Role of Arg163 in the N-glycosidase activity of neo-trichosanthin
Hui-Guang Li,
Shi-Zhen Xu,
Shen Wu1,
Li Yan,
Jian-Hui Li,
Richy N.-S. Wong2,
Qing-Li Shi2 and
Yi-Cheng Dong
Department of Protein Engineering, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 and
2 Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
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Abstract
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Three mutant crystals of neo-trichosanthin (n-TCS), R163K, R163H and R163Q, were obtained by the hanging drop vapor diffusion method. Structure determination indicated that there are no significant differences between the mutants and n-TCS except in the active pocket. All of them were also soaked in sodium citrate buffer (pH 4.5) containing 20% KCl and 10 mg/ml AMP. Structure determination suggests that in the active pocket of the crystals of R163K and R163H, parallel to the aromatic ring of Tyr70, each mutant possesses an adenine. The relationship between structure and function is discussed. Biochemical analysis reveals that the mutants R163K and R163H have N-glycosidase activity, while R163Q does not. This suggests that R163 is a crucial residue for the enzyme activity of n-TCS, and its role is providing proton.
Keywords: mutants/neo-trichosanthin/N-glycosidase activity/protein crystallography/RIPs
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Introduction
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Ribosome-inactivating proteins (RIPs) are a group of cytotoxins which possess a unique rRNA N-glycosidic activity. They are known to hydrolyze a single N-glycosidic bond between adenine and ribose at A4324 in the 28S rRNA of rat liver ribosomes. This reaction damages the ribosomes irreversibly with the consequent arrest of protein synthesis (Endo et al., 1987a
,b
, 1988
; Stirpe et al., 1988
). There are two types of RIPs, type I and type II. Type II RIPs consist of a catalytically active A chain linked to a carbohydrate-binding B chain. The B chain possesses lectin properties which facilities entry of the A chain into the cytoplasm of the cell (Stirpe et al., 1997). Trichosanthin (TCS) is a member of the type I RIPs and comprises 247 residues (Zhang and Wang, 1986
) with one large domain and one small domain. The active pocket responsible for its enzymatic activity is located in the cleft between the two domains (Gao et al., 1993
). TCS has long been used to induce mid-term abortion and to treat ectopic pregnancies, hydatidiform and trophoblastic moles in China (Huang, 1987
). In recent years, TCS has attracted increasing attention mainly because of its various pharmacological properties, including immunomudulatory, anti-tumor and anti-viral activities (Shaw et al., 1994
). TCS and other related proteins have been found to act selectively against HIV-infected cells in vitro by inactivating HIV replication in infected T cells and macrophages (McGrath et al., 1989
; Byers et al., 1990
).
Another area of research concerning RIPs is the search for specific inhibitors (Robertus et al., 1996
; Yan et al., 1998
; Olson and Cuff, 1999
) and novel RIPs (Leung et al., 1997
; Mulot et al., 1997
; Chow et al., 1999
; Lebeda and Olson, 1999
). A gene corresponding to an isoform of trichosanthin, denoted as neo-trichosanthin (n-TCS), has recently been isolated. N-TCS differs from TCS by 11 amino acids. TCS and n-TCS possess a similar rRNA N-glycosidase activity. Studies on the crystal structure of n-TCS have shown that there is little difference between TCS and n-TCS (Yan et al., 1999
). In the active pocket of RIPs, some residues, including Y70, E160 and R163, are invariant. Several mutants of n-TCS have been cloned and expressed. We have reported the crystal structure of Y70A n-TCS and analyzed its activity (Yan et al., 1999
). When Tyr70 is changed to Ala by site-directed mutagenesis, it loses its N-glycosidase activity. Studies on RIPs also demonstrate that R163 is crucial for n-TCS, and its strong positive charge may play a vital role in the N-glycosidase activity (Frankel et al., 1990
; Wu et al., 1998
). In order to study the function of R163 in N-glycosidase activity, we converted the positively charged residue Arg to Lys, His and Gln, respectively, to study their structurefunction relationship.
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Material and methods
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Crystallization and preparation of complex
Cloning, expression, purification and activity analysis of R163H, R163K and R163Q n-TCS will be published elsewhere. Crystallization of mutants were done by the hanging drop vapor diffusion method as described previously (Wu et al., 1998
). Solution A contains 40 mg/ml protein. Solution B is 100 mM NaAc-HAc buffer (pH 4.5) containing 20% KCl and 100 mM CaCl2. Five microliters solution A and 5 µl solution B were mixed in droplets, the reservoir solution was 1 ml solution B. Crystals were obtained after 5 days at 4°C.
