Tài liệu Báo cáo khóa học: The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain pptx

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

for this residue.
Keywords: ricin; ribosome; site-directed mutagenesis;
heterologous expression.
Ricin is a potent toxin found in the seeds of the Ricinus
communis plant. It is a member of a large family of
ribosome-inactivating proteins (RIPs) that exist in various
tissues of many plants, fungi and bacteria [1]. Ricin
consists of a catalytic A chain (RTA) joined by a single
disulphide bond to a lectin B chain (RTB) that facilitates
both cell surface binding and entry of the catalytically
active A chain into the cytoplasm [2]. RTA inactivates
eukaryotic ribosomes by catalysing the depurination of a
specific adenine residue of 28S rRNA, rendering the
ribosome unable to bind elongation factors and hence
abolishing protein synthesis [3]. The site of depurination
by RTA lies within a highly conserved purine-rich region
of 28S rRNA termed the sarcin–ricin loop. Both the
NMR structure [4,5] and X-ray structure [6] of an
oligoribonucleotide that mimics this region show it to be
a compact structure with a stem and four base loop,
GAGA, at its centre. Each of the four nucleotides in this
GAGA tetraloop are necessary for the action of RTA,
thus, implying that the N-glycosidase activity of RTA not
only requires specific interactions with the target adenine
but also direct recognition of other bases in the tetra-
nucleotide [7]. Unlike this tetranucleotide, the sequence of
the stem does not appear to be important as long as it is
at least three base-pairs in length [8]. Interactions between
RTA and the rRNA backbone in this area might be
required to maintain the tetraloop in the optimum

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

3
is the target for depurination by RTA) and RTA. The
Correspondence to J. M. Lord, Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, UK.
Fax: + 44 2476523701, Tel.: + 442476523598,
E-mail:
Abbreviations: ApG, adenyl (3¢fi5¢) guanosine; DMEM, Dulbecco’s
modified Eagles medium; FMP, formycin monophosphate; RIP,
ribosome-inactivating protein; RTA, ricin A chain; RTB,
ricin B chain; SRL, sarcin-ricin loop.
*Present address: Astex Technology, 436 Cambridge Science Park,
Milton Road, Cambridge, CB4 0QA, UK.
(Received 25 September 2003, revised 28 October 2003,
accepted 10 November 2003)
Eur. J. Biochem. 271, 153–162 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03914.x
models were based on the tetraloop structure of such a
hexanucleotide, where the conformation of the target
adenine had been altered, using the known structures of
complexes of RTA with FMP (formycin monophosphate)
and ApG (adenyl 3¢fi5¢-guanosine), to facilitate docking
intotheactivesiteofRTA.
Here, two of the models proposed [10] have been
examined (Fig. 1). The third model in which the hexanucle-
otide is bound to the active site of RTA in an open
conformation is not included in this study as, although
several favourable interactions between the hexanucelotide
and RTA are maintained, many additional interactions are
proposed that involve poorly conserved residues. Each of
the two models tested proposed that several arginyl residues
were involved in electrostatic interactions with the phos-

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

R258
R213
R180
N78
E177
D75
R134
Y123
Y80
N122
R258
R213
R180
N78
D75
E177
Y123
Y80
N122
R134
R258
R213
R180
N78
E177
D75
Y123
Y80
R134
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

MOLSCRIPT
[34,35].
154 C. J. Marsden et al. (Eur. J. Biochem. 271) Ó FEBS 2003
Creation of ricin A chain variants
Ricin A chain variants containing single amino acid sub-
stitutions were generated by PCR mutagenesis and standard
recombinant DNA techniques. Using the RTA expression
plasmid pUTA [14] as a PCR template, appropriate base
changes were introduced to encode for the single amino acid
substitutions R48A, R134A, R134Q, R213A, R213D,
R258A, R258D, N78S, D75A, D75S, D75N, N122A. All
substitutions were confirmed by DNA sequencing.
Expression and purification of ricin A chain variants
A single colony of Escherichia coli JM101 transformed with
the pUTA vector [14] containing the appropriate RTA-
variant sequence was used to inoculate 50 mL of 2YT and
grown overnight at 37 °C. This starter culture was used to
inoculate 500 mL of 2YT, and the culture was grown for 2 h
at 30 °C. Expression was induced by adding isopropyl thio-
b-
D
-galactoside to a final concentration of 0.1 m
M
for 4 h at
30 °C. Cells were harvested by centrifugation at 2740 g,
resuspended in 15 mL of 5 m
M
sodium phosphate buffer
(pH 6.5), and lysed by sonication on ice. Cell debris was
pelleted by centrifugation at 31 400 g at 4 °Cfor30min

