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
Trang 1The 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 G 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 tetratetra-nucleotide, 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 (C1G2A3G4A5G6, where A3
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: mlord@bio.warwick.ac.uk
Abbreviations: ApG, adenyl (3¢ fi 5¢) 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: AstexTechnology, 436 Cambridge Science Park,
Milton Road, Cambridge, CB4 0QA, UK.
(Received 25 September 2003, revised 28 October 2003,
accepted 10 November 2003)
Trang 2models 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¢ fi 5¢-guanosine), to facilitate docking
into the active site of RTA
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 G4of 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 G4in each of the two models have been examined
Experimental procedures
Materials (35S)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
R258
R213
R180
N78 E177
D75
R134
R258
R213
D75
E177 Y123
Y80
N122 R134
R258
R213
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].
Trang 3Creation 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 37C This starter culture was used to
inoculate 500 mL of 2YT, and the culture was grown for 2 h
at 30C Expression was induced by adding isopropyl
thio-b-D-galactoside to a final concentration of 0.1 mMfor 4 h at
30 C Cells were harvested by centrifugation at 2740 g,
resuspended in 15 mL of 5 mMsodium phosphate buffer
(pH 6.5), and lysed by sonication on ice Cell debris was
pelleted by centrifugation at 31 400 g at 4C for 30 min
and the supernatant loaded onto a 50 mL CM-Sepharose
CL-6B column (Amersham Biosciences) The column was
washed with 1 L of 5 mM sodium phosphate, pH 6.5
followed by 100 mL of 100 mM NaCl in 5 mM sodium
phosphate, pH 6.5 and RTA was eluted with a linear
gradient of 100–300 mMNaCl in the same buffer Fractions
containing RTA were pooled and stored at 4 C at a concentration of no more than 1 mgÆmL)1 Typical yields
of purified wild-type RTA and RTA variants were between
10 and 12 mgÆL)1unless 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 P41212
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 theN-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.
Data collection
Unit cell dimensions (A˚) a ¼ b ¼ 67.3, c ¼ 140.7 a ¼ b ¼ 67.5, c ¼ 140.6
R sym
a
Refinement
Non-hydrogen atoms 2492 (including 2 sulphate
406 water molecules)
2596 (including 2 sulphate, 1 acetate and 510 water molecules)
R cryst
b
Rmsds from ideal values
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.cR free is equivalent to R for a 4 % subset of reflections not used in the refinement [24].
Trang 4purified 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 mM
Tris/HCl (pH 7.6), 25 mM KCl, 5 mM MgCl2, in a total
volume of 20 lL Reactions were stopped by the addition
of 100 lL of 2· Kirby buffer [25] and 80 lL of H2O, and
rRNA was obtained by precipitation after two phenol–
chloroform extractions Four micrograms of rRNA were
treated with 20 lL of acetic-aniline for 2 min at 60 C,
and rRNA was precipitated and resuspending in 15 lL of
60% de-ionized formamide/0.1· TPE (3.6 mMTris, 3 mM
NaH2PO4, 0.2 mM EDTA) and heated at 65 C for
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 lg of RTB
(Vector Laboratories) and made up to a final volume of
2 mL with NaCl/Picontaining 0.1Mlactose and 2% (v/v)
2-mercaptoethanol This was dialysed for 24 h against 1 L
of NaCl/Picontaining 0.1Mlactose, followed by a further
36 h against 5 L of NaCl/Pi 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/Pi before eluting bound holotoxin (and
free RTB) in 5 mL of NaCl/Pi containing 75 mM
galactose Eluted protein was dialysed for 16 h against
1 L of NaCl/Pi to remove the galactose, before
quanti-fying against know quantities of RTA on a silver-stained
SDS/polyacrylamide gel using Molecular Dynamics
IMAGEQUANTversion 3.3
Cytotoxicity assay
Cells were plated out in a volume of 100 lL in 96-well plates
at a density of 1.5· 105cellsÆmL)1and incubated for 18 h at
37C Toxin dilutions (100 lL) in DMEM were added in
quadruplicate and the plates were incubated for 4 h at 37C
Protein synthesis was measured by incubating the plates for
90 min at 37C in the presence of 1 lCi of (35S)methionine
in 50 lL of NaCl/Pi 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 Supermix) to each well, plates were counted
in a Wallac 1450 MicroBeta Triluxliquid 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
D50 (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 CGAGAG (where A
Trang 5is the target for depurination by RTA) modelled into the
active site with the first base C1 of the hexanucleotide
forming a Watson–Crick pair with the last base G6 This
both closes the tetraloop and allows G2 and A5 to be
stacked upon this base pair forming a non-Watson–Crick
pairing (Fig 1A) The target adenine residue A3 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
G4 stacked on the G2-A5 pair of the hexanucelotide allowing two hydrogen bonds to be formed between the base G4and 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 G4and Asn122 can no longer be made
as G4is in an altered position making a continuous curved stack of aromatic groups, Tyr123, A, Tyr80, G In this
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; and ricin 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.
Trang 6model alone G4is 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 RTA N122A was reassociated with RTB and its cytotox i-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 Caatoms 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
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].
Trang 7involved 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) for 60 min at 30 C rRNA was isolated and 4 lg was
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 reprerepre-sent best-fitted curves (B) Dose
dependent cytotoxicity assay of ricin N78S Vero cells were challenged
with increasing concentrations of ricin (d), or ricin N78S (j) at 37 C
for 4 h Remaining protein synthesis after this time was measured by
the incorporation of (35S)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) or RTA N122A (j) for 60 min at 30 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) 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.
Trang 810-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].
Trang 9magnitude 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 contribucontribu-tion 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 G4 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) G4 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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