The effect of mutations surrounding and within the active site
on the catalytic activity of ricin A chain
Catherine J. Marsden, Vilmos Fu¨lo¨ p, Philip J. Day* and J. Michael Lord
Department of Biological Sciences, University of Warwick, Coventry, UK
Models for the binding of the sarcin–ricin loop (SRL) of 28S
ribosomal RNA to ricin A chain (RTA) suggest that several
surface exposed arginine residues surrounding the active site
cleft make important interactions with the RNA substrate.
The data presented in this study suggest differing roles for
these arginyl residues. Substitution of Arg48 or Arg213 with
Ala lowered the activity of RTA 10-fold. Furthermore,
substitution of Arg213 with Asp lowered the activity of RTA
100-fold. The crystal structure of this RTA variant showed it
to have an unaltered tertiary structure, suggesting that the
positively charged state of Arg213 is crucial for activity.
Substitution of Arg258 with Ala had no effect on activity,
although substitution with Asp lowered activity 10-fold.
Substitution of Arg134 prevented expression of folded pro-
tein, suggesting a structural role for this residue. Several
models have been proposed for the binding of the SRL to the
active site of RTA in which the principal difference lies in the
conformation of the second ÔGÕ in the target GAGA motif
in the 28S rRNA substrate. In one model, the sidechain
of Asn122 is proposed to make interactions with this G,
whereas another model proposes interactions with Asp75
and Asn78. Site-directed mutagenesis of these residues of
RTA favours the first of these models, as substitution of
Asn78 with Ser yielded an RTA variant whose activity was
essentially wild-type, whereas substitution of Asn122
reduced activity 37.5-fold. Substitution of Asp75 failed to
yield significant folded protein, suggesting a structural role

conformation for catalysis. Both the catalytic role of
RTA and its recognition and binding of the substrate
RNA have been investigated by chemical modification [9],
X-ray crystallography [10–12] and site-directed mutagen-
esis [13–17]. Chemical modification showed RTA to be
inactivated by treatment with the arginyl-specific reagent,
phenylglyoxal [9], although it is likely that the inactivation
observed in this study was primarily due to modification
of the crucial catalytic residue, Arg180 [18]. Crystal
structures of small molecules bound in the active site of
RTA have been solved [10,12,19], but to date there are no
structures of complexes of RTA with larger substrate
analogues.
The structure of the ribosome and of small RNA
oligonucleotides show that the target adenine is not in a
conformation that is compatible with binding to RTA [6].
Monzingo and Robertus [10] generated three models of
complexes of a hexanucleotide (C
1
G
2
A
3
G
4
A
5
G
6
,whereA

phdiester backbone of the hexanucleotide. The role of four
such arginyl residues (Arg48, Arg134, Arg213 and Arg258)
has been examined here. The most significant difference
between the two models is the identity of the residues
involved in interactions made between RTA and base G
4
of
the hexanucleotide (Fig. 1). In this study those amino acid
residues (Asp75, Asn78 and Asn122) around the active site
cleft of RTA that have a putative role in the binding of G
4
in
each of the two models have been examined.
Experimental procedures
Materials
(
35
S)Methionine was from Amersham, RTB was from
Vector Laboratories (Peterborough, UK) and microbridges
were from Crystal Microsystems (Oxford, UK).
R258
R213
R180
N78
D75
E177
R134
Y123
Y80
N122

A
B
Fig. 1. Models of hexanucleotide binding in the active site of RTA (based upon [10]). The structures are shown as stereo images with the
C
1
G
2
A
3
G
4
A
5
G
6
(where A
3
is the target for depurination by RTA) in red. C
1
of the hexanucleotide is at the bottom left and the target adenine
is at the top of each model. The sidechains of RTA are shown in blue. (A) Model 1 has the tetraloop bound in the active site of RTA with G
4
stacked upon the G
2
-A
5
pair and able to make interactions with Asn122. (B) Model 2 is a variation of model 1 with the tetraloop bound in a
conformation where G
4
stacks with Tyr80 and makes interactions with Asp75 and Asn78. Drawn with

and the supernatant loaded onto a 50 mL CM-Sepharose
CL-6B column (Amersham Biosciences). The column was
washed with 1L of 5m
M
sodium phosphate, pH 6.5
followed by 100 mL of 100 m
M
NaCl in 5 m
M
sodium
phosphate, pH 6.5 and RTA was eluted with a linear
gradient of 100–300 m
M
NaCl in the same buffer. Fractions
containing RTA were pooled and stored at 4 °Cata
concentration of no more than 1 mgÆmL
)1
. Typical yields
of purified wild-type RTA and RTA variants were between
10 and 12 mgÆL
)1
unless otherwise stated in the text.
Crystallization, X-ray data collection and refinement
of ricin A chain variants
Crystals were grown in the tetragonal space group P4
1
2
1
2
by the sitting-drop method using microbridges (Crystal

