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; elec
Trang 1activity of dimeric RNases
Eugenio Notomista1, Jose´ Miguel Manchen˜o2, Orlando Crescenzi3, Alberto Di Donato1,
Jose´ Gavilanes4and Giuseppe D’Alessio1
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` di Napoli Federico II, Napoli, Italy
4 Departamento de Bioquı´mica y Biologı´a Molecular I, Universidad Complutense, Madrid, Spain
The superfamily of pancreatic-type RNases [1] includes
several members capable of carrying out ‘special’
actions, i.e., actions other than catalytic, although
strictly dependent on their catalytic, RNA degrading
action [2] These actions could be linked to
physiologi-cal functions, as in the case of the angiogenic action of
angiogenins [3], or due to the mere reflection in the
laboratory assays, mirrors proposed in the experiment,
of unknown functions, as may be the case of the
anti-fertility action of seminal RNase [4] Particular
atten-tion has been given to the cytotoxic acatten-tion of some
RNases, especially because they often appear to be
selective for tumor cells [5]
Many studies have been devoted to the mechanism
of action of these antitumor RNases However, a
con-clusive understanding of why these RNases kill cells, especially why some of them selectively kill tumor cells, has not been obtained The correlation has been stressed [6] between the ability of certain RNases to display a cytotoxic action and their ability to evade the strong, neutralizing action of the cytosolic RNase inhibitor (cRI), a 50-kDa protein containing 16 leu-cine-rich repeat motifs [7,8] In fact, onconase from Rana pipiens eggs [9], and seminal RNase from 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: dalessio@unina.it
(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 RNases resistant, or considered to be resistant to cRI, were assayed for their effects on negatively charged membranes Furthermore, we analyzed the electrostatic interaction energy of the RNases complexed in silico with
a model membrane The results of this study suggest that close correlations can be recognized between the cytotoxic action of a dimeric RNase and its ability to complex and destabilize negatively charged membranes
Abbreviations
ANTS, 1,3,6-trisulphonate-8-aminonaphtalene; cRI, cytosolic RNase inhibitor; DMPG, dimyristoylphosphatidylglycerol; DMPS,
dimyristoylphosphatidylserine; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPX, p-xylenebispyridinium bromide; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EIE, electrostatic interaction energy; HP-RNase, human pancreatic RNase; kT, product of the Boltzmann constant by absolute temperature in K; PG, phosphatidylglycerol; RET, resonance energy transfer; SVT2, malignant murine fibroblasts.
Trang 2cells when they are engineered into cRI-resistant
RNases [6,11–14] These results led to the description
of the cytosolic RNase inhibitor as a sentry to guard
cells from, and inactivate, potentially cytotoxic foreign
RNases [6]
Recently however, it has been reported [15] that
HeLa cells deprived through RNA silencing of any cRI
in their cytosol, are still not affected by noncytotoxic
RNases This indicates that noncytotoxic, cRI-sensitive
RNases do not become cytotoxic when cRI, the
postu-lated ‘sentry’, is absent Furthermore, artificial dimers
of RNase A, obtained through lyophilization from
acetic acid, have been found to bind with high affinity
the cytosolic RNase inhibitor [16], but also to be
cyto-toxic on malignant cell lines [17] These results have
cast shadows on the prospect that the antitumor action
of RNases be based solely, or primarily, on its
resist-ance to the cytosolic RNase inhibitor
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, cytocyto-toxic 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 allowing, or not, an RNase to pass from one cell com-partment to another On the other hand, the RNase itself is expected to present to the membrane which it should permeate structural elements that determine recognition and favor its access through that mem-brane
For a systematic study of the interactions between membranes and cytotoxic RNases, we report here the effects on the stability of artificial membranes of nat-ural and engineered dimeric RNases with different degrees of cytotoxic activity Their electrostatic interac-tions with a model membrane are then compared through in silico analyses The results of these investi-gations indicate that close correlations can be recog-nized between the ability of an RNase to destabilize
a membrane, its attraction to a negatively charged membrane, and its cytotoxic action
Results
The RNases The RNases investigated in this study are described in Table 1 They are cationic proteins with cytotoxic activ-ity: BS-RNase [10] is a natural dimeric RNase;
RNase-AA [21], RNase-RNase-AA-G [21], and RNase-RNase-AA-GG [22] are dimeric variants engineered from bovine pancreatic RNase A; HHP-RNase [11] is a dimeric variant engine-ered from human pancreatic RNase (HP-RNase)
Effects on vesicle aggregation All RNases investigated are cationic proteins, with high isoelectric point values Thus we investigated the effects
Table 1 Natural and engineered RNases investigated in this study IC50is the RNase concentration producing half-maximal cytotoxicity on SVT2 malignant cells [5,11,14].