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

Microsystems, UK) and the conditions described for wild-
type RTA crystallization [11]. Data were collected at 100 K
and processed using the
HKL
suite of programs [20].
Refinement of the structures was carried out by alternate
cycles of
REFMAC
[21] and manual refitting using O [22],
based on the 1.8 A
˚
resolution model of wild-type RTA [11]
(Protein Data Bank code 1ift). Water molecules were added
to the atomic model automatically using
ARP
[23] at the
positions of large positive peaks in the difference electron
density, only at places where the resulting water molecule
fell into an appropriate hydrogen bonding environment.
Restrained isotropic temperature factor refinements were
carried out for each individual atom. Data collection and
refinement statistics are given in Table 1.
Assay of the
N
-glycosidase activity of ricin A chain
variants
The activity of each of the RTA variants was determined
by assessing their ability to depurinate 26S rRNA of
Table 1. Data collection and refinement statistics. Numbers in parentheses refer to values in the highest resolution shell.
R213D N122A

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

free
c
0.231 (0.257) 0.214 (0.192)
Reflections used 921 (24) 2,448 (188)
R
cryst
(all data)
b
0.157 0.179
Mean temperature factor (A
˚
2
) 24.8 19.8
Rmsds from ideal values
Bonds (A
˚
) 0.021 0.059
Angles (
o
) 1.9 3.0
DPI coordinate error (A
˚
) 0.145 0.060
PDB accession codes 1uq4, r1uq4sf 1uq5, r1uq5sf
a
R
sym
¼ S
j
S

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.

c
R
free
is
equivalent to R
cryst
for a 4 % subset of reflections not used in the refinement [24].
Ó FEBS 2003 Mutations affecting the activity of ricin A chain (Eur. J. Biochem. 271) 155
purified yeast (Saccharomyces cerevisiae) ribosomes. For
each reaction, 20 lg of yeast ribosomes were incubated at
30 °C for 1 h with the relevant RTA variant in 25 m
M
Tris/HCl (pH 7.6), 25 m
M
KCl, 5 m
M
MgCl
2
, in a total
volume of 20 lL. Reactions were stopped by the addition
of 100 lLof2· Kirby buffer [25] and 80 lLofH
2
O, and
rRNA was obtained by precipitation after two phenol–
chloroform extractions. Four micrograms of rRNA were
treatedwith20lL of acetic-aniline for 2 min at 60 °C,
and rRNA was precipitated and resuspending in 15 lLof
60% de-ionized formamide/0.1· TPE (3.6 m
M
Tris, 3 m

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

lactose, followed by a further
36 h against 5 L of NaCl/P
i
. Reassociated holotoxin was
separated from free RTA on a 0.5 mL immobilized
a-lactose column. The dialysate was loaded onto the
column three times and the column was then washed with
10 mL of NaCl/P
i
before eluting bound holotoxin (and
free RTB) in 5 mL of NaCl/P
i
containing 75 m
M
galactose. Eluted protein was dialysed for 16 h against
1L of NaCl/P
i
to remove the galactose, before quanti-
fying against know quantities of RTA on a silver-stained
SDS/polyacrylamide gel using Molecular Dynamics
IMAGEQUANT
version 3.3.
Cytotoxicity assay
Cells were plated out in a volume of 100 lLin96-wellplates
at a density of 1.5 · 10
5
cellsÆmL
)1
and incubated for 18 h at
37 °C. Toxin dilutions (100 lL) in DMEM were added in

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

aspartate (R213A and R213D, respectively) reduced the
D
50
(concentration giving half maximal depurination) by
10-fold and over 100-fold, respectively (Fig. 2A). Substitu-
tion of Arg258 with Ala (R258A) had no effect on activity
whereas substitution of Arg258 with Asp (R258D) lowered
the activity niinefold (Fig. 2B). Finally, substitution of
Arg48 with Ala (R48A) lowered the activity by 10-fold
(Fig. 2C).
Cytotoxicity of RTA variants containing arginine
substitutions
Each of the RTA variants described above were reassoci-
ated with RTB and the cytotoxicity of each of the resultant
ricin variants was compared to wild-type ricin. Ricin
R213A and ricin R213D were 12-fold and more than 2000-
fold less cytotoxic than ricin, respectively (Fig. 3A). Ricin
R258A was equally as cytotoxic as ricin (Fig. 3B) whereas
ricin R258D (Fig. 3B) and ricin R48A (Fig. 3C) were
10-fold less cytotoxic. The reductions in cytotoxcity
compared to native ricin were comparable to the decrease
in catalytic activity against ribosomes in vitro for each of
the substitutions except for ricin R213D, whose cytotoxi-
city was reduced by 20-fold more than the catalytic activity
of RTA R213D.
The X-ray structure of RTA R213D
In the absence of structural data for each of the RTA
variants discussed, it is possible that the reduction in activity
might be attributed to structural changes of the enzyme. The
most substantial decrease in catalytic activity was seen when

