(Received for publication, September 1, 1995)
From the
The A-chain of ricin is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine at position 4324 in 28 S rRNA. Of the 267 amino acids in the protein, 222 could be deleted, in one or another of 74 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA (Morris, K. N., and Wool, I. G.(1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4869-4873). The 45 amino acids that could not be omitted when the deletions were in sets of 20, 5, or 2 residues have now been deleted one at a time; 9 of these deletion mutants retained activity. A RNP-like structural motif in ricin A-chain that may mediate binding to ribosomal RNA has been identified.
Ricin is a cytotoxic RNA N-glycosidase that is
synthesized in the castor bean Ricinus communis. Proricin is a
polypeptide of approximately 65 kDa which is processed by removal of 12
amino acids to form an A-chain of 267, and a B-chain of 262, residues
linked by a disulfide bond(1) . The toxic ricin A-chain (RA) ()inhibits protein synthesis by inactivating ribosomes; the
inhibition is the result of the hydrolysis of the bond between the base
and the ribose of the adenosine at position 4324 (A4324) in 28 S
rRNA(2, 3) . RA is extraordinarily toxic: a single
molecule will inactivate 1500 ribosomes min
, indeed,
a single molecule resident in a cell is sufficient to kill
it(1) ; finally, this one covalent modification accounts
entirely for the cytotoxicity.
A value of an analysis of the mechanism of action of antibiotics and of ribotoxins is that it directs attention to components of the ribosome where efforts to comprehend functional correlates of structure are likely to be rewarded. That this one RA-catalyzed depurination inactivates the ribosome implies that this region of 28 S rRNA is crucial for the function of the particle and there is evidence that the ricin domain is involved in elongation factor-1 dependent binding of aminoacyl-tRNA to the ribosomal A-site and elongation factor-2 catalyzed GTP hydrolysis and translocation of peptidyl-tRNA to the P-site(4) .
A substantial effort has been made to relate the structure of RA to its mechanism of action spurred in part by the use of the protein in the construction of immunotoxins for cancer therapy(5) . The amino acid sequences of ricin and of several homologous proteins have been determined(6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and an atomic structure of ricin has been obtained from x-ray diffraction of crystals(16, 17, 18) . In the three-dimensional structure there is a prominent cleft that was proposed to be the active site of RA before the mechanism of action of the toxin had been defined, indeed, before the substrate had been identified(16) . Strong support for the suggestion has come from the observation that most of the amino acids that are invariant in related plant and bacterial toxins are clustered at the bottom of the putative active site cleft (18) ; from site-directed mutagenesis(19, 20, 21, 22, 23, 24) ; and from an analysis by systematic deletion of amino acids(25) .
Three series of amino acid deletions (of 20, 5, or 2 residues) that scanned RA were constructed: of 138 mutants, 74 retained the capacity to catalyze depurination of A4324 and in the latter 222 of the 267 residues in RA were omitted(25) . Four additional amino acids could be omitted if care was taken to construct the deletions so the amphiphilic character of helix D was preserved(26) . The resolution of the deletion map has now been extended as far as one can go by constructing single amino acid omissions encompassing the 45 residues that had produced inactive mutants when deleted as parts of larger sets.
Deletions were made in the RA cDNA using synthetic
oligodeoxynucleotides with 15 bases complementary to the 5` and 3`
sequences flanking the nucleotides to be deleted(25) . The
synthetic oligodeoxynucleotides (166 ng) were annealed to
single-stranded pRAIBI30 DNA (2 µg) in 10 µl of 20 mM
Tris-HCl, pH 7.4, 2 mM MgCl, and 50 mM NaCl. The reaction mixture was heated at 70 °C for 5 min,
allowed to cool to room temperature, and placed in an ice-water bath.
