The role of electrostatic interactions in the antitumor
activity of dimeric RNases
Eugenio Notomista
1
, Jose
´
Miguel Manchen
˜
o
2
, Orlando Crescenzi
3
, Alberto Di Donato
1
,
Jose
´
Gavilanes
4
and Giuseppe D’Alessio
1
1 Dipartimento di Biologia Strutturale e Funzionale, Universita
`
di Napoli Federico II, Napoli, Italy
2 Grupo de Cristalografı
´
a Macromolecular y Biologı
´
a Estructural, Instituto Rocasolano, Madrid, Spain
3 Dipartimento di Chimica, Universita
`

Bos taurus seminal vesicles [10], among the most stud-
ied natural cytotoxic RNases, do not bind cRI, and
are both totally resistant to its inhibitory action [8].
On the other hand, RNases with no cytotoxic action,
such as bovine or human pancreatic RNase, and a
very high affinity for cRI, acquire the ability to kill
Keywords
antitumor RNases; electrostatic interactions;
electrostatic interaction energy; RNases;
transport through membranes
Correspondence
G. D’Alessio, Dipartimento di Biologia
Strutturale e Funzionale, Universita
`
di Napoli
Federico II, Via Cinthia, I-80126 Napoli, Italy
Fax: +39 081 679159
Tel: +39 081 679157
E-mail:
(Received 12 April 2006, revised 31 May
2006, accepted 12 June 2006)
doi:10.1111/j.1742-4658.2006.05373.x
The cytotoxic action of some ribonucleases homologous to bovine pancre-
atic RNase A, the superfamily prototype, has interested and intrigued
investigators. Their ribonucleolytic activity is essential for their cytotoxic
action, and their target RNA is in the cytosol. It has been proposed that
the cytosolic RNase inhibitor (cRI) plays a major role in determining the
ability of an RNase to be cytotoxic. However, to interact with cRI RNases
must reach the cytosol, and cross intracellular membranes. To investigate
the interactions of cytotoxic RNases with membranes, cytotoxic dimeric

RNase catalytic activity is an absolute requirement
for the cytotoxic action of all cytotoxic RNases tested
[5]. Moreover, when the effects of cytotoxic RNases on
cellular RNA were studied, cytosolic rRNA [18] or
tRNA [19] were found to be the targets of cytotoxic
RNases. Thus, we can conclude that to exert its cyto-
toxic action, an RNase must reach the cytosol. Fur-
thermore, given that cRI is a cytosolic protein [6,7],
the characterization of an RNase as a cRI-resistant or
-sensitive RNase can only occur if the RNase reaches
the cytosol. These considerations lead to the conclu-
sion that to approach the cytosol, where their cyto-
toxic action is exerted, cytotoxic RNases must cross
not only the plasma membrane, but intracellular mem-
branes as well.
A study of the intracellular journey of cytotoxic,
dimeric seminal RNase has revealed that it is internal-
ized by various types of malignant cells, and has access
to endosomes, whereas noncytotoxic bovine pancreatic
RNase A is not internalized [18,20]. Interestingly,
when RNase A was made dimeric and cytotoxic by
site-directed mutagenesis, it gained access to endo-
somes [20]. It should be added that endocytosed sem-
inal RNase is found in endosomes both in malignant
and normal cells, but only in malignant cells does the
RNase reaches the trans-Golgi network, and then the
cytosol, where it degrades rRNA [18,20].
These data suggest that cell membranes can discrim-
inate RNases, and may play an important role in
determining the cytotoxicity of an RNase by simply