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

6
(where A
3
156 C. J. Marsden et al. (Eur. J. Biochem. 271) Ó FEBS 2003
is the target for depurination by RTA) modelled into the
active site with the first base C
1
of the hexanucleotide
forming a Watson–Crick pair with the last base G
6
.This
both closes the tetraloop and allows G
2
and A
5
to be
stacked upon this base pair forming a non-Watson–Crick
pairing (Fig. 1A). The target adenine residue A
3
projects
out of the tetraloop and is positioned in the same location as
indicated by the crystal structures of both the FMP and
ApG complexes [10], forming stacking interactions with the
aromatic groups of Tyr80 and Tyr123. The first model has
G
4
stackedontheG
2
-A
5

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-

taining arginine substitutions. Isolated yeast ribosomes (20 lg) were
incubated with RTA or RTA variants for 60 min at 30 °C. rRNA was
isolated and 4 lg was aniline-treated and electrophoresed on an ag-
arose/formamide gel. rRNAs were quantified from digital images using
IMAGEQUANT
software. The depurination was calculated by relating
the amounts of small diagnostic aniline-fragment [3] and 5.8S rRNA
and expressing values as a percentage. Symbols indicate the experi-
mental data, error bars represent the SD and solid lines represent best-
fitted curves. (A) RTA, d; RTA R213A, j, and RTA R213D, m.(B)
RTA, d; RTA R258A j, and RTA R258D, m; (C) RTA, d;RTA
R48A, j.
Ó FEBS 2003 Mutations affecting the activity of ricin A chain (Eur. J. Biochem. 271) 157
model alone G
4
is proposed to make two hydrogen bonds
with Asn78 and a further hydrogen bond with Asp75. In
order to test these models, substitutions were made at each
of the residues proposed to be involved – Asp75, Asn78 and
Asn122.
N
-glycosidase activity of RTA variants with substitutions
at Asp75 and Asn78
Substitution of either Asp75 or Asn78 with Ala resulted in
very low expression levels and purification of these variants
was not achieved. However, although a very low expression
level was again seen on substitution of Asp75 to Ser,
substitution of Asn78 to Ser (RTA N78S) produced a protein
that expressed as well as wild-type RTA and had equal
stability to digestion by trypsin as wild-type RTA (data not

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.

Cytotoxicity of RTA N122A
RTAN122AwasreassociatedwithRTBanditscytotoxi-
city was compared to ricin (Fig. 6B). Substituting Ala for
Asp at residue 122 in RTA reduced the cytotoxicity of RTA
by 30-fold (correlating well with the in vitro N-glycosidase
activity).
The X-ray structure of RTA N122A
In order to confirm that the reduction in catalytic activity of
RTA N122A could be attributed solely to the change in
charge and size of the sidechain of this single residue, the
crystal structure of RTA N122A was solved. The RMSD
from the C
a
atoms of the wild-type RTA crystal structure
[11] was 0.33 A
˚
indicating that the structure of RTA N122A
is essentially identical to recombinant wild-type RTA. The
electron density in the area of the substitution is shown in
Fig. 7. The positions of catalytic residues and all other
residues local to the substitution site do not differ from the
wild-type crystal structure.
Discussion
When the structure of ricin A-chain was solved to 2.5 A
˚
[26], it was suggested that a number of arginyl residues
were likely to be responsible for the binding of the rRNA
substrate. Several of these arginyl residues have subse-
quently been proposed to form an arginine-rich binding
motif [27]. Furthermore, when RTA is treated with the

