The structure of 3
'
-
O
-anthraniloyladenosine, an analogue of the 3
'-end of aminoacyl-tRNA
The structure of 3 ' - O -anthraniloyladenosine, an analogue of the 3 '-end of aminoacyl-tRNA
Barbara
Nawrot
,
Wolfgang
Milius
1
,
Andrzej
Ejchart
2
,
Stefan
Limmer
and
Mathias
Sprinzl*
Laboratorium für Biochemie,
1
Laboratorium für Anorganische Chemie and
2
Lehrstuhl für Struktur und Chemie der Biopolymere, Universität Bayreuth, D-95440
Bayreuth
,
Germany
Received December 5, 1996;
Accepted January 20, 1997
NAR-CCDC identifier CCDC-179
ABSTRACT
3
'
-
O
-Anthraniloyladenosine, an analogue of the 3
'
- terminal aminoacyladenosine residue in aminoacyl-tRNAs, was prepared by chemical synthesis, and its crystal structure
was determined. The sugar pucker of 3
'
-
O
-anthraniloyladenosine is 2
'
-
endo
resulting in a 3
'
-axial position of the anthraniloyl residue. The nucleoside is in
syn
conformation, which is stabilized by alternating stacking of adenine and benzoyl residues of the neighboring molecules in the crystal lattice. The conformation of the 5
'
-hydroxymethylene in 3
'
-
O
- anthraniloyladenosine is
gauche-gauche
. There are two intramolecular and two intermolecular hydrogen bonds and several
H-bridges with surrounding water molecules. The predominant structure of 3
'
-
O
-anthraniloyladenosine in solution, as determined by NMR spectroscopy, is 2
'
-
endo
,
gauche-gauche
and
anti
for the sugar ring pucker, the torsion angle around the C4
'
-C5
'
bond and the torsion angle around the C1
'
-N9 bond, respectively. The 2
'
-
endo
conformation of the ribose in 2
'
(3
'
)-
O
-aminoacyladenosine, which places the adenine and aminoacyl residues in
equatorial and axial positions, respectively, could serve as a structural element that is recognized by enzymes that interact with aminoacyl-tRNA or by ribosomes to differentiate between aminoacylated and non-aminoacylated tRNA.
INTRODUCTION
The ubiquitous 3'-terminal adenosine of tRNA serves as an acceptor for covalent attachment of the amino acid during enzymatic
aminoacylation of tRNA by aminoacyl-tRNA synthetases (
1
). Several enzymes, protein factors and ribosomes interact with aminoacyl- tRNA or peptidyl-tRNA during protein biosynthesis. In order to avoid inhibition by
non-aminoacylated tRNA, these macromolecules have to recognize the
aminoacyladenosine on the 3'-end of aminoacyl-tRNA. Indeed, differences in the equilibrium dissociation
constants for non-aminoacylated tRNA and aminoacyl-tRNA are, in some cases, up to five orders of magnitude (
2
). This raises the question about the differences in the structures of
aminoacylated and non-aminoacylated tRNA, which provide the structural basis for such efficient discrimination. This problem was addressed by numerous studies (
3
, and references therein). Experimental methods suitable for detection of gross
conformational changes did not indicate a large structural difference in tRNA
upon aminoacylation. Methods which specifically probed the structure of the CCA-end of tRNA indicated, however, a change in the conformation and metal binding properties in this part of the molecule upon aminoacylation (
3
,
4
). It has not been possible to determine the aminoacyl-tRNA structure by X-ray analysis or NMR spectroscopy. Such investigations are hampered by instability of the aminoacyl ester bond, which is
hydrolyzed at ambient temperature and pH 7, with a half-time between 20 and 120 min (
5
).
The nucleoside antibiotic puromycin resembles the 3'-end of aminoacyl-tRNA. Its structure has been determined by crystallization
and X-ray analysis (
6
), as well as by NMR in solution (
7
). The amino acid residue in puromycin is, however, linked to adenosine by an
amide bond, which may limit the value of this analogue as a structural model
for aminoacyl-tRNA. Similarly, the structure of 3'-
O
-acetyladenosine, which has been solved by X-ray crystallography (
8
), has only limited value as a structural model for 3'-
O
-aminoacyladenosine since it does not possess an [alpha]-amino group on the acyl residue.
