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* Max Planck Institute for Medical Research, Department of Biophysics, Heidelberg, Germany; RIKEN Harima Institute at Spring-8, Laboratory for Structural Biochemistry, Sayo, Hyogo, Japan; Actin Filament Dynamics Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Sayo, Hyogo, Japan; Dynamic NanoMachine Project, International Cooperative Research Project, Japan Science and Technology Corporation, Suita, Osaka, Japan; ¶ Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan; and || Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan
Correspondence: Address reprint requests to Dr. Toshiro Oda, RIKEN Harima Institute, Kouto 1-1-1, Mikazuki, Sayo 679-5148, Japan. Tel.: 81-791-58-2822; E-mail: toda{at}spring8.or.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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10 Å from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be 84° and 0.5 Å relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH)7 residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11. |
INTRODUCTION |
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Phalloidin is a well-known stabilizer of F-actin; it inhibits both release of phosphate as an ATPase product (Dancker and Hess, 1990
) and depolymerization of F-actin (Dancker et al., 1975; Estes et al., 1981). The aim of this study is to discuss one of the key mechanisms that stabilize F-actin by determining the position of bound phalloidin and analyzing its binding interactions to actin subunits. In the previous work, the position of phalloidin bound to F-actin was determined by modeling based only on the diffraction data from the phalloidin-F-actin complex (Lorenz et al., 1993). The method was model-dependent and therefore may not be free from bias. In the present study, we took a new approach to determining the position and orientation of phalloidin molecule in F-actin. We prepared well-orientated sols of F-actin and the phalloidin-F-actin complex and obtained x-ray fiber diffraction patterns from these sols. After extraction of layer-line amplitude data from the patterns, we determined the radial position of bound phalloidin by using a cylindrically averaged difference-Patterson map. Then, the axial and azimuthal positions relative to actin subunit were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection as described below in detail. Finally, we refined the orientation of bound phalloidin based on fiber diffraction data from the rhodamine-phalloidin-F-actin complex. Possible mechanisms for stabilization of the F-actin structure are discussed based on the binding interactions of phalloidin and actin. The new method reported here is applicable to other small molecules bound to F-actin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). The sols were incubated in superconducting magnets for a couple of weeks before the diffraction data collection to improve the orientation of F-actin in the sol specimens. The distribution of F-actin orientation as measured from the diffraction data was significantly improved by using a newly installed magnet with a field strength of 18.5-Tesla compared with those prepared with a magnet of 13.5-Tesla that we previously used. Typical solvent conditions were 30 mM NaCl, 10 mM Tris-acetate (pH 8), 1 mM CaCl2, 0.5 mM ATP, 1 mM 2-mercaptoethanol, and 1 mM NaN3. We also tried using KCl or C6H5COONa as monovalent salt, but we were unable to observe any systematic differences in the diffraction patterns.
Recording of x-ray diffraction patterns from F-actin sols
Diffraction patterns from these sols were recorded by using either of the following two systems. One is a rotating anode x-ray generator with a Cu target (RU-200; Rigaku, Tokyo, Japan) and image plates scanned off-line at a raster size of 100 µm (BA100, Fuji Film, Odawara, Japan). Diffraction data were recorded at a specimen-detector distance of 170 mm with an exposure time of 10 h. The other is a synchrotron source and an image plate detector (R-AXIS4, Rigaku, Osaka, Japan). Diffraction data were recorded at a specimen-detector distance of 400 mm, a wavelength of 1.00 Å, with an exposure time of 120 or 150 s at a SPring-8 beam-line, BL40B2. The diffraction patterns were processed as described by Yamashita et al. (1995): the four quadrants were averaged, the circular symmetric backgrounds were subtracted, and the resulting patterns were mapped into the reciprocal space.
Analysis of diffraction patterns and extraction of amplitudes
A program for layer-line deconvolution based on the two-dimensional profile-fitting procedure (K. Hasegawa, I. Yamashita, and K. Namba, unpublished) was used to extract layer-line amplitude distributions. The program recovers the intensity profiles along each layer-line whose position was calculated by given parameters (the helical pitch, the helical symmetry, the disorientation angle, etc.).
These parameters were refined as follows (Oda et al., 2001a): 1), the pitch of the 1-start helix from the position of 59 Å layer-line; 2), the helical symmetry from the positions of 51 Å and of 59 Å layer-lines; and 3), the disorientation angle from the width of 59 Å layer-lines. In almost all cases, the amplitudes were extracted by using either of the following symmetries: 13/6, 132/61, 67/31, 136/63, 69/32, and 28/13. Layer-lines that are contributed only by the Bessel functions having the order higher than the seventh were removed from the profile-fitting procedure, because the interval between nearest layer-lines are small and they were not well resolved. In the following calculation, these Bessel terms were neglected. The total number of layer-lines extracted was 48. In every case, the layer-line positions were not largely different from that of the well-known symmetry of 13/6. In this study, therefore, the layer-lines are denoted based on the symmetry of 13/6. Layer-line amplitudes were finally extracted from each diffraction pattern up to a resolution of 0.14 Å–1.