Data collection
Radiation, detector In-house CuKa MAXLAB BL-I711
and wavelength (A
˚
) DIP2030, 1.54184 MAR IP, 1.0213
Unit cell dimensions (A
˚
) a ¼ b ¼ 67.3, c ¼ 140.7 a ¼ b ¼ 67.5, c ¼ 140.6
Resolution (A
˚
) 28 - 1.9 (1.949 - 1.9) 46 – 1.4 (1.436–1.4)
Observations 53 233 274 010
Unique reflections 22 494 60 829
I/r(I) 21.6 (4.7) 43.2 (8.1)
R
sym
a
0.038 (0.133) 0.031 (0.121)
Completeness (%) 85.6 (43.6) 93.9 (99.3)
Refinement
Non-hydrogen atoms 2492 (including 2 sulphate
406 water molecules)
2596 (including 2 sulphate, 1 acetate
and 510 water molecules)
R
cryst
b
0.154 (0.154) 0.177 (0.176)
Reflections used 21 573 (720) 58 381 (4,486)
R

h
|I
h,j
–<I
h
>|/S
j
S
h
<I
h
> where I
h,j
is the jth observation of reflection h, and <I
h
> is the mean intensity of that reflection.
b
R
cryst
¼ S||F
obs
|–|F
calc
||/S|F
obs
| where F
obs
and F
calc
are the observed and calculated structure factor amplitudes, respectively.

M
NaH
2
PO
4
,0.2m
M
EDTA) and heated at 65 °Cfor
5 min. Ribosomal RNA fragments were separated on a
1.2% agarose, 0.1· TPE, 50% (v/v) formamide gel.
rRNAs were quantified from digital images of ethidium
bromide-stained gels, using
IMAGEQUANT
software, and
depurination was calculated by relating the amounts of
the small aniline-fragment and 5.8S rRNA and expressing
values as a percentage.
Reassociation and quantification of ricin A-chain variants
Purified RTA (100 lg) was mixed with 100 lgofRTB
(Vector Laboratories) and made up to a final volume of
2 mL with NaCl/P
i
containing 0.1
M
lactose and 2% (v/v)
2-mercaptoethanol. This was dialysed for 24 h against 1 L
of NaCl/P
i
containing 0.1
M

quadruplicate and the plates were incubated for 4 h at 37 °C.
Protein synthesis was measured by incubating the plates for
90 min at 37 °C in the presence of 1 lCi of (
35
S)methionine
in 50 lLofNaCl/P
i
per well. Proteins were precipitated
by washing three times with ice-cold 5% (v/v) trichloroace-
tic acid and, after the addition of 200 lL of scintillant
(OptiPhase ÔSupermixÕ) to each well, plates were counted
in a Wallac 1450 MicroBeta Trilux liquid scintillation
counter.
Results
N
-glycosidase activity of RTA variants containing
arginine substitutions
RTA variants in which arginyl residues 213 or 258 were
substituted with Ala, or Asp or in which Arg48 had been
substituted with Ala, expressed to levels equivalent to wild-
type RTA and were readily purified to homogeneity. Each
of these RTA variants had the same stability to digestion
by trypsin as wild-type RTA (data not shown). Substitu-
tion of Arg134 with either Ala or Gln resulted in barely
detectable expression levels and, as such, these mutants
could not be purified. To assess the effect of substitutions
made at each of the arginyl residues on catalytic activity of
RTA, the ability of each of the purified RTA variants to
depurinate yeast ribosomes was compared to that of wild-
type RTA. Conversion of Arg213 to either alanine or