As determined by NMR spectroscopy, the single-stranded ACCA sequence, which forms the 3'-end of tRNA, is continuously stacked to the RNA helix of the aminoacyl domain (
9
-
11
). Fluorescence measurements with tRNA containing a fluorescent adenosine
analogue formycin in position 76 revealed that the 3'-end of tRNA in solution has an ordered, stacked structure (
12
).
The crystal structure of an aminoacyl-tRNA in complex with elongation factor Tu (EF-Tu) and guanosine 5'-([beta],[gamma]-imino)- triphosphate has been recently determined
(
13
). In this structure the terminal 3'-adenine 76 of tRNA is not involved in stacking with the penultimate
cytosine 75, but instead it is placed in a cleft on the interface between
domains I and II of EF-Tu (
13
). Thus the possibility arises that the destacking of the 3'-terminal adenosine upon aminoacylation is the structural element
which determines the recognition of aminoacyl-tRNA by EF-Tu-GTP.
In the present work we investigate the structure of 3'-
O
-anthraniloyladenosine. This compound, which can be chemically synthesized (
14
), has a structure similar to 3'-
O
-aminoacyladenosine and is sufficiently stable to allow crystallization or
NMR studies. It has also been shown that the anthraniloyl group attached to the
adenosine 76 of tRNA mimics the structure of aminoacyl-tRNA and allows an efficient interaction of anthraniloyl-tRNA with EF-Tu-GTP (
15
).
MATERIALS AND METHODS
Modification of adenosine with isatoic anhydride was performed according to
Hiratsuka (
14
). Adenosine (1 mmol) was dissolved in 25 ml of water at 38oC. The pH was adjusted to 9.6 with 2 M NaOH, and isatoic anhydride (1.5
mmol) was added to the reaction mixture. The pH was maintained at 9.6 by
titration with 2 M NaOH for 2 h. The progress of the reaction was monitored by
thin layer chromatography on silica gel plates (Polygram sil G/UV
254
, Roth, Karlsruhe, Germany), in chloroform/methanol, (8:2, v/v). R
f
values are 0.38 and 0.73 for adenosine and 2'(3')-
O
-anthraniloyladenosine, respectively. The reaction mixture was cooled to 0oC, and the product was collected by centrifugation. 3'-
O
-anthraniloyladenosine was isolated from the crude reaction product by
crystallization from ethanol/water (1:4, v/v), and purified by silica
gel column chromatography (Kieselgel 60, Merck, Darmstadt, Germany) in
chloroform/methanol (9:1, v/v). The final purification was achieved by crystallization from ethanol/diethyl ether (5:5, v/v).
HPLC analysis was performed with a Beckman HPLC System
Gold using a Supelcosil LC-18S column, 250 * 4.6 mm, with a Supelcosil guard column, (Supelco, Bellafonte,
USA). Elution was done at 9oC, with a flow rate of 1 ml/min, with a gradient of 5 mM sodium phosphate
buffer pH 3.5 and methanol as follows: 0-5 min, 2.5% methanol; 5-10 min, 10% methanol; 10-65 min, 10-70% methanol gradient; 65-70 min, 70% methanol; 70-80 min: 70-2.5% methanol gradient.
Spectrophotometric detection was at 254 and 335 nm. 2'(3')-
O
-Anthraniloyladenosine 5'-phosphate was obtained under the same conditions as described
above, except that after reaction was complete the pH was adjusted to 7.0 with
1 N HCl. The product was isolated from the reaction mixture by chromatography
on a Sephadex A-25 column in a 20 mM sodium acetate, pH 5.0, and NaCl gradient from 10 to
250 mM. After desalting on Biogel P6 column the solution was lyophilized to
provide pure 2'(3')-
O
-anthraniloyladenosine 5'-phosphate as a white powder, which was characterized by HPLC,
NMR spectroscopy and mass spectrometry.