Trang 3of 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
of effects exerted by the RNases under investigation,
only to some extent less powerful
As previously reported [26], seminal RNase
(BS-RNase), a naturally dimeric RNase, exerts a very
strong effect on the aggregation of DMPG vesicles,
whereas monomeric RNases, namely RNase A and a
monomeric derivative of BS-RNase, exert no
signifi-cant effects However, the results illustrated in Fig 1
indicate that the quaternary structure of an RNase is
not a prerequisite for its ability to aggregate vesicles,
as RNase A does not acquire this ability upon
dimeri-zation into RNase-AA
The replacement in dimeric RNase-AA of one
negat-ively charged residue (Asp38) with an uncharged Gly
residue generates an RNase variant (RNase-AA-G)
capable of aggregating vesicles The result is even more
striking when two negatively charged residues (Asp38
and Glu111) are replaced with Gly residues, as in
RNase-AA-GG As shown in Fig 1, the effects on
DMPG vesicles of RNase-AA-GG are similar to those obtained with BS-RNase, the RNase with the most powerful aggregating effect When DMPS vesicles were used, similar effects were observed, but were less evi-dent (data not shown)
It should be added that CD spectra of the three RNase A dimers (RNase AA, -AA-G, and -AA-GG)
in the far-UV and near-UV regions were virtually iden-tical, with a slightly different spectrum for RNase-AA-G in the near-UV region Thermal denaturation, measured through the variation of ellipticity at
218 nm, gave an identical midpoint of denaturation at
62C 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 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.
Trang 4The 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
effects on the thermotropic behavior of the tested
membranes Surprisingly, also monomeric HP-RNase,
with no effect on membrane aggregation (see above),
was found to affect to a certain extent the bilayer
fluidity (Fig 2) Similar, albeit minor effects, were
observed when the experiments were carried out on
DMPS vesicles (data not shown)
Effects on membrane fusion
This parameter was evaluated by determining the
inter-mixing of DMPG phospholipids between unlabeled
vesicles and vesicles labeled with a donor⁄ acceptor pair
of fluorescent probes When the RNase under test
pro-moted intermixing, a decrease of the energy transfer
between the two probes was measured as resonance
energy transfer (RET) Under the test conditions (see
below), a RET value of 80% indicated that the RNase
under test did not promote any perturbations of the
vesicles bilayer
Figure 3 shows that all the RNases that promoted
membrane aggregation also engendered membrane
fusion Even native, monomeric HP-RNase, with no
effects on membrane aggregation, produced a modest
intermixing RNase-AA-IG instead, which induced a
low, but significant degree of membrane aggregation,
did not show any effects on vesicle intermixing No
effects of membrane fusion were observed with RNase
A and RNase-AA, or when DMPS vesicles were tested
Correlations between the effects of RNases on membranes and their electrostatic charges
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, DA360 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 membrane constructed with DMPG lipids (see below)
A series of EIE values were determined by varying the orientation of each RNase towards the membrane (Fig 4) The results of these analyses revealed that a single highest negative value of EIE (EIEmax) could be estimated for each dimeric RNase This value was found for all RNases at the unique orientation with
Table 2 Effects on membrane vesicles and properties of the dimeric RNases investigated in this study.
RNase
Aggregation (%)
Fluidity alteration (%)
Fusion (%) Positive net charge
EIEmax (kT)
Fig 3 Lipid mixing of DMPG vesicles induced by RNases.
Symbols: (e) BS-RNase; (+) RNase AA-GG; (n) HHP-RNase; (n)
HP-RNase; (h) RNase AA-G; (m) RNase AA; (·) RNase A.
Trang 5r 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-terC-ter-minal 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
angle ‘r’) and variation of the inclination (inclination angle ‘i’) with
respect to the plane of the membrane (parallel to the xy plane).
Cter and Nter are the C-terminal and N-terminal structural regions,
respectively, of the two A and B monomers of the dimeric RNase.
The black circle between the monomers indicates the mass center
of the dimer.
Trang 6RNases oriented with the negative end of the dipoles
toward the bottom, and the positive one toward the
top of the figure Figure S4 shows instead the
N-ter-minal surfaces colored by charge As expected, in the
orientation that provides the strongest attraction
between the RNase and the model membrane the
RNases dipoles are perpendicular to the negatively
charged membrane
In Table 2 the EIEmax values of the RNases
investi-gated in this study are tabulated and compared to the
RNases’ net charge values, and their ability to
desta-bilize negatively charged membranes A satisfactory
correlation is evident not only between the RNases’
EIEmax values and their positive net charges, but also
with their ability to destabilize a membrane
We then considered the possibility of a correlation
between the cytotoxic action of the investigated dimeric
RNases and their ability to destabilize and permeate a
membrane, which in turn appears to be linked to the
attraction of the RNase to a membrane, as evidenced
by the RNase negative EIEmaxvalues As a measure of
the RNase cytotoxicity we used the reciprocal of the
IC50parameter The values of IC50, 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 EIEmax values of
dimeric, cytotoxic RNases were plotted against the
IC50values 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 RNases capable of exerting this effect are mostly obscure As mentioned above, a primary if not exclu-sive role of the cytosolic inhibitor in the mechanism of antitumor RNases cannot be generalized The only firmly established fact is that the cytotoxic action of RNases is based on their RNA degrading activity [5] The cytosolic localization of target RNA(s), and previ-ous data on the intracellular journey of antitumor sem-inal RNase [20], indicate that cytotoxic RNases must
be capable of permeating specific membrane compart-ments to reach the cytosol Furthermore, antitumor seminal RNase has been found to alter the stability of negatively charged membrane vesicles, with no effects
on positively charged membranes [26]
It has long been known that subcellular membranes from malignant tissues have a higher content of negat-ively charged lipids when compared to normal tissues [28] More recently, it has been found [29] that some C2 domains, folded modules from proteins involved in signal transduction and vesicle trafficking, specifically associate to negatively charged membranes
As all these data are suggestive of a role of mem-branes, in particular of negatively charged memmem-branes,
in the mechanism of action of RNases, we investigated the ability of a series of RNases to destabilize model membranes as a representation of their potential to cross intracellular membranes through their journey
to the cytosol
We found that cytotoxic, dimeric RNases, natural or engineered, are able to destabilize negatively charged 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.
Trang 7until 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
to destabilize membranes with their specific
mem-brane-attractive region, and (b) their cytotoxic
action This finding strongly suggests that the
elec-trostatic surface potential of a defined 3D district of
these proteins has a role in determining their
cyto-toxic action
It has been reported that when negative charges are
abolished in noncytotoxic RNases, these become
cyto-toxic [30,31] This result, obtained with heterogeneous
protein products, only indicates that molecules with a
high content of positive charges are generally toxic to
cells However, a high content of basic residues may
not be the only basis of RNase cytotoxicity Two
RNases, homologous to the RNases investigated in
this study, have been isolated from human eosinophils,
namely eosinophil-derived neurotoxin (EDN) and
eosi-nophil cationic protein (ECP) They are RNases with a
high sequence identity (66%), a very high content of
basic residues and high isoelectric points (pI¼ 11.9
and 10.4, for EDN and ECP, respectively) Yet only
ECP, not EDN, displays cytotoxic activity on
eukary-otic and bacterial cells [32]
Given the recently reported cytotoxic activity of the
noncovalent dimers of RNase A [17], intriguing when
compared to their sensitivity to the cytosolic inhibitor
(see above), we tested their interaction with the model
membrane described above for RNase covalent
di-mers, and calculated their EIEmax values Two distinct
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 EIEmax values, a value of EIE was obtained
com-parable to those calculated for the other dimeric
RNase As shown in Fig 7, the EIEmax values
obtained for the two dimers satisfactorily correlate
with the dimers IC50 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 electrostatic features and structural element(s) for destabilizing and eventually permeating membranes, and that intracellular membranes play a decisive role
in defining the antitumor action of an RNase
Experimental procedures
Assays with membranes
Synthetic DMPG and DMPS were purchased from Avanti Polar Lipids (Alabaster, AL) Egg yolk phosphatidyl-glycerol (PG) was from Sigma (St Louis, MO) Unless indicated, lipid vesicles were prepared at 1 mgÆmL)1 phos-pholipid concentration in the indicated buffer, by extrusion through two 0.1-lm polycarbonate filters (Nuclepore, Costar, Cambridge, MA) in an Extruder (Lipex Biomem-branes Inc., Vancouver, Canada), as described previously [26,36–38]
Protein concentration was determined from absorbance measurements on a Beckman DU-7 (Palo Alto, CA) or Uvikon 930 (Kontron Instruments, Milan, Italy) spectro-photometers using the following extinction coefficients:
Fig 7 Correlation between antitumor activity on HL-60 cells (n) and ML-2 cells (h) and the highest negative EIE values for BS-RNase (BS) and RNase A noncovalent dimmers N d , the dimer with N-terminal swapping between subunits; C d , the dimer with C-terminal swapping between subunits Antitumor activity was plot-ted (see legend to Fig 6) as the reciprocal of IC 50 reported values [17] The square correlation coefficients (R2) are shown.
Trang 80.695 (E0.1%, 280 nm) for RNase A and its variants, 0.465
(E0.1%, 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-cence emission at 530 nm (donor emission) upon excitation
at 450 nm (4 nm slit width for both excitation and emission
beams), was continuously recorded on a SLM-Aminco 8000
spectrofluorimeter (Urbana, IL) Measurements were
per-formed in a thermostated cell holder at 37C The
percent-ages of RET were calculated according to the equation
(%RET)¼ (1–F ⁄ F0)· 100 where F and F0 are the
fluores-cence emission intensities at 530 nm upon addition of the
corresponding protein, and the value measured with vesicles
lacking the acceptor probe, respectively Normalization of
the measurements was performed by independent
experi-ments in the presence of 1% (v⁄ v) Triton X-100 [26,36,39]
Fluorescence depolarization measurements were
per-formed on a SLM-Aminco 8000 spectrofluorimeter
equip-ped with 10 mm Glan-Thompson polarizers (Urbana, IL)
Cells of 0.2-cm optical path were used Slit widths were
4 nm both for excitation and emission beams Labeling of
the vesicles with DPH (Aldrich, Milwaukee, WI) was
per-formed as previously described [40] The degree of
polariza-tion of the fluorescence emission of DPH was measured at
425 nm upon excitation at 365 nm, after equilibration of
the sample at the required temperature Independent
experi-ments demonstrated a negligible contribution of the protein
to the degree of polarization of the fluorescence probe
Leakage of vesicle aqueous contents induced by the
RNases was measured by the
1,3,6-trisulphonate-8-amino-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 (F0), and 100% leakage was the fluorescence intensity measured upon detergent addition [1% (v⁄ v) Triton X-100] (Fmax) Percentages of leakage were calculated as (%L)¼ (F–F0)⁄ (Fmax–F0)· 100 Protein fluorescence emission was measured on a SLM-Aminco 8000 spectrofluorimeter at 25C with 0.2 cm optical path cells CD measurements were performed on
a Jasco J-715 spectropolarimeter (Tokyo, Japan) with thermostated cylindrical cells
Determination of EIE
The EIE for RNase–lipidic membrane model complexes was carried out using delphi version 4 [42,43]
A model membrane in a crystal-like state was modeled containing 192 palmityl-stearyl-phosphatidylglycerol mole-cules per layer arranged in a parallelepiped with dimen-sions 117(l)· 108(w) · 54(h) A˚ The phospholipids were arranged in a compact hexagonal lattice with the fatty acid tails and the terminal glycerol moiety completely extended Each layer was composed of seven rows of 15 molecules separated by six rows of 16 molecules Within each row the distance between the P atoms of two adjacent phosphates was 8.88 A˚, whereas the minimum distance between the P atoms of adjacent rows was 8.98 A˚ The minimum distance between two P atoms of phosphates of the two different layers was 48.96 A˚
For each RNase several RNase–lipidic membrane model complexes were generated through an automated procedure which included the following steps:
(a) the model of the membrane was oriented with each layer parallel to the xy plane so that all the phosphorus atoms of each layer had the same z coordinate and the
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 (DGbindingR ⁄ M) of RNase (R) to a lipidic membrane
Trang 9(M) model complexes was calculated by the method of the
grid energy differences [42] through the equation:
EIE¼ DGbindingR=M¼ GR=M GR GM
where GR ⁄ M, GRand GMare the grid energies of the
com-plex, of the RNase alone and of the membrane alone,
respectively
The following delphi parameters were used for the
calcu-lations of the grid energy values: gsize¼ 250; scale ¼ 1.2;
indi¼ 4.0; exdi ¼ 80.0; prbrad ¼ 1.4; salt ¼ 0.1; ionrad ¼
2.0; bndcon¼ 2; maxc ¼ 0.0001; linit ¼ 800 Standard
for-mal charges at pH 7.0 were attributed to protein atoms,
whereas a charge of )0.5 was attributed to the nonester
oxygen atoms of each phosphate group in the membrane
(total charge of each phosphate group¼)1)
RNase structures and modeling
Protein–lipidic membrane complexes of BS-RNase, RNase
A, RNase AA-N, RNase AA-C and HP-RNase were
pre-pared using the crystallographic structures of these proteins
(PDB codes 1BSR, 7RSA, 1A2W, 1F0V and 1Z7X,
respect-ively) As in the 1Z7X structure of HP-RNase Lys1 is
lack-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 (http://www.expasy.org/spdbv); 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-viewer was used to optimize geometry of the hinge loop
after ligation Also in this case no clashes were detected at
the dimer interface of the models or in the surroundings of the hinge loop residues Likewise, the models of nonex-changing and exnonex-changing HHP-RNase were prepared start-ing from 1Z7X The programs pymol (DeLano Scientific LLC, San Francisco, CA), and swiss-pdbviewer were used
to inspect RNase structures and models
Acknowledgements
The authors are indebted to Dr Renata Piccoli, for critical reading the manuscript and helpful suggestions; and to Dr Valeria Cafaro, Aurora Bracale, Antonella Antignani and Sonia Di Gaetano for preparing some
of the RNase variants used in this study
This work was supported by the Italian Association for Cancer Research, the Ministry of University and Research (Italy) and the Ministerio de Educacion y Ciencia (Spain)
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