Structure–cytotoxicity relationships in bovine seminalribonuclease: new insights from heat and chemicaldenaturation studies on variantsConcetta Giancola1, Carmine Ercole1, Iolanda Fotticchia1, Roberta Spadaccini2, Elio Pizzo3,Giuseppe D’Alessio3 and 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 protein 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; S80-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; S80-mBS, R80S variant of

the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties.

FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 111

Introduction

The outstanding feature of bovine seminal ribonucle-

ase (BS-RNase), 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 abstractl 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.

Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.

112 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS

bovine 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

15N

(p.

p.m

.)

1H (p.p.m.)

A

B

Fig. 2. (A) Overlay of the 1H–15N-HSQC spectra of the monomeric

derivatives of BS-RNase (black) and S80-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.

C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease

FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 113

reoxidation of the intersubunit disulfides, followed by

incubation at 37 �C 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 59.4 �C for

BS-RNase and 59.0 �C for hA-BS-RNase, i.e. very

close to each other. In turn, significantly lower, and

comparable, Tm values of 54.3 �C 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 Tm values around 58.0 �C 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 DH0v: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 DH0v: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

12.5 25 50 100(µg·mL–1)

Cel

l su

rviv

al (

%)

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 ( ).

10 20 30 40 50 60 70 80 90

0

1A

B

Temperature (°C)

10 20 30 40 50 60 70 80 90Temperature (°C)

Fra

ctio

n u

nfo

lded

0

1

Fra

ctio

n u

nfo

lded

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 S80-hA-BS-

RNase (h). The unfolded fraction of protein was calculated as (Q –

Qmin) ⁄ (Qmax – Qmin); Q is the ellipticity at 222 nm at a given temper-

ature, and Qmax and Qmin are the maximum and minimum values of

ellipticity corresponding to the denaturated state and native state of

proteins, respectively.

Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.

114 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS

scanning 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 DH0cal 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 4 �C in the melting temperature

and of about 100 kJÆmol)1 in the value of DH0cal. 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. DH0cal and DH0

v:H:. values, both calcu-

lated from calorimetric profiles, are very similar and

the DH0cal=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 DH0v: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 DH0cal 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. Tm, denaturation

temperature; DH 0(Tm), calorimetric enthalpy change; DH0v:H:, van’t Hoff enthalpy change; DC0

p ðTmÞ, excess heat capacity change; DS 0(Tm),

entropy change; DG0298, denaturation Gibbs energy change at 298 K.

Tm (�C)

DH0(Tm)

(kJÆmol)1)

DH0v:H:

(kJÆmol)1)

DC0p ðTmÞ

(kJÆmol)1ÆK)1)

DS0(Tm)

(kJÆmol)1ÆK)1)

DG0298

(kJÆmol)1)

mBS 58.0 ± 0.5 428 ± 12 456 ± 20

408 ± 16a

5.3 ± 0.5 0.72 ± 0.03 36.9 ± 5.5

hA-mBS 58.5 ± 0.5 405 ± 13 397 ± 21

420 ± 17a

4.7 ± 0.6 0.69 ± 0.04 41.9 ± 6.3

S80-hA-mBS 53.9 ± 0.5 334 ± 10 330 ± 16

328 ± 13a

4.8 ± 0.4 0.57 ± 0.03 24.9 ± 3.7

S80-mBS 54.5 ± 0.5 331 ± 9 325 ± 18

316 ± 13a

5.4 ± 0.6 0.50 ± 0.03 25.4 ± 3.8

NCD BS-RNase 59.4 ± 0.5 619 ± 18 570 ± 23

528 ± 21a

– – –

NCD hA-BS-RNase 59.0 ± 0.5 646 ± 19 609 ± 20

588 ± 17a

– – –

NCD S80-hA-BS-RNase 54.3 ± 0.5 585 ± 17 597 ± 23

553 ± 22a

– – –

NCD S80-BS-RNase 53.6 ± 0.5 597 ± 18 590 ± 22

547 ± 21a

– – –

a DH0v:H: from CD measurements.

C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease

FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 115

recorded at the wavelength maximum, Imax, as a func-

tion of urea concentration (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 single-point

R80S variant. The results are reported in Fig. 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 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M

0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M

A B

C D

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 ) Imin) ⁄ (Imax ) Imin); I is the fluorescence

intensity at a given temperature, and Imax

and Imin are the maximum and minimum

values of fluorescence intensity

corresponding to the denaturated state and

native state of proteins, respectively.

Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.

116 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS

code 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 in Fig. 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 S80 variants 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 ⁄ 2 values were the denaturant concentrations

at half-completion of the transition.