Crystal structure of 3
'
-
O
-anthraniloyladenosine
3'-
O
-Anthraniloyladenosine crystallizes as a dihydrate in space
group P2
1
with two molecules in the asymmetric unit. In this respect 3'-
O
-anthraniloyladenosine exhibits a lower symmetrical crystal structure than
puromycin, which crystallizes orthorhombically with space
group P2
1
2
1
2
1
(
6
). Data pertinent for X-ray structure determination are compiled in Table
1
.
The resulting molecular structure of 3'-
O
-anthraniloyladenosine with its atom numbering scheme and solvent molecules is shown in Figure
3
a. There are two intramolecular hydrogen bonds: between N3 of purine and the 5' OH group of ribose and between the carbonyl group and the amino group of
the anthraniloyl residue. Two intermolecular hydrogen bonds per molecule are
between the amino group of the anthraniloyl residue and the 2'-oxygen atom of the neighboring molecule, and between the amino group of purine and the 2'-oxygen atom of the symmetrically equivalent molecule. Additionally, several hydrogen
bonds are possible with surrounding solvent molecules as indicated in Figure
3
b. Two intramolecular hydrogen bonds (H5'A-N3 and H11A-O6) influence the stability of the molecule in the adopted
conformation. There are no signs of protonation of either N1 of the adenine
base or the amino
group N11 of the anthraniloyl residue, as is observed for puromycin (
6
). Amino groups of adenine and anthraniloyl residues have a pyramidal
arrangement and consequently their hydrogen atoms are not located on the planes formed by the aromatic system planes. The sugar ring puckering is 2'-
endo
with endocyclic torsion angles [nu]
0
= -22.2(0.6)o, [nu]
1
= 35.8(0.5)o, [nu]
2
= -34.9(0.6)o, [nu]
3
= 23.3(0.6)o, [nu]
4
= -0.9(0.6)o. The pseudorotation phase angle P, calculated according to Saenger
(
16
), is 162o, thus in the range of 2'-
endo
ring puckering (144o <= P <= 180o), whereas the puckering amplitude [Phi]
m
is 36.4o (
17
).
The structure of 3
'
-
O
-anthraniloyladenosine in solution
The solution structure of 3'-
O
-anthraniloyladenosine was determined by NMR spectroscopy. In aqueous solutions both 2'-
O
- anthraniloyl isomers and 3'-
O
-anthraniloyl isomers exist in equilibrium with the predominant 3' isomer. This situation is similar to that of the 2' and 3' isomers of aminoacyladenosine (
20
).
In the low field part of the proton NMR spectra (8.27-6.76 p.p.m.) several resonances corresponding to the H2 and H8 protons of the
adenosine and protons of the phenyl residue occur. Upfield signals (6.39-3.82 p.p.m.) originate from aliphatic ribose protons. (For full resonance assignments, see `Supporting Information'.) In comparison to the unsubstituted adenosine molecule, all anthraniloyl derivatives of
adenosine show a downfield shift of ~1 p.p.m. for the ribose proton adjacent to the esterified hydroxyl
group. A similar effect, a downfield shift of ~0.3 p.p.m., is observed for neighboring protons (
21
,
22
). Resonance signals of 3'-
O
- anthraniloyladenosine are therefore at 5.62 and 5.11 p.p.m. for H3' and H2', respectively, whereas corresponding resonances for the
adenosine are at 4.40 and 4.77 p.p.m., respectively. Accordingly, in the case of 2'-
O
-anthraniloyladenosine the H1' proton gives rise to a signal at 6.39 p.p.m., whereas the 3'-
O
-anthraniloyladenosine H1' proton resonance
occurs at 6.16 p.p.m. The relative intensities of both signals reflect the
concentration of the isomers. The population ratios of the 2' isomer to the 3' isomer, expressed by an equilibrium constant K
eq
, are 0.50 and 0.46 for the 2'(3')-anthraniloyladenosine and the 2'(3')-anthraniloyladenosine 5'-phosphate, respectively.