The complementary DNA strand was synthesized with the annealed
oligodeoxynucleotides as primers by adding reagents to the original
annealing reaction to give the following concentrations in 13 µl:
23 mM Tris-HCl, pH 7.4, 5 mM MgCl
, 35
mM NaCl, 1.5 mM dithiothreitol, 0.4 mM dATP,
dCTP, dGTP, and dTTP, 0.75 mM ATP, 77 units/ml T4 DNA ligase,
and 77 units/ml T4 DNA polymerase. Incubation was in an ice-water bath
for 5 min, at room temperature for 5 min, and finally at 37 °C for
90 min. The reaction was stopped by adding 90 µl of 10 mM Tris-HCl (pH 8.0) and 10 mM EDTA. E. coli strain
SURE was transformed with 5 µl of the product of the synthesis
reaction. SURE cells are dut
, ung
so the template strand which contains
uracils is not replicated; thus, there is a strong selection against
the non-mutagenized strand of the
heteroduplex(29, 30) . Single colonies were selected
from among the transformants and grown overnight at 37 °C in 6 ml
of LB media with 50 µg/ml ampicillin. Plasmid DNA was isolated from
the remaining cells after lysis at 90 °C for 90 s in 700 µl of
50 mM Tris-HCl (pH 8.0), 8% sucrose, 5% Triton X-100, 5 mM EDTA, and 0.1 mg/ml lysozyme(31) . The cellular debris was
removed by centrifugation; plasmid DNA was precipitated by addition of
an equal volume of isopropyl alcohol and suspended in 100 µl of
water. The presence of the intended deletions was authenticated by
determining the sequence of nucleotides in the relevant region of the
RA cDNA using the dideoxynucleotide method(32) . Cells that
contained mutant plasmid RA cDNA were streaked on LB plates with 50
µg/ml ampicillin to obtain single colonies which were grown
overnight at 37 °C in 100 ml of LB with 50 µg/ml ampicillin.
This was to decrease the possibility that mutant plasmid DNA isolated
from the culture would contain wild-type plasmid DNA from contaminating
cells. Plasmid DNA was purified by banding in CsCl
gradients(26) . The sequence of nucleotides in the region of
the deletion in the RA cDNA was determined again to be certain that the
purified plasmid contained the correct mutation.
Figure 1:
The activity of deletion mutants of RA.
The analysis was by electrophoresis in polyacrylamide gels of total RNA
extracted from reticulocyte lysate translation reactions and treated
with aniline (+) or left untreated(-). The lysate was
programed (except in selected controls) with wild-type or mutant RA
cDNA transcripts. Lane 1, no addition; lane 2, RA
protein (7.4 10
M); lane
3, wild-type transcript; lane 4,
Phe-24; lane
5,
Ile-25; lane 6,
Val-28; lane 7,
Tyr-123; lane 8,
Gly-140; lane 9,
Asn-141; lane 10,
Glu-146; lane 11,
Ala-147; lane 12,
Ile-148; lane 13,
Pro-200; lane 14,
Asp-201; lane 15,
Ile-202; lane 16,
Ile-203; lane 17,
Val-204; lane 18,
Ile-205; lane 19,
Asn-141; lane 20,
Met-174 lane 21,
Asn-122. MN, oligoribonucleotide fragments that result
from micrococcal nuclease treatment of the reticulocyte lysate; RA, fragment from the 3` end of 28 S rRNA formed by aniline
treatment after depurination by RA. The mutants in lanes 7-9,
13, 14, and 17-21 are scored active (cf. Table 1). NB, in evaluating the results it is essential
to compare bands in the aniline treated and untreated
lanes.
Because of the extraordinary activity of
RA(1) , and because of the exquisite sensitivity of the
translation assay, it was important to rule out the possibility that
the apparent enzymatic activity of the mutants was due to contamination
with wild-type RA. This requires assurance that the mutant transcripts
are not contaminated with wild-type mRNA. The smallest amount of
contaminating wild-type DNA that would yield enough transcript to
result in detectable depurination of 28 S rRNA upon translation in the
reticulocyte lysate was determined. Transcripts were prepared from
mixtures having various ratios of the DNAs (a total of 250 ng) encoding
an inactive mutant (Arg-213) and the wild-type; one-fourth of the
total was translated in the lysate. Depurination was observed when the
amount of wild-type DNA was 25 pg (only 0.04% of the total in the
mixture) but not when it was 10 pg. Thus translation of transcripts
from extremely small amounts of cDNA can catalyze sufficient
depurination to be detected and this amount of cDNA would not be
discerned on sequencing gels. Therefore, the quality of the results is
conditioned by the purity of the mutant clones.
