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1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan
2 Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Mikazuki, Sayo, Hyogo 679-5198, Japan
Reprint requests to: Dr. Rikimaru Hayashi, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan; e-mail: hayashi{at}kais.kyoto-u.ac.jp; fax: 81-75-753-6128.
(RECEIVED August 2, 2001; FINAL REVISION October 3, 2001; ACCEPTED October 8, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.31102.
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Abstract |
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1 Å change in the distance between N
2 of His12 and N
1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule. Keywords: Crystal structure; ribonuclease A; active site; His119; thermal stability; accessible surface area
Abbreviations: ASA, accessible surface area • CpA, cytidilyl-3`,5`-adenosine • C>p, cytidine-2`,3`-cyclic monophosphate • RMSD, root-mean-square deviation • UpA, uridilyl-3`,5`-adenosine • U>p, uridine-2`,3`-cyclic monophosphate.
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Introduction |
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In this study, to investigate if the change in spatial positions of the two histidine residues is in fact caused by the replacement of Phe120 and if a conformational change does occur in the Phe120 mutant enzymes, the degree of the conformational changes—detailed conformational changes with respect to His12 and His119—as the result of mutagenic replacement of Phe120 were evaluated via an analysis of the crystal structures of F120A, F120G, and F120W mutant RNase A by X-ray crystallography at high resolution. Because a subtle conformational change could have an effect on catalytic activity, as has been previously suggested, wild-type RNase A was also crystallized under the same condition as other mutant enzymes, and its structure was used for a precise comparison with the mutant RNase A samples.
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Results |
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150 x 150 x 150 µm. We also attempted to crystallize F120L mutant RNase A, the activity, thermal stability, and carboxymethylation rate of which were also analyzed in our previous study (Chatani et al. 2001), but only a cluster of fine needle crystals was obtained in the similar condition as that of the wild-type RNase A. A single crystal was once obtained, but this was not reproducible and we could not obtain enough data.
The crystals of the wild-type and all mutant RNase A (F120A, F120G, and F120W RNase A) belonged to space group P3221, with similar values of unit cell dimensions (Table 1
). The highest resolution of data obtained in this study was 1.35 Å for the wild-type enzyme, 1.0 Å for the F120A and F120W enzymes, and 1.2 Å for the F120G enzyme. The statistics of the data collection were judged to be fit for use as listed in Table 1.
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30 water molecules were picked up and followed by continuous structural refinement. Ultimately, 115 to 238 water molecules were picked up. The fitness of electron density was evaluated with a model that was constructed at each step during the refinement. Manual corrections were performed by checking the 2Fo - Fc map and Fo - Fc map. All structures were refined up to a maximum resolution of 1.4 Å. The R-factor was 20% to 22% in all enzymes (Table 1
). Differences between Rfree and Rall were <5.5% in all cases. The electron density at position 120 in the F120A, F120G, and F120W mutant RNase A indicated that the mutagenic alteration of Phe120 to alanine, glycine, and tryptophan, respectively, had been accomplished. The completed coordinates of the wild-type, F120A, F120G, and F120W RNase A have been deposited to the Protein Data Bank (PDB) with the PDB codes 1FS3, 1EIC, 1EID, and 1EIE, respectively.
Overall backbone structures
The overall backbone structures of F120A and F120W RNase A were very similar to that of wild-type RNase A, except for the loop regions (Fig. 1), where the B-factors were higher than that of other regions of the wild-type and mutant RNase A (Fig. 2), indicating flexibility in the loop regions. The backbone structures of the C-terminal regions (residues 120 to 124) of F120A and F120W were well superimposed, but the backbone structure of the F120G RNase A deviated from that of the wild-type RNase A, despite good superimposition of the residual structure. The B-factors of the C-terminal region in F120G RNase A increased by 20 Å2 relative to that in wild-type RNase A, whereas those in F120A RNase A were only slightly larger than that in wild-type RNase A (Fig. 2).