The intensities that were extracted using the helical symmetry other than 67/31 were re-indexed according to the symmetry 67/31 based on the Bessel order. A helical pitch of 58.8 Å, a helical symmetry of 67/31, and layer-line amplitude data in a resolution range of 0.020–125 Å–1 were used for the determination of the bound phalloidin position and orientation.
Radial position determined from cylindrically averaged difference-Patterson map
A cylindrically averaged difference-Patterson map was calculated by using layer-line amplitude data obtained from F-actin and phalloidin-F-actin using the equation (MacGillavry and Bruins, 1948; Vainshtein, 1965) of
 | (1) |
To determine the radial position of bound phalloidin, the correlation function between the difference-Patterson map and Patterson maps calculated from the atomic coordinates of phalloidin models was calculated in an area surrounding the peak,
 | (2) |
calc and obs represent the value in a model Patterson map and the difference-Patterson map, respectively. The phalloidin molecule was placed (r) at every 1 Å and oriented (
, 
, 
) at every 20° around an approximate radial position determined from the peak position in the difference-Patterson map, and the radial position and orientation with the maximum correlation was determined.
Azimuthal and axial position of bound phalloidin from the cross-Patterson map in radial projection
The azimuthal and axial position of bound phalloidin relative to actin subunit in F-actin was determined by using single isomorphous replacement phasing and a cross-Patterson map in radial projection by the following procedures:
), two structure factors, Gnl(R)1 and Gnl(R)2, were obtained for each reflection, and the best structural factor was deduced as Gnl(R)e = (G–nl(R)1 + Gnl(R)2)/2. ; Vainshtein, 1965) at an interval of 1 Å in the z axis and 1° in the -axis,
 | (3) |
) and translation along the filament axis (z) to fit the two F-actin densities to each other. In other words, we can obtain the azimuthal and axial position of bound phalloidin relative to the actin subunit on the x axis in the filament.
Refinement of position and orientation of bound phalloidin
To refine the position and orientation of bound phalloidin, the following equation was used,
 | (4) |
, z, , , ) are the Fourier-Bessel functions derived from the experimental amplitudes with F-actin model phases and those calculated from the atomic coordinates of phalloidin, respectively (Klug et al., 1958). Q was calculated for each position (r, , z) and orientation (, , ) of phalloidin at every 0.5 Å and 10°, respectively, around the position determined above, and the minimum Q was searched through. Then, using a set of parameters (r, , z, , , ) as an initial guess, the translation and orientation were refined by Powell's method (Press et al., 1994). We used the atomic scattering factors modified for bulk-solvent correction (Holmes et al., 1990). |
RESULTS |
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) were determined to predict the positions of all the layer-lines and to reconstruct the two-dimensional intensity profile to extract the layer-line amplitudes. The pitch of the basic helix was 58.7(6) Å for F-actin and 58.7(2) Å for phalloidin F-actin, and these two pitches are identical within experimental error. On the other hand, the helical symmetry was distributed between 67 subunits/31 turns (2.161) and 132/61 (2.164) for F-actin and 69/32 (2.156) for the phalloidin F-actin complex. The symmetry change induced by phalloidin binding is consistent with the previous studies (Orlova et al., 1995). This shows that the binding of phalloidin to F-actin affects the twist of the F-actin helix. For analysis, we used only the diffraction patterns that had a disorientation angle <3°. This is important for obtaining the correct structural factors because the extracted layer-line amplitudes depend slightly on the angular disorientation of F-actin in the sols (Oda et al., 2001b).
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).
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) was constructed by using the layer-line amplitude data from F-actin and phalloidin-F-actin in a resolution range of 0.030–0.125 Å–1. Peaks appeared clearly on the map, and the positions were independent of the resolution range used. The peaks were interpreted as the vectors between bound phalloidin molecules, and the vector length indicated that the distance between the nearest-neighbor phalloidin molecules is 33 Å (Fig. 3 a).