Single colonies were selected from among transformants and grown overnight; the DNA was isolated and the sequence of nucleotides determined. The colonies with mutant DNA were streaked on agar plates so that single colonies could be selected easily. This is the colony purification procedure. These cells were grown, the DNA purified, and the sequence of nucleotides determined again. As a further precaution, cells were transformed with the DNA that had been isolated after colony purification from the nine active mutants and an additional cycle of selection and colony purification was carried out. None of these mutants lost their activity in the process.
The polymerase chain reaction was used to detect possible contamination of active mutants with wild-type DNA. Pairs of oligodeoxynucleotides were synthesized; one complementary to a sequence in both mutant and wild-type DNA, the second only to the wild-type. In control experiments with mixtures of wild-type and mutant DNA as little as 1 pg of the former could be detected after amplification. No wild-type DNA was detected in the reaction with the mutants specified above. Therefore, contamination is with less than 1 pg of wild-type DNA, much less than the amount (between 10 and 25 pg) required after transcription and translation to give detectable depurination. We conclude that the activity of the mutants cannot be accounted for by contamination with wild-type DNA.
Inspection of the amino acid sequences of 11 ribosome inactivating proteins that have the same mechanism of action as ricin led to the identification of 13 invariant residues(35) . In RA they are: Tyr-21, Phe-24, Arg-29, Tyr-80, Tyr-123, Gly-140, Ala-165, Glu-177, Ala-178, Arg-180, Glu-208, Asn-209, and Trp-211 (Fig. 2). Most of these amino acids are congregated in or near the putative active site cleft; three are in helix E. The identification of the invariant residues supported the assumption from the three-dimensional structure that the cleft was the active site and provided the first indication of which amino acids might be important for the function of the toxin.
Figure 2:
Ribbon representation of the -carbon
backbone of RA. In A, the proposed active site cleft is
located at the lower right on the front side.
-Helices are
represented by barrels and identified by uppercase letters (A-H);
-strands are represented by arrows and identified by lowercase letters (a-f); the numbers are positions of amino acids. The locations of the
amino acids that cannot be deleted without loss of the ability to
inactivate reticulocyte ribosomes are stippled. In B,
the amino acids that can be deleted without loss of the ability to
inactivate reticulocyte ribosomes are underlined and the
length of the line is proportional to the number of residues in the
deletion. Amino acids in
-helices (A-H) and
-strands (a-f) are designated by coiled and jagged lines and the 13 invariant residues in plant and bacterial toxins that
have the same mechanism of action as RA are in bold
type.
Several of the invariant residues were subsequently the object of site-directed mutagenesis. For example, mutations were made in Glu-177 and Arg-180(19, 20, 36) . These residues were chosen not only because they are invariant but also because they were thought to be directly involved in catalysis. The details of the chemistry of the hydrolysis of the N-glycosidic bond of A4324 in 28 S rRNA by RA are not known, however, a mechanism has been proposed based on an analogy with the cleavage by a nucleosidase of the bond between adenosine and ribose in AMP, a reaction that has been studied in some detail(37) . For RA the proposal (38) is that Arg-180 forms a hydrogen bond with N-3 of the substrate adenosine in a strained syn configuration approximating the transition state; this interaction may protonate the adenine ring transiently and thereby facilitate bond breakage. Glu-177 is thought to stabilize the developing oxycarbonium character of the ribose ring and, perhaps, also to polarize the water molecule that may be the nucleophile; the nitrogen-carbon bond is then displaced by nucleophilic attack of the water molecule on the C-1 of ribose. The conversion of the invariant Glu-177 to aspartic acid or to alanine reduced, by 0.08 and 0.18, respectively, but did not abolish enzymatic activity(19) . To rationalize the disappointingly trivial affect of the E177A mutation, it was suggested that either the carboxylate group at position 177 is not important for catalysis(19) , which seemed unlikely, or that Glu-208, which is also at the bottom of the cleft, can substitute for the missing functional group(20) . The E177D mutant may be less active than wild-type RA because the carboxylate of the shorter aspartic acid side chain is unable to reach an optimum position for catalysis and because it prevents Glu-208, by electrostatic repulsion, from moving into an effective position. The neutral alanine side chain in the E177A mutant, on the other hand, might not interfere with the positioning of the carboxylate of Glu-208. This possibility was tested by constructing an E177A,E208D double mutant which had, as predicted, virtually no activity; a result that supports the original explanation that Glu-208 can substitute for Glu-177 in the E177A mutant and affirms that a carboxylate function is important for catalysis. A control mutant, E208D, with no change at position 177, had as expected enzymatic activity equal to the wild type protein.