50
is the RNase concentration producing half-maximal cytotoxicity on
SVT2 malignant cells [5,11,14].
Abbreviations Natural or modified RNase Structure pI IC
50
(lgÆmL
)1
)
RNase A Bovine pancreatic RNase A Monomeric 9.8 > 200
RNase-AA A19P,Q28L,K31C,S32C-RNase A Dimeric 9.6 82
RNase-AA-G D38G-RNase-AA Dimeric 9.8 64
RNase-AA-GG D38G,E111G-RNase-AA Dimeric 9.9 27
HP-RNase Human pancreatic RNase Monomeric 10.1 > 200
HHP-RNase Q28L,R31C,R32C,N34K-HP-RNase Dimeric 9.9 26
BS-RNase Bovine seminal RNase Dimeric 10.3 22
Electrostatic interactions and antitumor RNases E. Notomista et al.
3688 FEBS Journal 273 (2006) 3687–3697 ª 2006 The Authors Journal compilation ª 2006 FEBS
of these RNases on the aggregation of negatively
charged dimyristoylphosphatidylglycerol (DMPG) and
dimyristoylphosphatidylserine (DMPS) vesicles. Aggre-
gation was measured from the increase of absorbance
at 360 nm due to the enhanced turbidity as engendered
by vesicle aggregation. The results with DMPG vesicles
are presented here, for the higher availability to outside
interactions of the negatively charged glycerol-linked
phosphate [23–25]. In DMPS vesicles, the bonding of
the serine carboxylate ion to the adjacent ammonium
group renders the phosphate negative charge less avail-
able [23–25]. In fact, when DMPS vesicles were tested
(data not shown), similar results were obtained in terms

218 nm, gave an identical midpoint of denaturation at
62 °C for all three RNase dimers, and identical far-UV
CD spectra of the thermally denatured states. These
data (not shown) indicate that the three proteins
apparently possess an identical folded structure under
the conditions used in the experiments with mem-
branes, hence their different effects on the model mem-
branes may not be ascribed to different conformations
of the three proteins.
Effects on bilayer fluidity
Figure 2 illustrates the effects of RNases on the phase
transition profile of DMPG vesicles labeled with 1,6-
diphenyl-1,3,5-hexatriene (DPH), a fluorescent probe
used for measuring fluorescence polarization. Bilayer
fluidity was measured by comparing the difference in
fluorescence anisotropy (Dr) as a function of pro-
tein : DMPG ratio.
Fig. 2. Effect of RNases on the thermotropic behavior of DMPG
vesicles. Symbols: (e) BS-RNase; (+) RNase AA-GG; (n)
HHP-RNase; (n) HP-RNase; (h) RNase AA-G; (m) RNase AA; (·)
RNase A.
Fig. 1. Aggregation of DMPG vesicles induced by RNases. Sym-
bols: (e) BS-RNase; (+) RNase AA-GG; (n) HHP-RNase; (n) RNase
AA-G; (h) RNase AA; (m) HP-RNase; (·) RNase A.
E. Notomista et al. Electrostatic interactions and antitumor RNases
FEBS Journal 273 (2006) 3687–3697 ª 2006 The Authors Journal compilation ª 2006 FEBS 3689
The data show that most investigated RNases affect
the bilayer fluidity of DMPG membranes, as evidenced
by a decrease of the amplitude of their thermotropic
transition. Only RNase A and RNase AA show no

In Table 2 the results described above on the effects
on membrane stability induced by the investigated
RNases are normalized to those obtained with BS-
RNase, taken as 100%. For the normalization the
highest values of Dr, DA
360
and percentage RET were
used for effects on bilayer fluidity, membrane aggrega-
tion and fusion, respectively (see above and Figs 1–3).
When the effects of RNases on negatively charged
membranes were compared with the net charge values
of the RNases, the comparison suggested (Table 2)
that the ability of RNases to destabilize lipid vesicles is
due to an electrostatic component, as the higher posit-
ive net charge of an RNase appears to enhance its
ability to aggregate, fuse, or affect bilayer fluidity of
the negatively charged membranes. However, a hydro-
phobic component may not be excluded in the RNase–
membrane destabilizing interactions. This can be
deduced: (a) from the RNases aggregating effects,
which could be generated by the relief of hydration
repulsion among vesicles occurring upon adsorption of
the RNases to the membrane bilayer; (b) from the
thermotropic behavior of lipids in the presence of
RNases, which can be assigned to lipid molecules
removed from participating in their gel-to-liquid phase
transition.
To further investigate this aspect, values of electro-
static interaction energy (EIE) were calculated for the
interactions between the dimeric RNases and a model