The complex crystals of R163H/Ade n-TCS and R163K/Ade n-TCS were prepared by the soaking method. The crystals were soaked in solution B containing 10 mg/ml AMP at 4°C. After 48 h the crystals were used for data collection.
Diffraction data collection
The diffraction data of the above crystals were collected on the Mar Research Area detector at the Crystallography Laboratory, Institute of Biophysics, Chinese Academy of Sciences. The Mar-Research software package was applied to data processing. The results are listed in Table I
.
Structure determination and refinement of R163H, R163K and R163Q n-TCS
The crystal of R163H is isomorphous to n-TCS, thus the initial model used was the refined structure of n-TCS (Yan et al., 1999
). The 2|Fo||Fc| and |Fo||Fc| difference electron density map was calculated by difference Fourier method with the collected diffraction data as Fo and the structure of n-TCS as Fc. Most of the residues fit well in the 2|Fo||Fc| map, except the last three residues of the C-terminus. When R163 is omitted from the 2|Fo||Fc| and |Fo||Fc| maps of the mutant R163H, the side chain of Arg163 expands like a ring. This suggests that R163 has been mutated to His. As a result, R163 is changed to His for further refinement using the program FRODO (Jones, 1985
). Finally, the positions of all residues in the peptide were checked and the side chains were adjusted in order to fit the map.
The structure refinements were carried out using the XPLOR software package (Brunger, 1992
). All atoms were given the same thermal factor (0.15 nm2). After a 40 step prepstage refinement, a simulated annealing refinement was carried out by decreasing the temperature in 25 K per step from an initial value of 3000 to 300 K. The crystallographic R factor was reduced from 0.295 to 0.250. A further 40 step energy restrained least square refinement and a 20 step restrained individual thermal factor refinement dropped the R factor from 0.250 to 0.221. 2|Fo||Fc| and |Fo||Fc| maps were calculated and inspected. Nearly all residues fit the map well except the last three residues of the C-terminus, which also show poor electron density in the crystal structure of n-TCS. Water molecule was assigned in the model if it could form a reasonable hydrogen bond with significant electron density in both the 2|Fo||Fc| and |Fo||Fc| maps. Since the crystals of R163K and R163Q n-TCS are isomorphous to n-TCS, Arg163 was omitted in the initial modeling of R163K n-TCS. The refinement of R163K and R163Q n-TCS was similar to that of R163H n-TCS.
Structure determination and refinement of R613H/Ade n-TCS and R163K/Ade
Using the refined structures of R163H and R163K n-TCS as initial models, the 2|Fo||Fc| and |Fo||Fc| difference electron density maps of crystals R163H and R163K n-TCS, which have been soaked in AMP solution, were calculated respectively. In each of their electron density maps, there was a heavy and clear electron density in the active pocket. However, the electron density only fit to put an adenine ring but not ribose or phosphate group. The refining processes are the same as that of R163H. Refinement statistics of all these crystals are shown in Table II
.
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Results
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After refinement, the R factor and r.m.s. deviation of bond length and bond angles of all structures are rational. His163, Lys163 and Gln163 fit their electron density maps well, and the adenines of R163K/Ade and R163K/Ade also have good electron density. Using His163 of R163H n-TCS as an example, such fitting between the models and electron densities is shown in Figure 1
.

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Fig. 1. The final (2|Fo||Fc|) electron density map of residue His163 of R163H n-TCS. The map is calculated with data 0.80.200 nm and contoured at the 1 level.