The contribution of the side chain of Arg-180 was assessed by converting it to lysine and histidine(20) . These mutations decreased enzymatic activity by 0.25 and 0.001, respectively. The relatively small loss in activity in the R180K mutant supports the assumption that it is a positive charge in the active site that is important for the chemistry of catalysis. The greater decrease in activity of the R180H mutant has been attributed to the likelihood that it is not protonated at physiological pH(20) .
The invariant aromatic residues Tyr-80, Tyr-123, and Trp-211 are thought to contribute to substrate binding(16, 18, 39) , although, no direct assessment of the binding of the substrate to the enzyme has as yet been made. The conversion of these residues to phenylalanine in separate mutants had relatively little effect on enzymatic activity (22, 23) , presumably because phenylalanine can also contribute to binding, as tyrosine and tryptophan are thought to contribute, by intercalating its aromatic ring between the bases of RNA. The conversion of Tyr-80 and Tyr-123 to serine, on the other hand, decreased enzymatic activity by 0.005 and 0.01, respectively(36) . The conversion of the invariant Asn-209 which is at the bottom of the cleft to serine had only a relatively small effect on enzymatic activity (23) . Arg-56 is near invariant (it is either arginine or lysine in the 11 toxins), is near the cleft, and might interact with bases in rRNA or with the phosphate backbone; however, an R56A mutation had no effect on the ability of the toxin to depurinate A4324(24) . In this vein, modification of arginine residues at positions 193, 196, 213, and 234, and 235 with phenylglyoxal had no effect on RA activity either (40) .
Thus, apart from the implication of Glu-177 and Arg-180 in the chemistry of catalysis, site-directed mutagenesis had not provided a great deal of help in the identification of functionally important residues. Perhaps for this reason another approach was adopted: yeast were transformed with an expression vector containing randomly mutated RA cDNA(41) . The assumption was that only yeast harboring a RA cDNA with an inactivating mutation would continue to grow. The sequence of nucleotides was determined for cDNAs isolated from seven yeast colonies that survived induction of expression of the toxin. The mutations were: E177D and E177K; W211R; G212W and G212E; S215P; and I252R. These results must be interpreted with caution since cells apparently can survive even if the mutant toxin is still active. For example, the E177D mutant retains activity (0.08 of wild-type) when assayed on ribosomes in a reticulocyte lysate(19) ; in addition, Ser-215 and Ile-252 can be deleted from RA without complete loss of activity (25) which leads to a strong presumption that their conversion to proline and arginine would not lead to a complete loss of activity either, a presumption strengthened by their being distant from the putative active site.
Another approach is to include all of the RA amino acids in the screen. In systematic deletion analysis one starts with a set of contiguous deletions, for example, of 20 amino acids as was done with RA(25) , each deletion adjacent to the preceding one. Subsequent sets increase the resolution of the deletion map by analyzing smaller omissions, first of 5 and then of 2 residues, and finally as here of one amino acid; each set after the first encompassing all of the residues whose omission had produced inactive mutants in the previous set. What recommends this approach is that it is blind to prejudices or preconceived ideas as to where the active site is and to which residues are important, one simply surveys them all. In the present study 9 of 45 single amino acid deletions retained activity (Fig. 3A). Thus in the four sets of amino acid deletions, we have identified 32 amino acids that cannot be dispensed with/without loss of activity. The 32 residues are congregated in or near the putative active site cleft (Fig. 3B). The 32 include 6 of the 13 invariant amino acids.
Figure 3: Three-dimensional structure of RA obtained with the program SETOR on a Silicon Graphics system using the coordinates from the x-ray defraction of the heterodimer. In A, the 9 amino acids whose deletion was tolerated are in dark blue. In B, the 32 amino acids that could not be deleted in any of the mutants without loss of RA activity are in red.
Helix E forms the backbone of RA (18) and has the
active site residues Glu-177 and Arg-180 which are at the bottom of the
cleft (Fig. 2A and Fig. 3). The largest
concentration of amino acids whose omission inactivates the enyme are
in helix E and the adjacent unstructured region, 16 of 17 residues
between positions 168 and 184; this is half of all of the essential
residues and it is noteworthy that 11 of the 17 are hydrophobic. The
single residue in this sequence that can be omitted is Met-174 which is
difficult to rationalize since it is very close to Glu-177; the
distance from C of Glu-177 to the C
of Met-174 is only 6.25
Å. Moreover, Met-174 appears to contribute to the stabilization
of the catalytic center. E is the longest helix in RA and has a
distinct bend of roughly 30° near its carboxyl-terminal end. This
bend positions Glu-177 and Arg-180 in the active site cavity. The bend
disrupts the local hydrogen bonding pattern in the helix (18) near the carboxyl carbon of Met-174; the latter is rotated
away from the helix axis by roughly 50° thereby allowing it to
hydrogen bond to the hydroxyl group of Tyr-21. Thus, the bend does not
depend on the Met-174 side chain and in the absence of the amino acid
the carboxyl carbon of Ile-175 may be able to compensate for the loss
of Met-174 and for a perturbance in the structure of helix E.
Phe-168 and Ile-172 in helix E are important (they cannot be
deleted), perhaps because they stabilize the core through hydrophobic
contacts with residues in the -sheet (Tyr-84, Phe-93, and Phe-117)
and in helix C (Tyr-123 and Leu-126)(18) . Phe-181 is another
essential nearby amino acid that interacts with the invariant Trp-211
and presumably their close packing is important to the geometry of the
active site. The essential Ile-184 also contacts Phe-181 and the
methylene carbons of the active site Glu-177 further stabilizing the
active center. Thus, the cluster of essential hydrophobic residues in
or near helix E appear to be important for the packing and hence the
geometry of the active site.
All of the amino acids in helices B, C, and D are dispensable, although helix D is a special case(25, 26) . Each of the amino acids in helix D can be deleted from one mutant or another provided care is taken that the deletion does not disrupt the amphipathicity of the helix(26) . The hydrophobic surface of helix D abuts helix E and the hydrophilic surface shields helix E from solvent. Thus it is not surprising that single amino acid deletions in helix D (Glu-146, Ala-147, Ile-148, and Ser-149) abolished RA activity (Table 1).
Many of the residues in helices A, G, F, and H can be deleted, although, a number are essential ( Fig. 2and Table 1). For example, Phe-24, Ile-25, Val-28, and the invariant Arg-29 in helix A cannot be omitted. These amino acids are relatively distant from Glu-177 and Arg-180 so it is unlikely they participate directly in the chemistry of catalysis; this suggests, but only because there is no other explanation, that they participate in substrate binding. Five of the nine residues in helix G (Pro-202, Ser-203, Thr-206, Leu-207, and Ser-208) are essential ( Fig. 2and Table 1); the role of these five residues in the structure and function of RA cannot be defined. The activity of RA does not survive deletion, even one at a time, of three residues (Trp-211, Gly-212, and Arg-213) in helix H (Table 1); however, Arg-213 can be chemically modified with phenylglyoxal, and presumably inactivated, without loss of RA activity(40) . The invariant Trp-211 is thought to participate in the binding of the adenosine in the RNA substrate(39) .
A relatively large number of
proteins that bind to RNA have a domain, referred to as the RNP motif (cf. (42) for citations and discussion). The element
has 90 to 100 amino acids and its most characteristic feature is two
conserved sequences of 6 and 8 residues, RNP1 and RNP2; there are also
a number of conserved, mostly hydrophobic, residues distributed in the
motif. It is important that some RNA binding proteins lack consensus
RNP1 and RNP2 amino acid sequences but have similar, structurally
significant, residues. The three-dimensional structures of the RNP
domains of two proteins, U1A (43) and hnRNP C(44) ,
have been determined and they share a
fold in which the four
-strands form an antiparallel
-sheet
packed against the two perpendicularly oriented
helices. In the
folded domain the amino acids of RNP1 and RNP2 are juxtaposed in the
central strands (
1 and
3) of the
-sheet and the charged
and aromatic side chains of these amino acids are thought to interact
with bound RNA through hydrogen bonds and stacking of rings. The
conserved residues of RNP1 and RNP2 are critical for the association
with RNA but do not confer specificity on binding; specificity is
believed to reside in the variable regions especially in the
unstructured loops. Binding studies ( (42) and references
therein) support this interpretation in that they indicate that the
-sheet of the RNP motif can make nucleotide sequence independent,
and hence nonspecific, contacts with RNA.
RA has an RNP-like
structural motif; different from the canonical but with sufficient
similarity to justify a comparison to the domains in U1A (Fig. 4). (We chose U1A for the comparison because the
coordinates for the structure are available whereas those for hnRNP C
are not.) The RNP-like motif of RA is composed of the following
secondary structure elements: a
A
b
c
d. . . .
E; this is to be contrasted with the related elements in U1A,
1
1
2
3
2
4. Thus the motif in RA differs from
the canonical, and particularly from that in U1A, in the order of, and
in the lack of continuity in the sequence of amino acids in, the
secondary structure elements; and perhaps most importantly in that RA
has neither RNP1 nor RNP2 consensus amino acid sequences. Recall,
however, that RNP1 and RNP2 sequences are not absolutely essential to
RNP motifs.
Figure 4: A comparison of the three-dimensional structures of the RNP motif in U1A and a similar domain in RA. The structure in U1A is white and in RA is green. The helices and strands in RA are lettered.
Despite these differences the tertiary structure of the
RNP domain in RA is in large measure congruent with the RNA binding
domain of U1A (Fig. 4). In particular the -sheet of RA
closely approximates that of U1A; the root mean square deviation of the
C
backbones is about 1 Å. The structural relationship of the
strands in the two
-sheets are as follows:
b of RA to
1
(RNP2) of U1A;
c of RA to
3 (RNP1) of U1A; and
d of RA
to
2 of U1A. Indeed, there is only one significant difference in
the
-sheets:
a of RA is parallel to
b, whereas in U1A
4 is antiparallel to
3.
In the comparison of the
structures, helix A of RA is displaced from the related helix
2 of U1A by 4 Å although they are parallel. It is possible
that the binding of RNA induces a change in the position of helix
A of RA bringing it closer to
a and thereby to more nearly
approximate the relationship of
2 and
4 of U1A. The long loop
between
A and
b of RA might facilitate this change in
structure. Helix
E of RA corresponds to
1 of U1A, in the
superimposition of the structures the two helices intersect at an angle
of about 20°. The intersection is at Met-174 of RA which is the
site of a bend in helix E (see earlier); this bend in
E brings the
carboxyl-terminal regions of the two helices close together.
We
judge that there is sufficient structural similarity of the RNP-like
domain of RA to the structure in U1A to suggest that the former is
involved in binding to 28 S rRNA. The -sheet of the motif might
mediate an initial, nonspecific, low affinity association of RA with
ribosomal RNA; this initial interaction might facilitate subsequent
homing in on a high affinity, specific site and the positioning of
A4324 in the catalytic center. This interpretation is supported by the
observation that a number of hydrophobic and aromatic residues in
d (VVGY), in
A (FI . . . VR), and in
E (FIICIQ.ISEAAR)
are essential for RA activity, indeed, these 3 structural elements have
more than half (20 of 32) of the essential residues in RA.
Two
hundred and three deletion mutants of RA have been constructed and 96
retain activity. In those 96, 235 of the 267 amino acids (88%) in the
protein were omitted. This is an indication that many of the residues
are neither absolutely essential for folding into some effective
conformation nor for catalysis. We recognize that in any single mutant
the deletion that was tolerated was short, only in one instance as long
as 20 residues and in the others 5 amino acids or less. Nonetheless, it
is surprising that residues that are hydrophobic and are buried in the
interior of the protein, or that are part of -helices or of
-strands, can be deleted without loss of activity. This unexpected
outcome reinforces the conviction that deletion analysis is a valuable
procedure for defining the minimal cohort of amino acids necessary for
substrate recognition and catalysis. The findings from deletions of
amino acids and from site-directed mutagenesis indicate that RA has a
great capacity for compensatory change in structure and, hence, a great
ability to preserve function; in short, the protein displays
considerable plasticity.