HP-RNase; (h) RNase AA-G; (m) RNase AA; (·) RNase A.
Electrostatic interactions and antitumor RNases E. Notomista et al.
3690 FEBS Journal 273 (2006) 3687–3697 ª 2006 The Authors Journal compilation ª 2006 FEBS
r ¼ 180° and i ¼ 90°. In this orientation, characterized
by the strongest electrostatic attraction of the RNases
with the membrane, all RNases pointed their N-ter-
minal regions towards the membrane. Figure 5 illus-
trates the results for some of the RNases (also
supplementary Fig. S1). It should be added that both
models of RNase A or HP-RNase dimers, either swap-
ping their N-terminal ends between subunits, or not
swapping dimers, led to virtually identical EIE values
(data not shown).
The analysis of the electrostatic field generated by
dimeric RNases showed that all dimers possess a posi-
tively charged end located at the N-terminal region
and a negative end located at the C-terminal one.
Hence, they can be described as dipoles with an orien-
tation parallel to the direction from the RNases C-ter-
minal surfaces (negatively charged) to the N-terminal
surfaces (positively charged). In the supplementary
material, Fig. S2 shows the shape of the electrostatic
field of BS-RNase seen from different directions.
Figure S3 compares the electrostatic field of different
Fig. 5. Plot of the EIE values as function of rotation (r) and inclination (i) angles of RNases. The hatched axis at the bottom and right of the
plots show schemes of some conformations with i ¼ 90° and r ¼ 180°, respectively.
Fig. 4. Scheme of the initial complex dimeric RNase–model mem-
brane used to generate the set of orientations for
DELPHI calcula-
tions through rotation of the RNase around its major axis (rotation

membrane, which in turn appears to be linked to the
attraction of the RNase to a membrane, as evidenced
by the RNase negative EIE
max
values. As a measure of
the RNase cytotoxicity we used the reciprocal of the
IC
50
parameter. The values of IC
50
, the RNase concen-
tration at which 50% of the cytotoxic effect is dis-
played, were obtained [11,21,22] by assaying the RNase
cytotoxic activity on the same cell type, namely SVT2
malignant fibroblasts. When the EIE
max
values of
dimeric, cytotoxic RNases were plotted against the
IC
50
values reported for these RNases (Fig. 6), a direct,
linear correlation was found between the two sets of
values, with a significant correlation coefficient (0.88).
This strongly suggests a positive correlation between the
attraction to a membrane of a dimeric RNase and its
cytotoxic activity.
Discussion
Although many years have gone by since an RNase
was found to have an antitumor action [27], the struc-
tural and functional determinants which render certain

membranes, as they strongly affect membrane aggrega-
tion, fluidity and fusion. When the RNases were tested
in silico with a model membrane by determining the
electrostatic energy of their interaction, we found that
their ability to destabilize membranes is correlated with
their electrostatic attraction to the model membrane.
In particular, such property was found to be strictly
dependent on the ability of the RNases to approach
the membranes with their N-terminal regions. Any
other orientations in their contacts with the mem-
branes produced a less attractive electrostatic interac-
tion. Hence RNases that tumble about a membrane
Fig. 6. Correlation between antitumor activity on SVT2 cells and
the highest negative EIE values for covalent dimeric RNases identi-
fied by the abbreviations listed in Table 1. Antitumor activity was
plotted as the reciprocal of IC
50
, the RNase concentration produ-
cing 50% cytotoxicity [11,14]. The square correlation coefficient
(R
2
) is shown.
Electrostatic interactions and antitumor RNases E. Notomista et al.
3692 FEBS Journal 273 (2006) 3687–3697 ª 2006 The Authors Journal compilation ª 2006 FEBS
until their most membrane-attractive structural ele-
ment, namely their N-terminal regions, interact with
the membrane, are the most capable of affecting and
permeating membranes.
Furthermore, our results also reveal a stringent
correlation between (a) the ability of dimeric RNases