The ratio of 2' isomer to 3' isomer for several aminoacyladenosine derivatives is, depending
on pH and temperature, in the range 0.36-0.47 (
21
). Thus, as in the case of adenosines esterified with naturally occurring amino
acids, 3'-
O
-anthraniloyladenosine is the predominant isomer in an equilibrium mixture
under similar conditions.
Furanose ring puckering of 3'-
O
-anthraniloyladenosine was analyzed by vicinal spin-spin coupling constants
3
J
HH
(
23
) (complete compilation of scalar coupling is given in supporting information
under Supplementary Material). As was determined for the crystal structure of 3'-
O
-anthraniloyladenosine, the 2'-
endo
conformer is also a predominant conformer in solution. For the corresponding 3'-
O
- anthraniloyladenosine 5'-phosphate, the 2'-
endo
conformer likewise prevails. 2'-
endo
populations of ~78% (J
1'2'
= 6.4 Hz) for the 2'-
O
-anthraniloyladenosine isomer and 90% (J
1'2'
= 7.2 Hz) for the 3'-
O
-anthraniloyladenosine isomer can be derived from the scalar coupling constants (
23
). For 2'-
O
-anthraniloyladenosine 5'-phosphate and 3'-
O
-anthraniloyladenosine 5'-phosphate, the 2'-
endo
populations are 68% (J
1'2'
= 5.7 Hz) and 90% (J
1'2'
= 7.2 Hz), respectively. Substitution of a 2'-OH group of adenosine or AMP with the anthraniloyl residue also
leads preferentially to the 2'-
endo
conformer, although a decrease in its population by several percent indicates
slightly different ring puckering.
The conformation around the C4'-C5' bond was determined using spin-spin coupling constants J
4'5'
and J
4'5''
(see supporting information available as Supplementary Material) (
24
). The sum of both coupling constants in the range of 5.0 Hz indicates the
gauche
-
gauche
as the preferred conformer for both 2'- and 3'-
O
-anthraniloyl isomers, ~80% of the population for both isomers.
Determination of the conformation around the glycosidic bond ([chi]) was performed on the basis of the two-dimensional nuclear Overhauser enhancement spectroscopy. As expected
for a low- molecular weight compound, negative cross peaks with respect to the
diagonal peaks were observed [positive nuclear Overhauser effect; cf. (
25
)] The nuclear Overhauser effect cross-peak intensities can be related to the distances between observed proton
spins (
25
). According to the reported intramolecular distances for a canonical A-DNA helix, with the torsion angle in the range of
anti
and pseudorotation angle for the 2'-
endo
conformer, the distances between the base and ribose protons H8-H2' and H8-H1' are 3.9 and 3.8 Å, respectively (
25
). These values determine the relative nuclear Overhauser effect cross-peak intensities to be approximately equal (I
H8-H1'
/I
H8-H2'
= 1.17). Similar data analysis for a Z-DNA molecule, which represents
syn
conformation around the glycosidic bond,
gives values of 4.1 and 2.3 Å for H8-H2' and H8-H1' distances, respectively. The predicted
relative magnitude of the NOESY cross-peak intensity ratio should then be on the order of 30. The observed NOESY
cross peak pattern involving the ribose protons H1' and H2', as well as the H8 proton of the adenine residue, for the mixture
of the 2'- and 3'-
O
-anthraniloyladenosine isomers is displayed in Figure
5
. The comparison of the relative cross peak intensity ratio for the 3'-
O
-anthraniloyladenosine and 2'-
O
-anthraniloyladenosine gives values of 1.23 and 3.93, respectively. These values
indicate a preferred
anti
conformation for both the 2' isomer and the 3' isomer. However, the value obtained for the 2'-isomer indicates that a small population of
syn
conformer is in fast exchange with the predominant
anti
conformer. Thus, the relative orientation of the base and the sugar moiety for
the anthraniloyl derivatives of adenosine in solution is not determined by
alternating stacking of the aromatic systems as observed in the solid state
(Fig.
4
).