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. Asp83 and Glu111 were not well superimposed, but these positional changes were not caused by the replacement of Phe120 because their positions are sensitively affected by crystallization conditions as seen by the coordinates of RNase A deposited to the PDB. The spatial position of amino acid residues at the position of 120 showed only slight differences among the wild-type and mutant enzymes as judged by root-mean-square deviation (RMSD) values: The RMSD values of the C
carbon at the position of 120 between wild-type RNase A and F120A, F120G, or F120W RNase A were 0.21 Å, 0.43 Å, and 0.30 Å, respectively. The RMSD values of the Cß carbons between wild-type RNase A and F120A or F120W enzymes were 0.28 Å and 0.57 Å, respectively. The benzene ring of Phe120 in the wild-type RNase A and the indole ring of Trp120 in F120W RNase A also orientated very similarly (
1 and 2 of His119 in the wild-type RNase A were 173° and -85°, respectively, and those in F120W RNase A were -168° and -90°, respectively). The B-factors of Trp120 in F120W RNase A and Phe120 in the wild-type RNase A remained the same. However, the B-factors increased as Phe120 was substituted with smaller hydrophobic residues, in the order of phenylalanine, alanine, and glycine (Fig. 2).
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), and their B-factors were also similar (Fig. 2).
The main-chains of His119 in the wild-type and Phe120 mutant RNase A coincided well, whereas side-chains of His119 in mutant RNase A deviated from the original positions, as shown in Figure 3. In addition, although His119 in the wild-type and F120W RNase A were in the A position and directed toward position 120, His119 in F120A and F120G RNase A were in the B position and directed toward position 118 (Fig. 4; see Discussion for A and B positions of His119).
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and 
angles of His119 in the wild-type (141° and -158°, respectively) and F120W RNase A (140° and -156°, respectively) were very similar, but the 1 and 2 angles of His119 in wild-type (156° and 87°, respectively) and F120W RNase A (-177° and -118°, respectively) were clearly different. It is interesting to note that the face of the imidazole ring in F120W RNase A appeared to turn inside out in the present model (Fig. 4), although this conclusion is tentative because it is impossible to distinguish N from C atoms of the imidazole ring by the present electron density map. When the imidazole ring of His119 in F120W RNase A was rotated by 180° from the original face in a manual operation using computer graphics, followed by energy minimization, the ring failed revert to the orientation that existed before the manual operation. On the other hand, an original hydrogen bond between N2 of His119 and O1 of Asp121, which is important in orientating the proper tautomeric form of His119 (Cederholm et al. 1991; Quirk et al. 1998; Schultz et al. 1998b), was lost in F120W even if the imidazole ring of F120W was rotated by 180° (Fig. 4), supporting the validity of our present proposed structure.
The position of His119 in F120A and F120G RNase A, which are in the B position, were compared with that of wild-type RNase A in B position determined by Zegers et al. (1994; PDB identification code 1RPH). The and angles of His119 (in the B position) in the wild-type (134° and -152°, respectively), F120A (146° and -155°, respectively), and F120G RNase A (151° and -151°, respectively) were all similar. However, the 1 and 2 angles of His119 for the wild-type (-54° and -51°, respectively), F120A (-72° and -45°, respectively), and the F120G RNase A (-66° and 126°, respectively) were different from each other. The His119 imidazole ring of F120G RNase A also turned inside out as did that of F120W (Fig. 4). Opposed to the case of F120W RNase A, however, this inside-out orientation was preferred to the original orientation of the wild-type RNase A, probably because of a hydrogen bond between N1 of His119 and a water molecule (Fig. 4). The B-factor of His119 in F120G RNase A was higher than that of wild-type and F120A RNase A.
