1Laboratory of Structural Biology and the MOE Laboratory of Protein Science, School of Life Science and Engineering, Tsinghua University, Beijing 100084, 2Department of Biochemistry, Chinese University of Hong Kong, Hong Kong and 3National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing, 100101, China
4 To whom correspondence should be addressed. e-mail: raozh{at}xtal.tsinghua.edu.cn; pcshaw{at}cuhk.edu.hk
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Abstract |
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Keywords: complex structure/mutants/N-glycosidase mechanism/ribosome-inactivating protein/trichosanthin
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Introduction |
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Trichosanthin (TCS) is a type I RIP isolated from the root tuber of Trichosanthes kirilowii Maximowicz (Zhang and Wang, 1986). It is a single-chain polypeptide with 247 residues and consists of two domains: an N-terminal domain (residues 1184) and a small C-terminal domain (residues 185247). The active pocket responsible for N-glycosidase activity is located in the cleft between the two domains (Gao et al., 1993
). TCS has various pharmacological properties, including immunomodulatory, anti-tumor and anti-viral activities (Shaw et al., 1994
). In particular, TCS and other related proteins, such as
-momorcharin (
MMC), have been found to act selectively against HIV-infected cells in vitro by inactivating HIV replication in infected T cells and macrophages. Clinical trials have been performed and the results showed an increase of CD4+ and CD8+ T cells in patients (Byers et al., 1994
; Kahn et al., 1994
).
In order to explore the N-glycosidase mechanism of RIPs, the structures of TCS, MMC and their complexes with substrate analogues such as FMP and NADPH have been reported (Ren et al., 1994
; Xiong et al., 1994
; Yang et al., 1994
; Huang et al., 1995
. These studies have provided the structural basis for understanding the mechanism of the N-glycosidase activity. TCS removes an adenine residue at position A4324 of mammalian 28S ribosome. Therefore, AMP has been used as a substrate analogue in crystallographic studies. However, previously reported attempts to prepare a TCS complex with AMP have been unsuccessful since TCS is able to interact with and hydrolyze AMP. A number of TCS mutants, including E160A, E160D, R163H, E189A and Y70A, have been used previously to prepare a complex with AMP (Li et al., 1999a
,b; Yan et al., 1999
), but in each case the AMP was hydrolyzed by TCS and the result was a complex with adenine.
In order to examine the effects of the charge and size of the side chain of residue 85 on the N-glycosidase activity, three mutants (E85Q, E85A, E85R) of trichosanthin were used to prepare a complex with AMP. Here we report the crystal structures of the three complexes and propose the mechanism of catalysis of N-glycosidase suggested by these complex structures.
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Materials and methods |
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Three site-directed mutants at position 85 of TCS were generated by polymerase chain reaction (PCR) mutagenesis using Pfu DNA polymerase (Strategene) on pET8C carrying the wild-type TCS sequence. The PCR product was cleaved by NcoI and BamHI and cloned to pET8C for expression. The mutation was confirmed by DNA sequencing. A single colony was inoculated into 2 l of LB medium containing 50 µg/ml ampicillin and 25 µg/ml chloramphenicol and the protein was induced by 0.4 mM IPTG at 37°C for 34 h. The protein was purified using an 8 x 2.5 cm CM-Sepharose CL6B column (Amersham Pharmacia Biotech) and 20 mM phosphate buffer with a 01 M NaCl gradient.
Crystallization and preparation of AMP complexes
Crystallization of three mutants was carried out by the hanging drop vapor diffusion method. Solution A contained 40 mg/ml protein and solution B contained 100 mM NaOAcHOAc buffer (pH 5.7) containing 20% KCl and 100 mM CaCl2. A 2 µl volume of solution A and an equal amount of solution B were mixed in droplets; the reservoir solution used was 0.2 ml of solution B. Crystals were obtained after 24 h at 291 K. The three complex crystals were prepared by the soaking them in a reservoir solution containing 10 mg/ml AMP at 291 K for 60 h.
Data collection and processing
Diffraction data were collected using an MAR345 image plate with a Rigaku RU2000 Cu K rotating anode X-ray generator operating at 48 kV and 98 mA (
= 1.5418 Å). The three complex crystals all belong to the space group P212121. The data were processed with the programs DENZO and SCALEPACK (Otwinowski and Minor, 1997
). The crystal parameters and data collection statistics are listed in Table I.
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The three complex crystals are isomorphous with wild-type TCS, hence the starting model used for molecular replacement was the refined structure of TCS (Gao et al., 1993) with E85 replaced by G85. The three complex structures were all solved by the difference Fourier method. The structure refinements were carried out using the CNS software package (Brünger et al., 1998
) and all manual rebuilding of coordinates was performed using the program O (Jones et al., 1991
).
In the electron density maps of both E85Q TCS and E85A TCS complexes, clear electron densities were observed in the active pocket (Figure 1). The AMP molecule can be fitted into the electron density smoothly. In the electron density maps of the E85R TCS complex, however, the shape and size of the electron density in the active pocket were sufficient only to accommodate an adenine molecule. The adenine molecule was built into the electron density.
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Results |
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After refinement, the R-factors and r.m.s deviations of bond length and bond angles of the three complexes are reasonable. The distribution of main-chain torsion angles was calculated using the program PROCHECK (Laskowski et al., 1993). All main-chain torsion angles lie within the allowed regions of the Ramachandran plot. Residue 85 fits the electron density map well in each of the three mutant structures. Equally, AMP and adenine show a good fit with the electron density in their respective complex structures. The fitting of AMP in the electron density map for E85Q TCS/AMP is shown in Figure 1a.