Transacylation rate between 2
'
-
O
-anthraniloyladenosine and 3
'
-
O
-anthraniloyladenosine
Kinetics of the transacylation reaction between 2'-
O
-anthraniloyladenosine and 3'-
O
-anthraniloyladenosine were measured by
1
H NMR spectroscopy using H1' peak integrations for mole fraction determination. Equilibrium constant
(K), equilibrium rate (k)
and transacylation rates (k
3' -> 2'
and k
2' -> 3'
) were determined according to the equation A(t) = A
e
+ (A
o
-A
e
)e
-kt
, where A
o
and A
e
are mole ratios of 2'-
O
-anthraniloyladenosine at time 0 s and at equilibrium, respectively. There, the intensity increase of resonance signals due to the 2' isomer was analyzed.
Structural features of crystalline puromycin (6), 3'-
O
-acetyladenosine (8) and 3'-
O
-anthraniloyladenosine
3'-
O
-acetyladenosine
3'-
O
-anthraniloyladenosine
puromycin
Glycosidic torsion angle ([chi])
syn
syn
anti
C4'-C5' bond conformation ([gamma])
gauche-gauche
gauche-gauche
gauche-gauche
Sugar ring pucker
2'-
endo
2'-
endo
3'-
endo
Position of C1'-N bond
equatorial
equatorial
axial
Position of C3'-O bond
axial
axial
equatorial
The equilibrium rate k = k
3' -> 2'
+ k
2' -> 3'
(at p
2
H 7.2 and 4oC) determined in this way was (1.15 * 0.09) x 10
-4
s
-1
, which is almost four orders of magnitude less than the equilibrium rate
reported for 2'(3')-
O
-phenylalanyladenosine [1.01 s
-1
at p
2
H 7.3 and 25oC; (
21
)] The equilibrium constant K = (k
3' -> 2'
)/(k
2' -> 3'
), which gives the molar ratio of 2' and 3' isomers at equilibrium, amounts to 0.43 +- 0.02, which is very close to the value observed for 2'(3')-
O
-phenylalanyladenosine [0.40; (
21
)]. The transacylation rates k
3' -> 2'
and k
2' -> 3'
are (0.34 * 0.07) * 10
-4
s
-1
and (0.81 +- 0.04) * 10
-4
s
-1
, respectively. The distinctly slower migration of the anthraniloyl residue as
compared to transacylation rates of 2'(3')-
O
-phenylalanyladenosine (
21
) is due to the more stable ester bond connecting the anthraniloyl residue with
the adenosine moiety and also due to the presence of the aromatic amino
function with a pKa 4.95, which is lower than that of the aliphatic [alpha]-amino group.
DISCUSSION
3'-
O
-anthraniloyladenosine is the first aminoacyl derivative of adenosine which
was crystallized and its structure determined. Adenosines esterified with
proteinogenic amino acids are labile. The ester bond is susceptible to rapid hydrolysis and a transacylation between 2'- and 3'-hydroxyl groups. The presence of the vicinal hydroxyl
group is important for both reactions. In one case it accelerates hydrolysis of
the ester bond (
26
), in the other it is the prerequisite for transacylation between the 2' and 3' positions. Due to attachment to an aromatic ring, the carboxyl
and amino groups of anthranilic acid have a different reactivity as compared to
[alpha]-amino acids. As a consequence, the rate of hydrolysis and rate of
transacylation are more than four orders of magnitude lower than in the case of
adenosines esterified with [alpha]-amino acids. This allows the study of the structure of 2'(3')-anthraniloyladenosine by crystallography or
NMR.
It was reported that anthranilation of adenosine or adenosine 5'-phosphate by isatoic anhydride leads exclusively to 3'-
O
- anthraniloyladenosine derivatives (
14
). We demonstrate in this work that this is not the case. All available, free
hydroxyl groups of adenosine or adenosine 5'-phosphate can be esterified, with preference for the 3'-hydroxyl group. We were successful in isolation of
pure 3'-
O-
anthraniloyladenosine, since the structure of this isomer is stabilized by
alternative stacking of adenine and phenyl rings in the crystal lattice. This
allows the purification of the 3'-isomer by crystallization. We did not succeed, however, in
purification of a single isomer of 2'(3')-
O
-anthraniloyladenosine 5'-phosphate, which could not be prepared in crystalline form.