a substitution was made at Arg213 (R213D). The crystal
structure was solved to determine whether the reduction in
activity of this RTA variant could be attributed solely to the
change in charge and size of the sidechain of this single
residue. The structure of RTA R213D is essentially identical
to that of recombinant wild-type RTA with an root mean
square deviation (RMSD) from the Ca atoms of the wild-
type crystal structure [11] of 0.33 A
˚
. The electron density in
the area local to the substitution is shown in Fig. 4. The
positions of catalytic residues and all other residues local to
the substitution site do not differ significantly from the wild-
type crystal structure.
Binding of the GAGA tetraloop to RTA
In order to better understand the specific interactions that
RTA makes with the GAGA tetraloop, the models of
RTA–substrate interactions proposed by Monzingo
and Robertus [10] have been examined. The first model
examined here has the sequence C
1
G
2
A
3
G
4
A
5
G

pair of the hexanucelotide
allowing two hydrogen bonds to be formed between the
base G
4
and the sidechain of Asn122. In the second model
(Fig. 1B) the structure of the hexanucelotide is, on the
whole, unchanged and the majority of the interactions that
were seen in the first model are maintained. However, the
interaction between G
4
andAsn122cannolongerbemade
as G
4
is in an altered position making a continuous curved
stack of aromatic groups, Tyr123, A
3
, Tyr80, G
4
.Inthis
Fig. 3. Dose dependent cytotoxicity assay of ricin variants, ricin R213A
and ricin R213D towards Vero cells. Vero cells were challenged with
increasing concentrations of toxin at 37 °C for 4 h. Remaining protein
synthesis after this time was measured by the incorporation of
(
35
S)methionine. Symbols indicate the mean of four replicate samples,
error bars represent the SD. (A) ricin, d, ricin R213A, j;andricin
R213D, m.(B)ricin,d; ricin R258A, j, and ricin R258D, m;(C)ricin,
d; ricin R48A, j.
Fig. 2. Assessment of the N-glycosidase activity of RTA variants con-

shown). Further substitutions were made at Asp75 to Asp,
Arg and Gln. However, all were expressed at very low levels,
and purification to homogeneity, to allow quantitative
activity assays of these RTA variants, was not achieved. To
assess whether substitution at Asn78 with Ser changed the
catalytic activity of RTA, the N-glycosidase activity of RTA
N78S against yeast ribosomes was determined and compared
to that of wild-type RTA (Fig. 5A): there was less than a
twofold reduction in activity between them.
Cytotoxicity of RTA N78S
RTA N78S was reassociated with RTB and its cytotoxicity
was compared to ricin (Fig. 5B). Ricin containing RTA
N78S was approximately twofold less cytotoxic than wild-
type ricin. This difference in cytotoxicity was comparable
to the small reduction in N-glycosidase activity seen for
RTA-N78.
N
-glycosidase activity of RTA variants with
substitutions at Asn122
Conversion of Asn122 to Ala (RTA N122A) produced an
RTA variant that expressed to high levels (equivalent to
wild-type RTA). RTA N122A had equal stability, based on
sensitivity to trypsin digestion, as the wild-type protein (data
not shown) and was readily purified to homogeneity. To
assess whether the substitution at Asn122 for Ala had
caused any change in the catalytic activity of RTA, the
N-glycosidase activity of RTA N122A against yeast ribo-
somes was determined and compared to that of wild-type
RTA (Fig. 6A). The reduction in activity of this RTA
N122A was 37.5-fold.

arginyl specific reagent, phenylglyoxal, it is readily inacti-
vated leading to the proposal that this inactivation
R213D R213D
Fig. 4. Electron density of RTA R213D in the vicinity of residue 213. The backbone and sidechains of the R213D substitution are shown as stereo
images in thick ball and stick and the position of the Arg213 side-chain of the wild-type enzyme is overlayed and shown in thin ball and stick. The
SIGMAA [33] weighted 2mF
o
-DF
c
electron density using phases from the final model is contoured at 1 r level, where r represents the rms electron
density for the unit cell. Contours more than 1.4 A
˚
from any of the displayed atoms have been removed for clarity. Drawn with
MOLSCRIPT
[34,35].
158 C. J. Marsden et al. (Eur. J. Biochem. 271) Ó FEBS 2003
involved the modification of arginyl residues 196, 213, 234
and 235 [9]. Of theses residues, only Arg213 is in the
vicinity of the active site of RTA. In order to establish the
role of arginyl residues around the active site of RTA in
the binding of rRNA, a number of RTA variants have
been constructed. Four such arginyl residues were selected
as targets for site-directed mutagenesis. We appreciate that
the mutagenic approach we have taken does not distin-
guish between a role for particular residues in substrate
binding or the catalytic reaction itself. Both roles are
required for RTA catalysis and hence for the cytoxicity of
ricin. However, it does not seem unreasonable to assume,
at least as a broad generalization, that residues lying within
the active site cleft are involved in the reaction catalysed,