The TCS in the three complex structures is very similar to uncomplexed wild-type TCS. Superposition of C backbones shows r.m.s deviations of 0.41 Å between E85Q TCS/AMP and TCS, 0.31 Å between E85A TCS/AMP and TCS and 0.33 Å between E85R TCS/adenine and TCS. The main differences in conformation occur in the last three residues of the C-terminus.
Interactions between AMP or adenine and TCS in the active pocket
In the E85Q TCS/AMP complex, a network of hydrogen bonds between TCS and AMP helps to stabilize the substrate in its location in the binding pocket of TCS (Figure 2a). The N3 nitrogen of AMP interacts with OE1 of Glu160 and NH1 of Arg163 indirectly through a water molecule. The O atom of Glu189, OE2 of Glu160 and O3' of AMP interact with each other through a water molecule. The N6 nitrogen of AMP makes two hydrogen bonds with OE1 of Gln85 and O of Ile71. The OH of Tyr70 makes two hydrogen bonds with O3P and O5' of AMP. There is another hydrogen bond between O4 of AMP and N of Tyr111.
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In the E85R TCS/adenine complex, the adenine molecule is located in the bottom of the active pocket. The interactions between adenine and TCS are clearly different from those observed in the E85Q TCS/AMP and E85A TCS/AMP complex structures. In the E85R TCS/adenine complex, the N3 atom of adenine forms a direct hydrogen bond with NH1 of Arg163. This interaction is similar to that between NADPH and TCS in the TCS/NADPH complex (Xiong et al., 1994). The orientation of adenine in the E85R TCS/adenine complex is slightly different to that in the TCS/NADPH complex, resulting in the formation of more hydrogen bonds between adenine and TCS (Figure 2c).
The main hydrogen bonds between substrate analogues and TCS in four known complex structures are listed in Table II. The superimposed map of related residues and substrate analogues in the four structures is shown in Figure 1. The AMP molecule adopts very similar positions in the binding pockets of both E85Q and E85A TCS mutants. The adenine molecules of both the E85R TCS/adenine and TCS/NADPH complexes are similarly oriented in the binding pocket. However, the position of AMP in the binding pocket is clearly different from the adenine position, which suggests the presence of two binding sites in the active pocket.
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Residue E85 is not highly conserved among the RIP family members. According to the structure determination of three E85 mutant complexes, their overall structures are very similar. However, two different types of complex were obtained. The first type of complex, obtained for E85Q and E85A mutants, has a complete AMP molecule bound in the active pocket. The second type of complex, obtained for the E85R mutant, has an adenine molecule bound in the active pocket. This adenine molecule is the product of hydrolysis of AMP by TCS, as reported for the complex structures of other TCS mutants (Li et al., 1999a,b). On preparing the complex crystal by the soaking method, the TCS/adenine complex was obtained when residue 85 was charged and the TCS/AMP complex was obtained when residue 85 was uncharged. This suggests that residue 85 could have important implications for N-glycosidase activity. The size of the side chain of residue 85 appears to have no effect on the molecule bound in the active pocket.
Protonation occurs at N3 of adenine
Two different hypotheses about protonation of the adenine substrate have been reported to date. First, Huang et al. proposed that atom N7 of adenine is protonated by Glu85 of TCS (Huang et al., 1995). However, in the study of the interactions between TCS and tubercidin, Wu et al. observed that the N-glycoside bond in tubercidin is cleaved by TCS (Wu et al., 1998
). In tubercidin, the equivalent to atom N7 in adenine is a saturated carbon that cannot be protonated (Wu et al., 1998
). Second, Ren et al. proposed that atom N3 of adenine is the most likely protonation site on the basis of the hydrogen bond between N3 and guanidinum of Arg163 (Ren et al., 1994
). However, this arginine group is unlikely to protonate adenine owing to its high pKa value (Ren et al., 1994
).
From the E85Q TCS/AMP and E85Q TCS/AMP complex structures, the N3 atom of adenine as a protonation site is possible. A stable water molecule is located at the top of the binding pocket, where it forms hydrogen bonds with N3 of AMP and guanidinum of Arg163. This water molecule is ideally placed to act as a proton donor when the AMP molecule enters at the top of the active pocket. Following protonation of the N3 nitrogen of AMP, Arg163 would be able to stabilize the partial negative charge on the water molecule. The substrate would then move to the bottom of the binding pocket, into the position occupied by NADPH in the TCS/NADPH complex structure. This proposed mechanism of protonation is shown in Figure 3.
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From the above structural comparison between TCS/AMP, TCS/adenine and TCS/NADPH complexes, it is reasonable to suggest that the N-glycosidase activity can be divided into two steps, recognition and hydrolysis. First the substrate is recognized and bound to the enzyme at the top of the active pocket, in a similar position to AMP in the E85Q TCS/AMP and E85A TCS/AMP complex structures. The position of adenine in the E85R TCS/AMP complex is the position for catalysis, which is similar to NADPH in the TCS/NADPH complex structure. Residue 85 with or without charge has an important effect on N-glycosidase activity. However, the size of the side chain of residue 85 has no effect on substrate binding.
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Acknowledgements |
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References |
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Received December 10, 2002; accepted May 20, 2003.