The crystal structure of 3'-
O
-anthraniloyladenosine has some remarkable features, which are different
from the structure of the antibiotic puromycin, but similar to the structure of
3'-
O
-acetyladenosine (Table
2
). The most significant is the 2'-
endo
pucker of the ribose in the case of the 3'-
O
-anthraniloyl and the 3'-
O
-acetyl derivatives. In contrast, puromycin has a 3'-
endo
conformation both as a crystal (
6
) as well as in solution (
7
). We demonstrate in this work that the 2'-
endo
sugar pucker is a predominant conformation for 3'-
O-
anthraniloyladenosine in solution as well. The ribose ring puckering defines the
orientation of the glycosidic bond, which is equatorial for the 2'-
endo
conformation, but axial for the 3'-
endo
conformation. This difference may be important for the recognition of the aminoacylated adenosine on the 3'-end of tRNA during various steps in translation. Recently the
structure of the EF-Tu in complex with a GTP analogue and aminoacyl-tRNA was determined (
13
). The terminal adenosine of aminoacyl-tRNA in this structure is not stacked to the penultimate cytosine of the
CCA-end, but instead it is extended to a lipophilic pocket of the protein.
Since in tRNA that is not aminoacylated, the CCA-end forms a continuous stack (
9
,
10
,
12
), it is possible that aminoacylation of the 3'-terminal ribose causes destacking of adenosine from the CCA-end providing a signal for recognition.
The mechanism leading to this destacking may involve the interaction of the 2' hydroxyl group of the ribose moiety with the vicinal ester-carbonyl group of the aminoacyl residue. Such an interaction
occurs, e.g. during migration of the aminoacyl residue between 2' and 3' positions of the terminal adenosine with an obligatory orthoester
acid intermediate (
27
). Correspondingly, it was demonstrated for the interaction of tRNA with
aminoacyl-tRNA synthetase that subtle structural variations on the 3'-terminal ribose lead to conformational changes in the tRNA-synthetase complex (
28
,
29
). Aminoacylation of tRNA and the presence of a free vicinal OH-group probably lead to structural changes in the CCA-end and formation of 2'-
endo
conformation on the terminal ribose (
30
).
In A-type RNA the nucleobase is held in axial position, and the ribose adopts the 3'-
endo
conformation. In this case the 3'-substituent is placed to the equatorial position. Such a structure was observed for puromycin, in which the aminoacyl residue,
attached by an amide bond to the 3'-position, is equatorial and the ribose possesses a 3'-
endo
conformation. This may be due to the lack of interaction of the amide-carbonyl with the vicinal 2'-hydroxyl group. In agreement with this interpretation
aminoacyl- tRNA in which the aminoacyl residue is attached via an amide bond to the 3' position of the terminal adenosine does not form stable complexes
with EF-Tu-GTP (
31
).
ACKNOWLEDGEMENTS
This work is dedicated to Professor Max Herberhold on the occasion of his 60
th
birthday. We would like to thank to Melanie Beikman for help with preparation
of the manuscript. The work was supported by the
Deutsche Forschungsgemeinschaft
, Sp-243/8-1 and the Human Frontier Science Programme, RG-369/93.
Supplementary data available
The crystallographic data were deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-179. Copies are available, free of charge, on request from: The Director,
CCDC, 12 Union Road, GB-Cambridge CB1E2 (fax: +1223 336033).
Tables of the proton chemical shifts and the vicinal scalar coupling constants
3
J
HH
of aminoacylated adenosine derivatives (2'-
O
-anthraniloyladenosine, 3'-
O
-anthraniloyladenosine, 2'-
O
-anthraniloyl-AMP and 3'-
O
-anthraniloyl-AMP) can be found as supporting information for this manuscript (one page).
Ordering information is given on any current masthead page.
See supplementary material
available in NAR Online.
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