Cavities or pockets
Cavity and pocket sizes of wild-type and three mutant enzymes, and RNase A in which His119 occupies B position (1RPH), were analyzed by CASTp program (Liang et al. 1998) with a solvent probe of 1.4 Å. As a result, although no cavity was detected, 8 to 12 pockets were found in all crystal structures, as shown in Table 2. The pockets were dispersed all over the protein surface. Phe120 of wild-type (1FS3), Phe120 of wild-type (1RPH; Zegers et al. 1994), Ala120 of F120A, Gly120 of F120G, and Trp120 of F120W belonged to the pockets 7, 10, 8, 9, and 11, respectively (Table 2).
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Discussion |
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-electron stacking between Phe120 and substrates (Zegers et al. 1994; Tanimizu et al. 1998). However in the present case, the kcat value of F120W mutant RNase A—in which the indole ring of Trp120 is orientated similarly with the benzene ring of Phe120 in wild-type RNase A, and therefore -electron stacking between the indole ring of Trp120 and the substrate is possibly made—decreases, without supporting this suggestion. Under these considerations, it can be concluded that the positional change of His119 induced by the mutagenic substitution of Phe120 is a major cause in decreasing the activity.
The positional change of His119 by mutagenic replacement of Phe120
It has been suggested that His119 position of RNase A fluctuates between the A and B positions in the aqueous solution. In the crystal state, the location of His119 is visible in A and/or B positions, depending on the crystallization conditions (Borkakoti et al. 1982; Wlodawer et al. 1982; Martin et al. 1987). Although the wild-type and mutant RNase A were crystallized under similar conditions, in which it would be expected that His119 of all enzymes assumed the A position, the His119 imidazole rings of wild-type and F120W RNase A assumed the A position; those of F120A and F120G RNase A, the B position. A hydrogen bond between His119 and Asp121 in the wild-type enzyme (de Mel et al. 1992) is not essential in favoring the A position because His119 in the F120W mutant does not contain this hydrogen bond but still assumes the A position. An increase in salt concentration or a decrease in pH favors the B position of His119 in the wild-type RNase A (de Mel et al. 1994b; Fedorov et al. 1996), indicating that a possible ionic interaction between the -electron of Phe120 and the imidazole ring of His119 makes the A position of His119 preferable. Such ionic interaction could be present between the electrons of the indole ring of Trp120 in F120W but not in the F120A and F120G enzymes, which favor the B position over the A position in the crystal. However, this is not the case for an aqueous solution, in which His119 of all the Phe120 enzymes would fluctuate between the A and B positions because the transition energy is very low (de Mel et al. 1994b). Thus, in the substrate-free state, the location of His119 at the A or B position is a crystallographic artifact and can be ignored in the case of an aqueous solution.
When wild-type RNase A is complexed with d(CpA) and 3`-CMP, the His119 assumes the A and B positions, respectively (Zegers et al. 1994), indicating that the His119 position changes during the reaction process, that is, the A position during transphosphorylation and the B position for the case of the hydrolysis reaction (Borkakoti 1983). However, this is inconsistent with the present result because kcat of the F120W and F120A mutant RNase A was affected more in the hydrolysis reaction than in the transphosphorylation, whereas the kcat for the F120G enzyme was more affected in the transphosphorylation reaction than in the hydrolysis reaction (Chatani et al. 2001). These results show that the preference of the His119 positions is not reflected in the total enzyme activity. It is reasonable to conclude that His119 exists in one position during the two continuous reactions, consistent with a recent proposal made by Schultz et al. (1998b).
The possibility has been suggested that the face of His119 in F120W RNase A is rotated by 180° from that of wild-type enzyme, which is probably caused by the loss of a hydrogen bond between Asp121 and His119. The hydrogen bond is thus important in preventing the rotation of the imidazole ring but is not significant in maintaining the proper pKa of His119 because the pKa of His119 in F120W RNase A was at the same level (Tanimizu et al. 1998). But the flipping of His119 should be confirmed with an additional experiment at higher resolution or by nuclear magnetic resonance study for concluding the role of the hydrogen bond between Asp121 and His119.