[Urea]1 ⁄ 2 (M)

CD

[Urea]1 ⁄ 2 (M)

Fluorescence

mBS 5.70 ± 0.20 5.70 ± 0.08

NCD BS-RNase 3.00 ± 0.06

5.60 ± 0.04

3.00 ± 0.06

5.70 ± 0.03

S80-hA-mBS 4.10 ± 0.15 4.20 ± 0.20

NCD S80-hA-BS-RNase 1.60 ± 0.05

4.50 ± 0.15

1.60 ± 0.20

4.60 ± 0.20

hA-mBS 5.31 ± 0.03 5.36 ± 0.15

NCD hA-BS-RNase 2.20 ± 0.04

5.60 ± 0.01

2.62 ± 0.03

5.64 ± 0.01

S80-mBS 4.76 ± 0.01 5.04 ± 0.03

NCD S80-BS-RNase 2.07 ± 0.02

5.12 ± 0.02

2.17 ± 0.03

5.09 ± 0.02

0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M

0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[Urea] M

A B

C D

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)

S80-hA-BS-RNase. The unfolded fraction of

protein was calculated as (Q ) Qmin) ⁄(Qmax ) Qmin); Q is the ellipticity at 222 nm

at a given temperature, and Qmax and Qmin

are the maximum and minimum values of

ellipticity corresponding to the denaturated

state and native state of proteins,

respectively.

C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease

FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 117

exchange 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 N2MI2transition 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 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0F

ract

ion

un

fold

ed

[Urea] M

A

0 2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n u

nfo

lded

[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 S80-hA-BS-RNase. The Arg80–

Ser18 and Ser20–Gln101 hydrogen bonds, which are detectable

only in BS-RNase, are indicated.

Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.

118 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS

that 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 coli cells 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 4 �C, 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

by M=M isomers: they were incubated at 37 �C for at

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

interchain disulfide bridges was confirmed by SDS ⁄PAGE,

under nonreducing conditions. The NCD forms were kept

at 4 �C 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 inhibition 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 37 �C. 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

Setaram micro-DSC instrument at 1 �CÆmin)1. DSC data

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 4–80 �C. The excess

heat function hDC0Pi. 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]:

DH0v:H: ¼ aRT2

mDC0PðTmÞ=DH0ðTmÞ ð1Þ

where a originates from the stoichiometry of the reaction

(a = 4 or a = 6 for monomer or dimer denaturation,

respectively), Tm is the maximum of the DSC peak,

DC0PðTmÞ 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

C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease

FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 119

monomers, 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 ¼DH0ðTmÞTm � 298

Tm

� �� DC0

pðTmÞ

ðTm � 298Þ þ 298 DC0pðTmÞ ln

Tm

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–15N-

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 4 �C, andnormalized by subtraction of the buffer spectrum. Molar

ellipticity per mean residue [h] in deg cm2Ædmol)1 was 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

bandwidth and 20 nmÆmin)1 scan rates. Thermal denatur-

ation curves were recorded in temperature mode at 222 nm,

with heating of the protein solution from 4 �C to 80 �Cand a scan rate of 1.0 �CÆmin)1. The enthalpy changes were

calculated using origin 7.5 to fit CD melting curves by the

van’t Hoff equation:

@ lnK

@ 1=Tð Þ

� �P

¼ �DH0

Rð3Þ

where K is the equilibrium constant and R is the gas con-

stant.

The urea-induced transition curves at 4 �C 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 4 �C. 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 concentra-

tion was 0.2 mgÆmL)1, corresponding to 14 lm for the

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 4 �Cwith 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 4 �C. Thevalues 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 state, I2

the dimeric intermediate and U the unfolded monomer,

respectively [16]. Fitting of the data was performed with the

matlab 5.3 package. The model gives the Gibbs energy

changes, DG1 and 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 struc-

tures deposited at the PDB. In particular, the NMR struc-

ture of mBS (PDB code 1QWQ) was used for S80-mBS,

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

Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.

120 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS

help with thermodynamic analysis, and P. A. Temussi

for critical reading and suggestions. Financial support

from MIUR (FIRB RBNE03B8KK) is also acknowl-

edged.

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Supporting information

The following supplementary material is available:

Fig. S1. Overlay of the 1H–15N-HSQC of the mono-

meric derivatives of: mBs (black), hA-mBS (magenta)

and S80-hA-mBS (green).

Fig. S2. Cytotoxicity of BS-RNase and its variants, all

assayed at a final concentration of 100 lgÆmL)1, on

3T3 cells after 48 h of incubation.

Fig. S3. DSC profiles of monomers of BS-RNase

variants: (A) mBS; (B) hA-mBS; (C) S80-mBS; (D) S80-

hA-mBS.

Fig. S4. DSC profiles of NCDs of BS-RNase variants:

(A) BS-RNase; (B) hA-BS-RNase; (C) S80-BS-RNase;

(D) S80-hA-BS-RNase.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

this journal provides supporting information supplied

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from supporting information (other than missing files)

should be addressed to the authors.

Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.

122 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS