Báo cáo Y học: Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin a-sarcin potx

Arginine 121 is a crucial residue for the specific cytotoxic activity of
the ribotoxin a-sarcin
Manuel Masip, Javier Lacadena*, Jose
´
Miguel Manchen
˜
o, Mercedes On
˜
aderra, Antonio Martı
´
nez-Ruiz†,
A
´
lvaro Martı
´
nez del Pozo and Jose
´
G. Gavilanes
Departamento de Bioquı
´
mica y Biologı
´
a Molecular, Facultad de Quı
´
mica, Universidad Complutense, Madrid, Spain.
a-Sarcin, a cyclizing ribonuclease secreted by the mould
Aspergillus giganteus, is one of the best characterized
members of a family of fungal ribotoxins. This protein
induces apoptosis in tumour cells due to its highly specific
activity on ribosomes. Fungal ribotoxins display a three-
dimensional protein fold similar to those of a larger group of

microbial noncytotoxic RNases, represented by RNases T1
and U2. This similarity involves the three catalytic residues
and also the Arg121 residue, whose counterpart in RNase
T1, Arg77, is located in the vicinity of the substrate phos-
phate moiety although its potential functional role is not
known. In this work, Arg121 of a-sarcin has been replaced
by Gln or Lys. These two mutations do not modify the
conformation of the protein but abolish the ribosome-
inactivating activity of a-sarcin. In addition, the loss of the
positive charge at that position produces dramatic changes
on the interaction of a-sarcin with phospholipid membranes.
It is concluded that Arg121 is a crucial residue for the
characteristic cytotoxicity of a-sarcin and presumably of the
other fungal ribotoxins.
Keywords: Aspergillus protein; cytotoxin; protein –lipid
interaction; ribotoxin; a-sarcin.
a-Sarcin is the best characterized member of a family of
fungal cytotoxic ribosome-inactivating proteins (ribotoxins)
that cleave one single phosphodiester bond at an evolu-
tionarily conserved sequence of the larger rRNA [1], impair-
ing the binding of elongation factors EF-1 and EF-2 [2].
This well known exquisite ribonucleolytic action in cell-free
systems has been also recently observed in intact cells,
namely human rhabdomyosarcoma cells, where the toxic
effect produces cell death via apoptosis [3]. a-Sarcin is
internalized by the target cells via endocytosis involving
acidic endosomes and the Golgi [3,4]. This traffic might be
related to the known ability of a-sarcin to interact with
model membranes. In fact, besides its specific ribonuclease
activity, this highly polar protein binds to acidic lipid vesicles

promoting their aggregation and fusion [5,6] and resulting in
translocation of the protein across the model membranes [7,8].
Ribotoxins of the a-sarcin family display a high degree of
sequence similarity although they are secreted by a variety
of different moulds [9–11], i.e. restrictocin and mitogillin,
two other members of this family, display 85% sequence
identity to a-sarcin, which is only one residue longer
[12–14]. These ribotoxins belong to a larger group of micro-
bial extracellular RNases, represented by the noncytotoxic
RNases T1 and U2 [10,11,15], based on their sequence and
three-dimensional structure similarities. Thus, a-sarcin [15],
restrictocin [16] and RNase T1 [17] display an identical
architecture and connectivity of the secondary structure
elements in the three proteins. The catalytic mechanisms of
a-sarcin and RNase T1 are similar [18–20] and related to
that of bovine pancreatic RNase A [21]. They behave as
cyclizing RNases, the overall reaction being composed of
two steps, cyclization followed by cleavage of the 2
0
,3
0
-
cyclic product formed [18,21–23]. In RNase T1, His92 acts
as the general acid and Glu58 as the general base during the
first step of the reaction. The hydrolysis of the cyclic
derivative is performed by the same groups, but their roles
are reversed [23]. In a-sarcin the same roles are fulfilled by
His137 and Glu96, although differences between the two
enzymes regarding the individual pK
a

values of these
catalytic residues and optimum pH have been observed
[19,20]. Analysis of the three dimensional structure of
RNase T1 complexed with different inhibitors or substrate
analogues has revealed the presence of some other side
chains in the vicinity of the substrate phosphate moiety
[22,24]. This is the case for His40 and Arg77, their counter-
parts in a-sarcin being His50 and Arg121 [10,11] occupying
geometrically conserved positions [15,25] (Fig. 1). Site-
directed mutagenesis experiments have revealed that His40
Correspondence A. Martı
´
nez del Pozo or J. G. Gavilanes,
Departamento de Bioquı
´
mica y Biologı
´
a Molecular I, Facultad de
Quı
´
mica, Universidad Complutense, 28040 Madrid, Spain.
Fax: 1 34 91 3944159, Tel.: 1 34 91 3944158,
E-mail: or
Enzymes: mitogillin and restrictocin (RNMG_ASPRE, P04389,
EC 3.1.27 ); ribonuclease A (RNP_BOVIN, P00656, EC 3.1.27.5);
ribonuclease T1 (RNT1_ASPOR, P00651, EC 3.1.27.3); ribonuclease
U2 (RNU2_ASTSP, P00654, EC 3.1.27.4); a-sarcin (RNAS_ASPGI,
P00655, EC 3.1.27.10). All accession codes are for the SWISS-PROT
data base.
*Present address: Facultad de Biologı

´
a, Universidad SEK, 40003
Segovia, Spain.
†Present address: Centro de Investigaciones Biolo
´
gicas, CSIC,
Vela
´
zquez 144, 28006 Madrid, Spain.
Note: a web page is available at
(Received 2 July 2001, revised 4 September 2001, accepted 1 October
2001)
Abbreviations: ApA, adenylyl(3
0
!5
0
)adenosine; Myr
2
GroPGro,
dimyristoylglycerophosphoglycerol; Nbd-Myr
2
GroP Etn, [N-(7-nitro-
2-1,3-benzoxadiazol-4-yl)-dimyristoylglycerophosphoethanolamine;
rhodamine-GroP Etn, N-(lissamine rhodamine B sulfonyl)-
diacylglycerophosphoethanolamine; RNase, ribonuclease.
Eur. J. Biochem. 268, 6190–6196 (2001) q FEBS 2001
of RNase T1 [23,26,27] and His50 of a-sarcin [20] are
involved in catalysis rather than in substrate binding. The
role of Arg77 has not yet been established as all attempts to
isolate any RNase T1 Arg77 mutant have been unsuccessful

[23]. Hydroxylamine treatment has been used to produce
point mutations in recombinant mitogillin produced by yeast
[28]. Only a few transformants survived upon induction,
suggesting that the affected residues, one of them Arg120
(the counterpart of Arg121 in a-sarcin), has a crucial role in
the ribonucleolytic catalysis [28].
The work herein presented deals with the production,
purification and characterization of two a-sarcin mutants
where Arg121 has been substituted by Lys (R121K) or Gln
(R121Q). The results obtained show that this residue is
involved in the specific ribonucleolytic activity of ribotoxins
against ribosomes and also in the interaction with
membranes.
EXPERIMENTAL PROCEDURES
DNA manipulations
All materials and reagents were of molecular biology grade.
Cloning procedures and bacteria manipulations were carried
out according to standard methods [29] as described pre-
viously [20,30,31]. Oligonucleotide site-directed mutagen-
esis was used to replace Arg121 with Lys (R121K) or Gln
(R121Q) as described previously [20,30–32]. The muta-
genic primers used were 5
0
-CCTGGCCCGGCGAAGGTCA
TCTACACC-3
0
for R121K and 5
0
-CCTGGCCCGGCG
CAGGTCATCTACACC-3

0
for R121Q (the site of mutation
is underlined). The E. coli strains used were BW313
{(HfrKL16 pol45 [LysA (61– 62)] dut1 ung1 thi1 relA1] to
obtain the uridine-rich ssDNA, DH5aF
0
({[F
0
] endA1 hsdR17
(r
–
K
m
–
K
) supE44 thi-1 recA1 gyrA (NaI
R
) relA1 D(lac-
ZYA-argF ) U169 deoR [B80 dLac D(lacZ ) M15]}) to
obtain the expression constructs, and BL21(DE3)(F
0
ompT[lon] hsd
B
(r
–
B
m
–
B
)) to produce the proteins. The

thioredoxin producing plasmid (pT-Trx) [33] was a generous
gift of S. Ishii, from the Riken Tsukuba Life Science Center
(Riken, Japan).
Proteins production and purification
BL21(DE3) cells cotransformed with pT-Trx and the corre-
sponding a-sarcin mutant plasmid were used to produce the
mutant proteins, which were purified as described for the
wild-type protein [30,34]. Fungal wild-type a-sarcin was
produced and purified as previously reported [30,35,36].
Polyacrylamide electrophoresis, tryptic digestions, acid
hydrolysis of proteins, peptide maps, and amino-acid
analysis were also performed according to standard pro-
cedures described previously [20,30,35,37].
Spectroscopic characterization
Proteins were dissolved in either 50 m
M sodium phosphate,
pH 7.0, or 50 m
M sodium acetate, pH 5.0, both containing
0.1
M NaCl, as required and centrifuged at 14 000 g for
5 min. Absorbance measurements were carried out on a
Uvikon 930 spectrophotometer at 100 nm
:
min
21
scanning
speed, at room temperature, and in 1-cm optical-path cells.
CD spectra were obtained on a Jasco 715 spectropolarimeter
Fig. 1. Diagrams corresponding to the three-dimensional structure
of a-sarcin constructed from the atomic coordinates deposited in the

PDB (accession code [1DE3], Images were
generated by the
MOLMOL program [52]. (A), Ribbon diagram of the
whole protein molecule where selected residues (His50, Glu96, His137,
Arg121, in black; Tyr48, Trp51, Tyr106, in grey) are shown. (B), Detail of
the residues not labelled in part A. (C), Active site residues of a-sarcin
(black)/RNase T1 (grey), after fitting of the backbone atoms of the
residues involved in the five-strands central b sheet of both proteins. The
protein view is maintained through the three diagrams.
q FEBS 2001 Role of Arg121 on cytotoxicity of a-sarcin (Eur. J. Biochem. 268) 6191
at 0.2 nm
:
s
21
scanning speed; 0.1- and 1.0-cm optical-path
cells were used in the far- and near-UV wavelength range,
respectively. Mean residue weight ellipticities were
expressed in units of degrees
:
cm
2
:
dmol
21
. Extinction coef-
ficients E
0.1%
(280 nm, 1-cm optical path) were calculated
from the corresponding absorbance spectra and amino-acid
analyses. Thermal denaturation profiles were obtained by

measuring the temperature dependence of the ellipticity at
220 nm in the range of 25–85 8C; the temperature was
continuously changed at a rate of 0.5 8C
:
min
21
. The tem-
perature values at the midpoint of the thermal transition (T
m
)
were calculated assuming a two-state unfolding mechanism
[38–40]. Fluorescence emission spectra were obtained on a
SLM Aminco 8000 spectrofluorimeter at 25 8C in 0.2-cm
optical-path cells, as described previously [20].
Ribonucleolytic activity
The specific ribonucleolytic activity of a-sarcin against intact
ribosomes was followed by detecting the release of the 400
nucleotide a-fragment [1,41,42] from a cell-free reticulo-
cyte lysate (Promega) as described previously [20,30,31].
The production of the 400 nucleotide a-fragment was
visualized by ethidium bromide staining after electrophor-
esis on 2.4% agarose. The activity of the purified proteins
against polyadenylic acid was assayed after subjecting the
samples to an electrophoretic procedure in 15% polyacryl-
amide gels containing 0.1% SDS and 0.3 mg
:
mL
21
poly(A).
This method, designated as a zymogram, was based on one

previously described [31,43]. Proteins exhibiting ribonuclease
activity appear as colourless bands after proper destaining
treatment. Hydrolysis of adenylyl(3
0
!5
0
)adenosine (ApA)
by a-sarcin and the mutant variants was performed as
described elsewhere [18] at pH 5.0. The reaction products
were resolved by HPLC with a phosphate-methanol gradient
as described [18,44]. All of these assays were performed
with the corresponding controls to test potential nonspecific
degradation of the substrates, which did not occur under the
conditions used.
Protein–lipid interactions
Dimyristoylglycerophosphoglycerol (Myr
2
GroPGro) was
purchased from Avanti Polar Lipids Inc. Vesicles were
formed by hydrating a dry lipid film with Tris buffer (15 m
M
Tris, pH 7.5, containing 0.1 M NaCl and 1 mM EDTA) for
60 min at 37 8C. The lipid suspension was subjected to five
cycles of extrusion through two stacked 0.1 mm (pore
diameter) polycarbonate membranes [45]. The average
diameter of the vesicle population was 100 nm (85% of the
vesicles in the range 75–125 nm), as determined by electron
microscopy studies [45]. Phospholipid concentration was
determined as described [46]. Aggregation of phospholipid
vesicles was monitored by measuring the increase in the

absorbance at 360 nm of a suspension of Myr
2
GroPGro
vesicles in Tris buffer (30 m
M final lipid concentration)
after addition of a small aliquot of a freshly prepared
solution of protein [5]. Intermixing of membrane lipids was
measured by fluorescence energy transfer assays [47] as
described [45,48]. A decrease in the donor-to-acceptor,
[N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylglycero-
phosphoethanolamine (Nbd-Myr
2
GroP Etn) and N-(lissamine
rhodamine B sulfonyl)-diacylglycerophosphoethanolamine
(rhodamine-GroP Etn), respectively, fluorescence energy
transfer indicates lipid-mixing between membranes. Bind-
ing of the proteins to the lipid vesicles was analysed by
measuring the free protein concentration in the supernatant
obtained by centrifugation (160 000 g for 20 min, Beckman
Airfuge) of protein –vesicle mixtures at different protein/
lipid ratios. In this particular case, the buffer used was
15 m
M Mops, pH 7.0, containing 0.1 M NaCl and 1 mM
EDTA. The protein concentration was calculated by
densitometering the Coomasie-stained SDS-gels resulting
from subjecting supernatant aliquots to SDS/PAGE. A
calibration plot, volumogram obtained (density or quantity
of a spot calculated from its volume made of the sum of all
pixel intensities composing the spot obtained with a photo
documentation system UVI-Tec using the software facility

UVISOFT UVI BAND) vs. protein concentration (determined
by amino-acid analysis of acid-hydrolysed protein samples)
was used for these calculations. Other experimental details
were as reported previously [45,48,49]. Control assays
without protein were always performed.
RESULTS
Purification and structural characterization
Both mutant variants of a-sarcin, R121K and R121Q, were
purified to homogeneity according to their behaviour on
SDS/PAGE, and displayed a single immunoreactive band
when stained with anti-(a-sarcin) Ig in Western-blots ana-
lysis. Yields were about 5.0 mg per litre of E. coli culture
for the R121K variant but much lower for R121Q (less than
0.3 mg per L). Amino-acid analysis as well as tryptic map of
the purified mutants were consistent with the residue
substitutions planned. The calculated E
0.1%
(280 nm, 1-cm
optical path) were 1.30 and 1.12 for R121K and R121Q,
respectively, in reasonable agreement with the value of 1.34
reported for the wild-type protein [37]. The far-UV CD
spectra of both mutant variants were indistinguishable from
that already reported for the natural fungal a-sarcin
[20,30,36,37] (Fig. 2A). Small differences were observed
in the near-UV wavelength range (Fig. 2B). Only minor
differences were also observed in terms of protein
Fig. 2. Circular dichroism spectra of fungal wild-type a-sarcin (X)
and its R121K (W) and R121Q (K) mutant variants. (A) Far- and (B)
near-UV. Mean residue weight ellipticities (u
MRW

) are expressed in
units of degrees
:
cm
2
:
dmol
21
.
6192 M. Masip et al. (Eur. J. Biochem. 268) q FEBS 2001
fluorescence emission. Thus, the fluorescence emission of
R121K coincided with that of the wild-type protein while
R121Q showed identical Trp emission but an increased
(1.5-fold) tyrosine contribution (Fig. 3).
R121K and R121Q displayed a decreased stability in
comparison to the wild-type protein. Denaturation of these
proteins was studied by analysing the thermal variation
of their far-UV circular dichroism properties (Fig. 4). The
T
m
value for the wild-type protein was 62 8C at pH 5.0,
the optimum pH for both enzyme activity and stability
[19,20,50]. The T
m
of the R121K and R121Q mutant
variants was 56 8C and 53 8C, respectively, which corre-
sponded to a decreased stability of 8.36 and 12.54 kJ
:
mol
21

,
respectively, according to the calculated DDG values.
Enzymatic characterization
Neither of the two mutants produced the a-fragment result-
ing from the specific ribonucleolytic activity of these fungal
ribotoxins against ribosomes. Forty nanograms of the wild-
type protein was enough to completely release the a-fragment
from the larger rRNA under the standard assay conditions
[20,30,31], but no cleavage was observed even when 100 ng
of the mutant variants were assayed. In addition, although
wild-type a-sarcin degrades polyadenylic acid on a zymo-
gram assay, R121K and R121Q did not cleave the homo-
polynucleotide even when 500 ng of protein were assayed,
the lowest detection limit of this assay being 50 ng of wild-
type protein [20]. A quantitative analysis of the ribonuclease
activity of a-sarcin has been developed by using the
dinucleotide ApA as substrate [18]. Both mutant variants
hydrolysed ApA. They displayed a K
m
value similar to
that of the wild-type protein although they exhibited a
lower catalytic efficiency (Table 1), thus suggesting that the
mutated residue is involved in catalysis rather than in
substrate binding.
Interaction with phospholipid vesicles
The interaction of a-sarcinwithmodelphospholipid
vesicles through electrostatic and hydrophobic interactions
is well documented. The ribotoxin promotes aggregation of
vesicles and mixing of phospholipids from different bilayers
[5,6]. The mutant variant R121K promoted the same effects

than the wild-type protein and these became saturated at the
same protein/phospholipid molar ratio, although R121K
displayed a slightly higher affinity for the target vesicles
(1.3-fold that of the wild-type protein) (Fig. 5). However,
Fig. 3. Fluorescence emission spectra of wild-type a-sarcin (WT) and its R121K and R121Q mutant variants. (1), Fluorescence emission
spectra for excitation at 275 nm; (2), fluorescence emission spectra for excitation at 295 nm (tryptophan contribution) normalized at wavelengths
above 380 nm where the tyrosine emission is negligible; (3) calculated difference spectra of (1) minus (2) (tyrosine contribution). Fluorescence
emission is expressed in arbitrary units considering the intensity at the wavelength of the emission maximum of the wild-type protein for excitation at
275 nm as 1.0.
Fig. 4. Thermal denaturation profiles of fungal wild-type a-sarcin
(X) and its R121K (W)andR121Q(K)mutantvariants.
Measurements were performed by continuously recording the mean
residue weight ellipticity at 220 nm, expressed in units of
degrees
:
cm
2
:
dmol
21
.
Table 1. Activity of a-sarcin and its mutant variants against ApA at
pH 5.0. Kinetic parameters (^ SD) determined from the transesterifi-
cation of ApA by linear regression analysis of double reciprocal plots
from three different determinations [18].
Protein K
m
(mM) k
cat
(s

21
) k
cat
/K
m
(M
21
:
s
21
)
Wild-type 40 ^ 4 (27.0 ^ 1.0) Â 10
25
6.7 ^ 0.7
R121K 36 ^ 4 (1.2 ^ 0.2) Â 10
25
0.3 ^ 0.1
R121Q 27 ^ 4 (7.9 ^ 0.3) Â 10
25
2.3 ^ 0.4
q FEBS 2001 Role of Arg121 on cytotoxicity of a-sarcin (Eur. J. Biochem. 268) 6193
R121Q exhibited a low affinity, 0.2-fold that of the wild-
type a-sarcin, which corresponded to a very low affinity to
aggregate vesicles and promote lipid-mixing between
bilayers (Fig. 5).
DISCUSSION
Both purified mutant forms of a-sarcin display the same
global fold as the wild-type protein according to the spec-
troscopic and structural characterization performed. Thus,
the modified activities of these mutants to cleave RNA

substrates or to interact with phospholipid vesicles cannot be
considered as a consequence of an altered native confor-
mation. In fact, no secondary structure variations have been
detected and only slight differences were observed in terms
of extinction coefficient and environment of aromatic amino
acids (near-UV circular dichroism and fluorescence
emission spectra). Tyr48, Trp51 and Tyr106 are located in
the vicinity of the mutated 121st position (their a-carbons
are about 11 A
˚
distant in the same side of the central b sheet
of the protein) (Fig. 1) [15]. Thus, such differences might be
attributed to changes in the local environment of these
residues. In fact, the side-chain of Trp51 has been proven to
be responsible for most of the ellipticity signal of a-sarcin
observed in the near-UV wavelength region but it does not
show fluorescence emission [51]. Therefore, the near-UV
ellipticity variations observed for both mutant variants arise
from local changes affecting Trp51, while the change on the
fluorescence of R121Q, 1.5-fold increased Tyr emission, is
related to Tyr48 and/or Tyr106.
Both mutant variants were isolated in a very different
yield (< 5mg
:
L
21
and < 0.3 mg
:
L
21

for R121K and
R121Q, respectively), but this cannot be related to a differ-
ent protein stability. Although mutation results in a less
stable protein form, the stability change DDG is similar in
both cases, 28.36 and 212.54 kJ
:
mol
21
for R121K and
R121Q, respectively.
The cytotoxic action of a-sarcin can be dissected into two
different steps. First, the protein must enter the cells,
crossing the phospholipid bilayer barrier. This inter-
nalization occurs in the target cells by endocytosis [3],
which would require a membrane interaction. Further, the
toxin cleaves ribosomes, impairing protein biosynthesis
and producing cellular death via apoptosis [3]. Conse-
quently, both abilities have been studied for R121Q and
R121K.
Substitution of Arg121 by Lys or Gln abolishes the
activity of a-sarcin to release the a-fragment from
ribosomes. This lack of ribonuclease activity against the
specific substrate is also observed against the homopoly-
nucleotide poly(A). These results indicate that this residue
plays an important role in the rRNA catalytic cleavage by
a-sarcin, which would not be simply related to the bearing
positive charge. Both mutant variants of a-sarcin cleave
ApA, with slight variation in the K
m
value but k

cat
being
reduced by about one order of magnitude (Table 1). A very
similar result has been obtained before for the H50Q variant,
while mutation of either Glu96 or His137, residues acting as
the general acid and base on the catalysis by a-sarcin [19,20],
rendered proteins with no detectable activity against ApA
[20]. Cleavage of ApA by a-sarcin is a low-specificity
reaction requiring high amounts of protein and long incu-
bation times [18]. Thus, using this dinucleotide substrate
only allows us to obtain information about the minimal
requirements needed for a-sarcin to cleave a phosphodiester
bond but it appears to be a more sensitive assay than those
using rRNA or poly(A). From this point of view, this Arg
residue although not essential for catalysis would contribute
to position the substrate in the optimum conformation for
cleavage. In fact, in the crystal structure of restrictocin the
active site residues His49, Glu95, Arg120 and His136
(equivalent to His50, Glu96, Arg121 and His137 in a-sarcin)
cluster together and point towards a tetrahedral-shaped
electron density which was interpreted as an inorganic
phosphate group derived from the crystallization buffer [16].
A very similar situation occurs with Arg77 in the active site
of RNase T1, where the phosphate group can accept
hydrogen bonds from Arg77(N1H/NhH) [22– 24], although
its role in catalysis has not been elucidated because no
mutant forms at this position have been obtained [23]. Thus,
Fig. 5. Effect of fungal wild-type a-sarcin (X) and its R121K (W)
and R121Q (K) mutant variants on phosphatidylglycerol vesicles.
(A), Protein binding to the vesicles. Protein bound is expressed as

percentage of the total protein present in the assay vs phospholipid/
protein molar ratio. (B), Aggregation of lipid vesicles. Aggregated
vesicles scatter more light than the un-aggregated vesicles and the
process can be measured from the resulting apparent DA
360
promoted
by the proteins in a vesicle suspension. Results are expressed as relative
DA
360
values, considering the maximum increase produced by the wild-
type protein as unit, vs. protein/phospholipid molar ratio. (C), Lipid-
mixing from different bilayers. This assay is performed with a mixture
of fluorescence-labelled and unlabelled vesicles (1 : 9 ratio, labelled to
unlabelled, respectively). The lipid mixing results in dilution of the two
fluorescence probes and consequently in decrease of the relative
fluorescence energy transfer (RET). Results are expressed as relative
RET values, considering the maximum decrease produced by the wild-
type protein as unit, vs. protein/phospholipid molar ratio. In all cases,
the results shown are the average of three independent experiments.
6194 M. Masip et al. (Eur. J. Biochem. 268) q FEBS 2001
in the absence of the Arg guanidinium group, the protein
would not be able to accommodate polymeric RNA, such
as rRNA or poly(A), in the proper conformation within the
active site. ApA, a much smaller substrate, would fit more
easily into the cleavage pocket although not under optimal
structural constraints. This would explain the observed
reduction in k
cat
. In fact, it has been already proposed
that the mechanism of action of a-sarcin against ribo-

somes might differ in some details from that against dinuc-
leotides [18,19]. The behaviour of a-sarcin against ApA as a
function of pH, altogether with the characterization of the
individual pK
a
values of the active site residues, were
consistent with the existence of two mechanisms for the
hydrolysis of ApA with different optimum pH and
different orientations for the dinucleotide within the active
site [19].
The ability of R121K and R121Q variants to interact with
phospholipid model membranes was studied by analysing
their ability in aggregating vesicles and mixing phospho-
lipids from different vesicles. The results obtained indicate
that the loss of a positive charge in the position corre-
sponding to Arg121 side-chain has a dramatic effect on the
a-sarcin–membrane interaction. Regarding to this, the region
around Trp51, located at around 11 A
˚
from position 121,
is involved in the interaction with the lipid bilayer, as
this Trp residue is located in the hydrophobic core of the
bilayer following protein– vesicle interaction [51]. The
membrane affinity of R121Q is largely diminished, while
that of R121K is not modified (Fig. 5A). The decreased
binding affinity of the R121Q mutant is correlated with a
reduced ability to aggregate lipid vesicles that requires the
formation of vesicle–protein–vesicle complexes [45]. Conse-
quently, the lipid-mixing from different bilayers within the
vesicle aggregates also occurs in a very reduced extent

(Fig. 5C).
From the results obtained, it can be concluded that
Arg121 of a-sarcin, a conserved residue in its fungal ribo-
toxins family, plays a crucial role in the cytoxicity shown by
this protein. Firstly, because this residue is required for the
specific ribonuclease activity of the protein against ribo-
somes. and secondly, because this residue is essential for the
binding of the protein to the membranes which would be a
required step in crossing the cell membrane barrier via
endocytosis. This essential character of Arg121 for mem-
brane binding may be a consequence of its essential role in
the ribonucleolytic mechanism. The dramatic membrane
affinity decrease due to the loss of only one positive charge
in a protein with 24 basic residues and 17 acid residues [12]
and its location at the ribonucleolytic active site may suggest
some kind of relationship. It would not be unreasonable to
assume that proteins evolved to interact with nucleic acids,
such as RNases, would have developed structural deter-
minants to recognize polyphosphate lattices, i.e. RNA, and
this ability, in some cases, may allow a recognition of
phospholipid surfaces, considered as two-dimensional
phosphate networks.
ACKNOWLEDGEMENTS
This work has been supported by grant BMC2000-0551 from Direccio
´
n
General de Investigacio
´
n (Ministerio de Ciencia y Tecnologı
´

a), Spain.
M. M. is recipient of a fellowship from Ministerio de Educacio
´
n,
Cultura y Deporte, Spain.
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