noncovalent dimers have been described for RNase A,
in which the dimeric structure is stabilized through
the exchange between subunits of either their N-ter-
minal, or their C-terminal ends [33–35]. We found
that the behavior with the model membrane of the
dimer in which the C-terminal ends are exchanged
between subunits is perfectly superimposable to the
behavior described in this report for any covalently
dimeric RNase. Its highest negative value of EIE was
calculated to be )28 kT. The dimer, in which the sub-
units exchange their N-terminal ends, behaved differ-
ently. It did not present a single, unique value of
highest negative EIE, but two weak negative values of
)13 and )10 kT. We noted that by summing up the
two EIE
max
values, a value of EIE was obtained com-
parable to those calculated for the other dimeric
RNase. As shown in Fig. 7, the EIE
max
values
obtained for the two dimers satisfactorily correlate
with the dimers IC
50
values as reported by Matousek
et al. [17]. Thus, a role of electrostatic membrane
attraction has been identified also for noncovalent,
cRI sensitive RNases.
In conclusion, the data presented here lead to the
proposal that cytotoxic RNases must possess specific

ted (see legend to Fig. 6) as the reciprocal of IC
50
reported values
[17]. The square correlation coefficients (R
2
) are shown.
E. Notomista et al. Electrostatic interactions and antitumor RNases
FEBS Journal 273 (2006) 3687–3697 ª 2006 The Authors Journal compilation ª 2006 FEBS 3693
0.695 (E
0.1%
, 280 nm) for RNase A and its variants, 0.465
(E
0.1%
, 278 nm) for BS-RNase.
The absorbance variation at 360 nm produced by the
addition of the proteins to a vesicle suspension was con-
tinuously measured on a Beckman DU-640 spectrophoto-
meter equipped with a high performance temperature
controller. In all assays, controls with no protein were
always carried out.
Intermixing of membrane lipids was analyzed by fluores-
cence RET assays [39]. The RET assay monitors the relief
of fluorescence energy transfer between a donor ⁄ acceptor
pair as the two probes dilute from labeled into unlabeled
bilayers. A vesicle population containing 0.6% (v ⁄ v) N-(lis-
samine rhodamine sulphonyl)-diacylphosphatidylethanol-
amine as acceptor and 1% (v ⁄ v) N-(7-nitro-2-1,3-
benzoxadiazol-4-yl)-dimyristoylphosphatidylethanolamine
as donor (Avanti Polar Lipids) was mixed with unlabeled
vesicles at 1 : 9 molar ratio. The increase of the fluores-

naphtalene (ANTS) ⁄ p-xylenebispyridinium bromide (DPX)
system [26,38,41]. PG vesicles contained 12.5 mm ANTS,
45 mm DPX, 20 mm NaCl and 10 mm Tris buffer pH 7.5.
Unencapsulated material was separated from the
ANTS ⁄ DPX containing vesicles by gel filtration on a
Sephadex G-75 column (Sigma) equilibrated in 10 mm Tris
buffer pH 7.5 containing 0.1 m NaCl and 1 mm EDTA.
Fluorescence emission at 510 nm was measured upon exci-
tation at 386 nm on a SLM-Aminco 8000 spectrofluorime-
ter. Internal calibration of the assays was as follows: 0%
leakage corresponded to the fluorescence signal of the
vesicles before protein addition (F
0
), and 100% leakage
was the fluorescence intensity measured upon detergent
addition [1% (v ⁄ v) Triton X-100] (F
max
). Percentages of
leakage were calculated as (%L) ¼ (F–F
0
) ⁄ (F
max
–F
0
) · 100.
Protein fluorescence emission was measured on a
SLM-Aminco 8000 spectrofluorimeter at 25 °C with 0.2 cm
optical path cells. CD measurements were performed on
a Jasco J-715 spectropolarimeter (Tokyo, Japan) with
thermostated cylindrical cells.