4 lg was aniline-treated and electrophoresed on an agarose/forma-
mide gel. rRNAs were quantified from digital images using
IMAGE-
QUANT
software. The depurination was calculated by relating the
amounts of small aniline-fragment and 5.8S rRNA and expressing
values as a percentage. Symbols indicate the experimental data, error
bars represent the SD and solid lines represent best-fitted curves. (B)
Dose dependent cytotoxicity assay of ricin N122A. Vero cells were
challenged with increasing concentrations of ricin (d) or ricin N122A
(j)at37°C for 4 h. Remaining protein synthesis after this time was
measured by the incorporation of (
35
S)methionine. Symbols indicate
the mean of four replicate samples, error bars represent the SD.
Ó FEBS 2003 Mutations affecting the activity of ricin A chain (Eur. J. Biochem. 271) 159
10-fold, consistent with the role predicted for this residue by
modelling, but in contrast to an earlier study [29]. The
apparent discrepancy with this earlier study is probably due
to the type of the assays used. The earlier study [29] used
single point assays which may not have been sufficiently
sensitive to observe a 10-fold decrease in activity that is
readily observed in the dose–response assay used here.
Arg258 is another variable residue that Monzingo and
Robertus [10] suggested might form an ion-pair with the
phosphodiester backbone of rRNA, specifically with that of
the second guanine in the GAGA motif. Olson and Cuff [28]
also proposed that this residue was involved in substrate
binding, interacting with the loop-closing guanine base (the
first G after the GAGA) and, due to it’s variable nature,

be readily tested by site-directed mutagenesis due to its
structural role.
Arg213 is a weakly conserved residue, the amino acyl
residue at this position being positively charged in
nearly 60% of ribosome-inactivating proteins [28]. The
Monzingo and Robertus models [10], and the 29mer
oligonucleotide-binding model of Olson and Cuff [28], show
this residue forming an ion-pair with the phosphodiester
backbone of the first cytosine residue in the CGAGAG
tetraloop motif. Furthermore, an RTA mutant in which
Arg213 had been deleted was found to be inactive [31].
Substitution of Arg213 with Ala reduced activity 10-fold
against both purified ribosomes and whole cells, while
reversal of the charge at this site reduced activity a further
10-fold. This additive effect of changing the charge of residue
213 strongly suggests that it is the charged nature of Arg213
that is responsible for effects on enzyme activity rather than
any structural changes induce by the substitutions. This is
confirmed by the finding that the structure of RTA R213D is
identical to that of the wild-type enzyme except for the
sidechain of residue 213. Thus, Arg213 forms a significant
electrostatic interaction with the substrate ribosome, prob-
ably via interaction with the phosphodiester backbone.
Individual substitution of arginyl residues around the
active site cleft of RTA resulted in different effects on the
activity of RTA. Whereas, one had no effect on the activity
of the enzyme (R258A), others reduced activity by one
(R213A, R258D, R48A) or by two (R213D) orders of
N122A
Y80

were studied by modelling a hexanucleotide into the active
site [10] whose structure was based upon the crystal
structure of a GNRA loop which had been solved [19]. A
series of RTA variants were made based on two of the
models proposed by Monzingo and Robertus [10]. Site-
directed mutagenesis was used to make RTA variants with
substitutions at either Asp75 or Asn78, both of which are
highly conserved in the RIP family and were proposed to
make interactions with the second G
4
in the GAGA
tetraloop in the second of the models (Fig. 1B). Whereas
none of the substitutions to Ala, Ser, Asp, or Arg were
tolerated at Asp75 implying that this residue might play an
important structural role, RTA N78S had the same catalytic
activity as wild-type RTA suggesting that this residue may
not play a significant role in substrate binding, although it
remains possible that a substituted serine could still make
the hydrogen bond normally made by Asn78. In the first
model (Fig. 1A) G
4
is proposed to interact with Asn122
and, in agreement with this, substitution of Ala for Asp at
this site was shown to lower the N-glycosidase activity
37.5-fold, and cytotoxicity of the reassociated holotoxin by
30-fold, without affecting the overall structure or the
position of either active site residues or residues in the
vicinity of the substitution. That the RTA N78S variant had
no effect on activity, but the RTA N122A variant reduced
catalytic activity over 30-fold compared to wild-type RTA,

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