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Least square superposition of R163H, R613K and R163Q with native n-TCS was performed. The main-chain (except the last three residues) r.m.s. deviation of R163H n-TCS and n-TCS is 0.0207 nm, R163K n-TCS and n-TCS is 0.0152 nm, and R163Q n-TCS and n-TCS is 0.017 nm. This suggests that there are no considerable structural differences among R163H, R163K and R163Q n-TCS and native n-TCS. The most significant difference occurs at the site of mutation. The last three amino acids of n-TCS do not interact with any other residues or water molecules. They have free mobile action and have very poor electron density. This is consistent with their high thermal factors. Located at the inner part of n-TCS, R163 is one of the crucial amino acids of n-TCS. The mutation of R163 leads to changes in the vicinity. The main-chain r.m.s. deviation of R163H n-TCS and n-TCS at the active pocket (residues 6971, 109111, 122, 155163 and 189193) is 0.0241 nm, this shows that R163H n-TCS does not change dramatically at the active pocket when compared with n-TCS. However, hydrogen bonds change greatly around the active pocket (Figure 2
). The conservative hydrogen bond between NH1 of R163 and OE1, OE2 of E160 disappeared naturally. In n-TCS, the main chain of E189 O, OE2 of E160, NH1 of R163 interact with each other through a water molecule X316, which is located among them. While in R163H, X316 does not form a hydrogen bond with the side chain of His, but only interacts with E160 and E189. There is a new hydrogen bond formed between ND1 of H163, and O of S159, which did not exist between n-TCS and R163Q n-TCS, due to the mutation from R163 to H163.
The main-chain r.m.s. around the active pocket (as indicated above) between R163Q n-TCS and n-TCS is 0.0219 nm. This indicates that they are similar in structure. Hydrogen systems around the active pocket also change (Figure 3
). The NE2 of Q163 replaces NH1 of R163, and forms a hydrogen bond with E160 OE2. There is also a water molecule X286 between Q163 and Y70, at the active pocket, which does not exist in n-TCS. There are hydrogen bonds between OE1 of 163Q, main chain O of N68 and X286, NE2 of Q163 which interacts with E189O, E160 OE1 through X287. The hydrogen bonds between E160 O and Q164 N, V69 O and S159 OG remain unchanged.
The structures of R163H n-TCS and R163Q n-TCS show little difference with a r.m.s deviation of 0.0235 nm, but the r.m.s. deviation around the active pocket (as indicated above) is 0.0308 nm. This suggests that the structure around the active pocket has changed considerably. Though the side chains of R163H and R163H are shorter than R163, the resulting structures of the two forms are quite different (Figure 4
). In R163H n-TCS, the side chain of Y70 moves toward the active pocket. While in R163Q, there is a water molecule X286 in the active pocket due to the strong negative charge of the main chain atom 163 O. This water molecule fills the vacant position left by the mutation of R163 to Q163, and pushes the side chain of Y70 outward. The maximum distance between main chain Y70 of R163H and R163Q reaches 0.097 nm.

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Fig. 4. Superposition of backbone of residues 6573 and 158163 of R163H (thin line) and R163Q n-TCS (thick line).
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Several groups have studied crystal structures of RIP/substrate-like (Ren et al., 1994
; Xiong et al., 1994
; Yang et al., 1994
; Wu et al., 1998
). Day et al. (1996) obtained an R180H/AMP ricin A complex at pH 7.5. After crystals of R163H and R163K n-TCS were soaked in the reservoir solution containing 10 mg/ml AMP, structure determination demonstrates that there is an adenine located in the active pocket. This suggests that AMP is hydrolyzed by R163H or R163K n-TCS, releasing ribose and a phosphate group while adenine stays in the active pocket (Figure 5
). In the structure of R163K/Ade, the side chains of Tyr70, Tyr111 and adenine form an aromatic stack, with adenine sandwiched between the two aromatic rings. Adenine has two hydrogen bonds with the peptide and one hydrogen bond with the water molecule. They are N6 and L71 O, N7 and E85 OE1, N9 and X318. These hydrogen bonds and the aromatic stack help stabilize the adenine in the active pocket.

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Fig. 5. The final (2|Fo||Fc|) electron density map of R163K/Ade n-TCS around the active pocket. The map is calculated with data 0.80.195nm and contoured at 1 level.
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Superposition of R163K and R163K/Ade n-TCS was done: they have a r.m.s. of 0.018 nm. This means the structures of R163K and R163K/Ade n-TCS have not changed greatly (Figure 6
). Only residues near the active pocket produce notable changes. The aromatic ring of Tyr70 rotates about 50° around the CC axis, paralleling itself to the purine ring of the adenine. The side chain of Glu85 also moves toward the active pocket by 0.051 nm. In the active pocket of R163K, there are four water molecules, X318, X340, X349 and X350; while in the active pocket of R163K/Ade, all water molecules, X340, X349 and X350, are being squeezed out, except X318.

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Fig. 6. Superposition of R163K n-TCS (thin line) and R163K/Ade n-TCS (thick line) around the active pocket.