z axis was perpendicular to the center of the membrane
(Fig. 4);
(b) the major axis of the dimeric RNase was made coinci-
dent with the z axis of the system, hence orthogonal to the
membrane plane (Fig. 4);
(c) the dimeric RNase was rotated around the axis z (by
rotation angle r); values of EIE were recorded at 30° inter-
vals of r from 0 to 360°;
(d) the inclination of the dimer major axis with respect to
the z axis, hence to the membrane plane, was changed (by
inclination of angle i); EIE values were recorded at 30°
intervals of i from 0 to 180°;
(e) steps (c) and (d) were repeated until i reached the maxi-
mum value (180°).
It should be noted that after each rotation or inclination
the RNase dimer was translated: (a) to keep the mass cen-
tre of the protein (marked with a black dot in Fig. 4) onto
the axis z, and (b) to conserve the minimal distance
between protein and membrane at 3.5 A
˚
.
The EIE, i.e., the electrostatic contribution to the binding
energy (DG
bindingR ⁄ M
) of RNase (R) to a lipidic membrane
Electrostatic interactions and antitumor RNases E. Notomista et al.
3694 FEBS Journal 273 (2006) 3687–3697 ª 2006 The Authors Journal compilation ª 2006 FEBS
(M) model complexes was calculated by the method of the
grid energy differences [42] through the equation:
EIE ¼ DG

ing, this residue was modeled using the 7RSA structure as
template. Protein–lipidic membrane complexes of RNase
AA, RNase AA-G, RNase AA-GG, and HHP-RNase were
prepared using models of these dimers. The models of
RNase A covalent dimers in the nonexchanging conforma-
tion were prepared using the 7RSA structure and the struc-
ture of nonexchanging BS-RNase (1R3M) as template.
Briefly, two molecules of RNase A were superimposed to
the subunits of BS-RNase using the fit tools of swiss-
pdbviewer ( hence residues
19, 28, 31, 32, and when necessary 38 and 111 of RNase A
molecules were mutated to the corresponding residues of
BS-RNase, forcing the mutated side-chains to adopt the
conformation present in BS-RNase. The models were opti-
mized for energy minimization using the gromos imple-
mentation of swiss-pdbviewer (50 cycles of steepest descent
followed by 50 cycles of conjugate gradients and 50 cycles
of steepest descent). No clashes were detected at the dimer
interface of the models or in the surroundings of the
mutated residues. The models of RNase A covalent dimers
in the exchanging conformation were prepared using the
7RSA structure and the structure of exchanging BS-RNase
(1BSR) as template. The server swissmodel [44–46] was
used to prepare the homology model of exchanging RNase
AA dimer based on the exchanging structure of BS-RNase.
The hinge loop, i.e., the loop which adopts different confor-
mations in the exchanging and nonexchanging BS-RNase
dimers, was cut from the optimized homology model and
ligated to the models of nonexchanging RNase A dimers as
described above. The gromos implementation of swiss-pdb-

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Supplementary material
The following supplementary material is available
online:
Fig. S1. Structure of the BS-RNase–model membrane
complex with the highest negative EIE value. Lipids
are shown as van der Waals spheres and colored
according to the atom type. Solvent accessible surface
is shown for BS-RNase. The two subunits are colored
blue and red. In (A) the solvent accessible surface is
transparent to show secondary structure elements.
Fig. S2. Electrostatic field of BS-RNase at pH 7.0.
Blue meshes correspond to potential values ¼ +2
kTÆe
)1
and red meshes to potential values ¼ )2kTÆe
)1
.
Secondary structure elements are shown in green.
Fig. S3. Electrostatic field at pH 7.0 of monomeric and
dimeric RNases. The proteins have the same orienta-
tion with the ‘basic surface’ toward the top of the fig-
ure. Cyan meshes correspond to potential values ¼
+2 kTÆe
)1
and red meshes to potential values ¼ )2
kTÆe
)1
. The solvent accessible surfaces of the RNases