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,

while those positively charged residues surrounding but
outside the active site are probably involved in binding the
negatively charged RNA substrate.
Arg48 is a variable residue that lies in a loop on the edge
of the active site cleft of RTA that has been implicated in
substrate binding by modelling studies [28]. Substitution of
this RTA residue with alanine reduced catalytic activity
Fig. 5. Assessment of the N-glycosidase activity and cytotoxicity of
RTA N78S and ricin N78S. (A) N-glycosidase activity of RTA N78S.
Isolated yeast ribosomes (20 lg) were incubated with RTA (d)or
RTA N78S (j)for60minat30 °C. rRNA was isolated and 4 lgwas
aniline-treated and electrophoresed on an agarose/formamide gel.
rRNAs were quantified from digital images using
IMAGEQUANT
soft-
ware. 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 repre-
sent the SD and solid lines represent best-fitted curves. (B) Dose
dependent cytotoxicity assay of ricin N78S. Vero cells were challenged
with increasing concentrations of ricin (d),orricinN78S(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.
Fig. 6. Assessment of the N-glycosidase activity and cytotoxicity of
RTA N122A and ricin N122A. (A) N-glycosidase activity of RTA
N122A. Isolated yeast ribosomes (20 lg) were incubated with RTA
(d)orRTAN122A(j)for60minat30°C. rRNA was isolated and

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,

could perhaps be responsible for the differing binding
affinities of RIPs for their substrates. Substitution of Arg258
with Ala had no effect on activity, implying that this residue
is not necessary for the activity of RTA. This is supported
by the earlier observation showing that deletion of residues
258–262 did not inactivate RTA [30]. However, although
this residue is not essential for activity, reversing the charge
of this sidechain resulted in a 10-fold reduction in activity,
consistent with this residue being close to the phosphodi-
ester backbone of the substrate but not making an essential
interaction with it.
Arg134 is highly conserved in RIPs, and it has been
proposed that it could make a forked interaction with both
of the phosphodiester backbones of the target adenine and
of the preceding guanine residue in the GAGA motif [10].
Substitution of Arg134 with Ala or Gln abolished expres-
sion of folded protein, and consequently it was concluded
that Arg134 played a critical structural role. Although
Arg134 is predicted to be able form an ionic interaction
with the phosphodiester backbone of the substrate, the
structure of RTA also shows that this residue is at the
centre of a hydrogen bonding network, forming H-bonds
with Glu127, Asn209 and Glu208. Arg134 also forms a p-
stacking interaction with Tyr123, a residue directly
involved in substrate binding. Substitution of residues
Asn209, Glu208 and Tyr123 permitted RTA folding and
reduced activity between threefold and 10-fold, suggesting
that they form only modest interactions with the substrate
[14,18]. Thus, while it remains possible that Arg134 makes
a significant contribution to substrate binding, this can not

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

N122A
Y80
Fig. 7. Electron density of RTA N122A in the vicinity of residue 122. The backbone and sidechains of the N122A substitution are shown as stereo
images in thick ball and stick and the position of the Asn122 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].
160 C. J. Marsden et al. (Eur. J. Biochem. 271) Ó FEBS 2003
magnitude. It then appears that, in addition to catalytic site
residues, a number of the Arg residues located around the
active site cleft play a significant role in the activity of RTA.
As none of these arginyl residues have been implicated in
catalysis, the reduction in activity of each of these RTA
variants is probably due to a loss of electrostatic interactions
that are critical for the stable binding of the substrate RNA
molecule in the active site pocket. Although the contribution
of each individual residue is relatively small, the cumulative
effect of these residues would make a substantial contribu-
tion to substrate binding. This contribution is probably
relatively nonspecific in nature such that these residues
probably make a major contribution to binding, but do not
greatly contribute to specificity.
Further interactions of the substrate RNA with RTA

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,

is consistent with the first of the models proposed by
Monzingo and Robertus [10].
In order to understand more fully the precise role of
Asn122 in the catalytic activity of RTA, it would be of
value to solve the structure of RTA N122A in the
presence of small nucleotides such as ApG that can be
diffused into RTA crystals. RTA cleaves a single
N-glycosidic bond from among over 4000 in 28S rRNA
[32]. Ultimately, complete understanding of the specificity
of RTA for its ribosomal RNA substrate, and the role
that recognition and binding of the RNA stem and loop
might play in this, may only be fully achieved when
crystal structures of complexes of RTA with much larger
RNA fragments become available.
Acknowledgements
This work was supported by the UK Biotechnology and Biological
Sciences Research Council grant 88/B16355 and Wellcome Trust
Programme Grant 063058/Z/00/Z. VF is a Royal Society University
Fellow.
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