The face of His119 (B position) in the F120G RNase A crystal is also rotated by 180°. When the crystal structure of the F120G enzyme in which His119 assumes the A position is determined, this will clarify the issue of whether the rotation is also caused by the loss of the hydrogen bond. However, when the side-chain of His119 is rotated around 1 and 2 to dihedral angles of 159° and -137°, respectively, which are the rotation angles from the B position to the A position in the case of wild-type RNase A, and the imidazole ring then turns inside out (180° rotation), no hydrogen bond was formed between Asp121 and His119. Therefore, the rotation of His119 in F120G is possibly caused by the loss of the hydrogen bond. Even if N2 of His119 in the A position and the O1 of Asp121 of F120G RNase A is sufficiently close for forming a hydrogen bond, the high flexibility of His119 in F120G RNase A (see B-factor in Fig. 2) prevents the formation of a hydrogen bond between His119 and Asp121.
Relationship between the distance from N2 of His12 to N1 of His119 and activity
It has been known that the two nitrogen atoms N2 of His12 and N1 of His119 participate in catalysis as an acid and base (Cuchillo et al. 1997; Raines 1998). We previously estimated that positional changes in His12 and/or His119 lower activity in Phe120 mutant enzymes from our results of carboxymethylation experiments, and in this study, we found a correlation between the deviation in distance from N2 of His12 and N1 of His119 in the free form and enzyme activity (Table 3): As the absolute deviation increases, the activity decreases. Here, it should be noted that the highest deviation was only 1.3 Å (F120G), which results in a decrease in 93.3% of kcat for CpA. Although the scale of the deviation was much smaller than expected, the similar correlation is also observed in the other sets of mutant enzymes, that is, D121N and D121A and in semisynthetically prepared F120Y and F120L RNase A (it should be noted that the pKa of His12 and 119 of these mutant enzymes are the same as that of the wild-type enzyme; de Mel et al. 1994a; Schultz et al. 1998b), all of the deviations of which were <1.0 Å (Table 3). The obvious correlations shown in Table 3 indicate the possibility that a 1-Å deviation might drastically reduce activity. This conclusion is very surprising, because the flexibility of the active site of enzymes, the so-called induced fit, has been known to the active site of RNase A from the nuclear magnetic resonance and X-ray crystallographic studies, in which the active site conformation changed by substrate binding (Arus et al. 1982), and the temperature factors were lowered by the low temperature, accompanied by the loss of activity (Rasmussen et al. 1992). Even if a 1-Å deviation could be diminished by the induced fit, the obvious correlation between the deviation in distance from N2 of His12 and N1 of His119 in the free form of enzymes and enzymatic activity indicates that the diminished deviation might still drastically reduce activity, namely, such conformational flexibility does not cover the 1-Å deviation to result in a dramatic decrease in activity. The active site of RNase A seems to involve two contrary features at the same time: flexibility and strictness. Attempts to determine the crystal structures of complexes with substrate analogs are underway to analyze the degree of the deviation after the induced fit to accept the substrate.
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2 of Asp121. Because loop regions are usually flexible, as evidenced by their high B-factor (Fig. 2), the observed conformational differences in the loop regions of the present mutant enzymes are within experimental error. Thus, it can be concluded that the decrease in activity in mutant RNase A at Phe120 can be ascribed not to conformational changes in the loop region but rather to the shift of His119. At pH 2.2, the loop region of RNase A moves away from its C terminus, accompanied by a significant disorder in the C-terminal region (Ratnaparkhi and Varadarajan 1999). This shows that the conformation of the loop and the C-terminal regions cooperatively changes as the result of the mutation at Phe120, located in the C terminus.
Relationship of B-factor at C-terminal region with activity and stability
A larger decrease in the activity of F120G RNase A than would have been expected may be owing to not only the positional change in His119 but also an increase in the B-factor of His119 (Fig. 2). An increase in flexibility lowers the probability of juxtaposing His119, leading to a decrease in activity.
