-value is a measure of the dependence of DG on denatur-ant concentration [31], and is proportional to the amount
of protein surface area exposed upon unfolding [32]
Molecular modeling
The structures of monomeric and dimeric variants of BS-RNase were calculated from the NMR and X-ray tures deposited at the PDB In particular, the NMR
whereas for the dimeric NCD form three crystallographic structures were used, corresponding respectively to the NCD BS-RNase derivative (PDB code 1TQ9), to the N-dimer of RNase A (PDB code 1A2W) and to the PM8 human pancreatic RNase variant (PDB code 1H8X) The atomic coordinates of the above-mentioned protein struc-tures were used as a template to predict the 3D structure of the variants, using modeller 8v5 [33] The quality of the structural models was evaluated with modeller, using the score of variable target function method [34] Model analy-ses were performed with molmol [35] and pymol [36]
Acknowledgements
C Ercole is a fellow of the Department of Chemistry
‘Paolo Corradini’ of University of Naples ‘Federico II’, supported by a training grant from the ‘Compag-nia di San Paolo di Torino’ We thank T Tancredi for help with NMR spectra acquisition, L Petraccone for