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Tyr70 is one of the few residues around the active pocket which is invariant throughout the RIP family. In all of the R163 mutated structures of n-TCS, Tyr70 does not form hydrogen bonds with any other residues, but its aromatic ring stretches out into the solution. The average thermal factor for the side chain of Tyr70 is higher than two aromatic rings (Phe83, Tyr111) in the active pocket, which implies the side chain of Tyr70 is very flexible. In the structure of R163K/Ade, the side chain of Tyr70 undergoes a major change in conformation. The aromatic ring of Tyr70 rotates about 50° around the CßC
axis, together with the aromatic ring of Tyr70 and the substrate forms an aromatic stack. The aromatic ring of Tyr70 is essential for the enzyme activity of n-TCS. In our former study (Yan et al., 1999
), when Tyr70 was converted to Ala, it neither binds the AMP molecule nor catalyzes the N-glycosidase reaction.
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Discussion
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R163Q n-TCS crystals in reservoir solution were soaked with 10 mg/ml AMP. Structure determination revealed no electron density map around the active pocket. We also incubated R163Q with AMP at different molar ratios (1:10, 1:15 and 1:30) at various reactive times (0.5, 2, 8, 48 and 72 h) according to the conditions of Chen et al. (1996). Reaction products were analyzed using C18 reverse-phase column on HPLC. No adenine can be detected. This suggests R163Q n-TCS does not possess N-glycosidase activity. Studies on RIPs show that R163 of n-TCS plays a crucial role in N-glycosidase activity. The mechanism proposed by Wu et al. (1998) states that the proton of the guanidinium group of Arg163 is shared by N3 of AMP. Because of the conjugated effect of adenine, the electron flows from C1' to N9, resulting in the formation of an oxycarbonium ion, which could be stabilized by the carboxy group of Glu160. The nucleophilic attack of a water molecule results in the cleavage of the N-glycosidase bond. Biochemical analysis shows R163H and R163K still possess N-glycosidase activity, but their enzyme activities are lower than that of n-TCS (R163K shows a 1.5-fold decrease in activity; R163H has a much lower activity), while R163Q has no detectable N-glycosidase activity.
What are the reasons that account for the decreased activities of R163K and R163H n-TCS? The reasons are conjectured as follows:
(a) Structural changes of the active pocket. Since both K163 and H163 have shorter side chains than R163, the distance between proton donors (NZ of K163 and NE2 of H163), and N3 of AMP is increased. Only AMP molecules with a high kinetic energy can reach the interactive position and continue further with the reaction. Because of the spatial resistance, it is difficult for AMP to reach the active pocket. This greatly improves the activating energy and leads to a low velocity.
(b) Changes of biochemical characteristics. When R163 is mutated to His or Lys, the proton donating abilities of the mutants are weakened. The pKa of Arg is 12.5, and that of Lys and His are 10.5 and 6.0, respectively. Since the N-glycosidase activity is a nucleophilic reaction, a weak positive charge will lead to a lower velocity. All these factors suggest that R163 is the crucial residue of n-TCS and its major role in the N-glycosidase reaction is protonation of the substrate. It will retain its N-glycosidase activity only if its positive charge is maintained. Studies on mutants of ricin A also show similar results (Frankel et al., 1990
).
In the final structure of R163K/Ade, the distance between N3 of Ade and NZ of K163 is 0.532 nm, which is larger than the length of a hydrogen bond. In the R163H/Ade structure, the distance between N3 of Ade and NE2 of H163 is even larger (0.620 nm). This is consistent with our hypothesis of N-glycosidase activity. The position of Ade reflects the final position of the reaction product, not necessarily the substrate position where the substrate is being cleaved. When a new AMP molecule comes near, the Ade is expelled. The AMP moves deep into the active pocket and the reaction is repeated. When there is no or very little AMP in solution, Ade will occupy the position situated at the active pocket where it can be stabilized by interactions with the surrounding molecules (e.g. Tyr70 or Tyr111).
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Acknowledgments
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We thank Dr Ji-Jie Chai and Prof. Yu-Gang Hu for their help in data collection. This work was supported by the National Science Foundation of China (Grant No. 3968003) and partially supported by grants from University Grant Committee of Hong Kong (RGC/96-97/28) and Hong Kong Baptist University Faculty Research Grant (FRG/95-96/50).
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Notes
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1 To whom correspondence should be addressed;email: wushen{at}sun5.ibp.ac.cn 
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Received February 1, 1999;
revised July 13, 1999;
accepted July 27, 1999.