The flexibility of the C-terminal residues also affects conformational stability, because the B-factor around the C-terminal region increases as Phe120 is replaced by smaller hydrophobic residues in the order of F120G > F120A > wild type > F120W. This is the same order with entropy values of thermal denaturation. It is therefore entirely possible that the entropy change is the result of a change in flexibility in the C-terminal region, which is, in turn, the result of the replacement of Phe120.
Correlation of accessible surface area with thermal stability
A linear correlation between the accessible surface area (ASA) of a cavity and thermal stability has been reported for some proteins (Eriksson et al. 1992; Takano et al. 1997; Coll et al. 1999). The Phe120 mutant RNase A used herein showed a linear correlation between the change in ASA of the pocket to which the amino acid residue at 120 belongs (
ASApartial) and H for thermal denaturation (Chatani et al. 2001), with a correlation coefficient of 0.92 (Fig. 5a). A better correlation was obtained between the change in total ASA of all pockets (ASAtotal) and H, with a correlation coefficient of 0.97 (Fig. 5b). In addition, a good linear correlation exists between ASAtotal and the ASA of amino acid residue itself occupying the position 120 (ASAamino acid at 120; Miller et al. 1987), with a correlation coefficient of 0.99 (Fig. 6). These facts indicate that the change in volume at the position 120 as the result of the mutation affects not only the neighborhood of the substituted position but also the entire protein molecule, and these widespread conformational deviations cause the changes in enthalpy and entropy, thus destabilizing the protein structure. Phe120 appears to interact with a number of other amino acid residues as a part of the network of noncovalent bonds to build up the strict conformation of RNase A, which produces not only the maximum activity but also the optimum conformational stability of RNase A.
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Materials and methods |
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Crystallization
The purified enzyme solution was diluted with water 1000-fold and concentrated to 24 mg protein/mL. Wild-type and mutant RNase A were crystallized by the hanging-drop vapor-diffusion method according to Schultz et al. (1998a) with minor modifications: A 6-µL drop containing the protein solution and the reservoir solution in a ratio of 1:1 were equilibrated against 1.0 mL of the reservoir solution, which contained 100 mM sodium acetate buffer (pH 6.0), 30% to 35% (v/v) of saturated ammonium sulfate (35% for F120W RNase A, 34% for F120A RNase A, and 30% for wild-type and F120G RNase A), and 50% (v/v) of saturated NaCl by incubating at 25°C.
Data collection
X-ray diffraction data of F120W and F120A mutant RNase A were collected with an ADSC Quantum 4R CCD detector system at SPring-8 BL44B2 (Hyogo, Japan). X-ray diffraction data for the wild-type and F120G mutant RNase A were collected with a RIGAKU RAXIS IV++ imaging plate detector system at SPring-8 BL40B2 (Hyogo, Japan). During the data collection, the crystals were cooled to 100 K with a stream of nitrogen gas. Data from CCD detector system and the imaging plate detector system were processed and scaled using the MOSFLM program in Collaborative Computational Project No. 4 (1994) and the DENZO program (Otwinowski and Minor 1997), respectively. Details of the data collection are listed in Table 1.
Refinement of the crystal structure
The molecular structure was determined by the molecular replacement method using the X-PLOR program (Ver. 3.81; Brünger 1992). The coordinate of the PDB code 1RNM, which was stripped of all solvent molecules, was used as a starting model. The geometry of the main-chain and side-chains was analyzed using the PROCHECK program (Laskowski et al. 1993). The model was adjusted manually in the O program (Jones et al. 1991). After several cycles of least-square refinements and manual adjustments, water molecules were added to the model. In analyzing conformational change as the result of the mutagenic replacement of Phe120, the structures of wild-type and mutant RNase A were superimposed by use of backbone atoms using the Swiss-PdbViewer program (Guex and Peitsch 1997).
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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