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Axial and azimuthal position of phalloidin in the F-actin model
The best phase angles were obtained for reflections of F-actin by using the Harker (1956) diagram on the basis of the bound phalloidin with its mass center located at the radial position described above (9.5 Å) on the x axis (hereafter termed the experimental structural factor of F-actin). At the same time, the observed amplitudes were combined with phases calculated from the Holmes et al. (1990) F-actin model coordinates, with the actin mass center put on the x axis (the model structural factor). A cross-Patterson map in radial projection was calculated from these two sets of structural factors (Fig. 3 d). The map was interpreted as the spatial correlation in –z projection in real space between the two F-actin filaments. The peak nearest the origin was located at = 84° and z = 0.5 Å, showing that F-actin with experimental phases fit that with model phases by rotation of 84° and translation of 0.5 Å. Namely, bound phalloidin was located at a position of 84° -rotation and 0.5 Å z-translation when an actin subunit in the filament was placed on the x axis. The deduced position of bound phalloidin was only slightly dependent on the conformational differences of the F-actin models used, even though these models have different local structures within the radial region being considered.
In conclusion, bound phalloidin was located within a radial range of 8.5–11.5 Å, an azimuthal range of 75°–100°, and an axial range of –2 Å to 2 Å relative to the actin subunit.
Refinement of the phalloidin position
Finally, the position and orientation of bound phalloidin were refined by using the Q function defined in Eq. 4. The results are shown, not as an atomic model of phalloidin, but as its electron density at 8 Å resolution in the F-actin model (Fig. 4). The calculated layer-line amplitude profiles are also compared with the observed ones (Fig. 5). By adding the phalloidin model, the crystallographic R-factor between the two profiles decreased from 12.0% to 9.5%, when the model phase of Lorenz et al. (1993) was used. Similar results were obtained by using the other F-actin models. The final position was almost identical irrespective of the F-actin model phases used. Also, it did not depend much on the phalloidin conformation. Phalloidin is located close to the loop of 198–201 of the lower actin subunit, the loop of 73–75, and the sheet including 197 of the diagonal subunit, but is rather distant from the upper subunit. This result basically supports the phalloidin position determined by Lorenz et al. (1993), although the refined position is somewhat closer to the filament axis.
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DISCUSSION |
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and was not sensitive to small conformational differences of the F-actin models used for phasing. The binding site is close to residues that are known to be required for F-actin-binding of phalloidin. It is close to the loop of 198–201 of the lower actin subunit, the loop of 73–75, and the sheet including 179 of the diagonal subunit, but is rather distant from the upper subunit. It is reasonable that phalloidin cannot bind to Tetrahymena actin because it has relatively low similarity in the amino acid sequence to skeletal muscle actin. Overall identity is 74.6%, but one large difference between the two exists in the region of residues 190–200, which is hydrophilic in skeletal muscle actin but hydrophobic in Tetrahymera actin (Hirono et al., 1987). Phalloidin cannot bind to a double-site mutant yeast F-actin, R177A/D179A (Drubin et al., 1993). The side chains of Arg177 and Asp179 reside at 6 Å from the mass-center of phalloidin both in the Lorenz model and in the Holmes model. Since the size of the phalloidin molecule is 6 Å, it may be placed in contact with Arg177 and Asp179. However, the Arg177 side chain may not be extended toward phalloidin, but rather the side chains of Arg177 and Asp179 form tangential contacts with the phalloidin molecule. This is because phalloidin can bind to actin with a single mutation R177D and stabilizing F-actin (Schüler et al., 2000), and because the Arg177 may interact with Ser199 of the adjacent subunit, On the other hand, Met119, Gln117, and Met355 of F-actin react with bound affinity-labeling derivatives of phalloidin (Vandekerckhove et al., 1985). The side chains of Met119 and Gln117 are located 20 Å from the mass-center of phalloidin, and thus the cross-linking results are accounted for by the arm-length of the derivatives. However, Met355 is too far to be cross-linked with a derivative that binds specifically to the phalloidin-binding site. The apparent positive result might be due to cross-linking with a phalloidin molecule that binds to a weak subsidiary binding site on F-actin. This might have happened because the experiment was carried out at fivefold molar excess of the phalloidin derivative.
Phalloidin position and orientation in F-actin using some local information
Phalloidin is composed of two rings. One ring is Cys3-Pro(OH)4-Ala5-Trp6. This part is essential for the toxicity of phalloidin. For example, phalloidin loses its toxicity by the replacement of Ala5 with Gly, replacement of cis-Pro(OH)4 with trans-Pro(OH), or alkylation of indole NH in Trp6 (Wieland, 1986). Recent structural analysis showed that these residues form either a ß-turn type-I structure in DMSO (Kessler and Wein, 1991) as well as in the crystal of Leu7 Ala-phalloidin (Zanotti et al., 2001), or a ß-turn type-II structure in water at pH 3 (Kobayashi et al., 1995). The transition between the two conformations does not occur in a short timescale accessible by molecular dynamics simulations. This suggests that this ring has a unique and rigid conformation under each condition, which is required for the toxicity (Kobayashi et al., 1995; Zanotti et al., 2001).
The other ring is Ala1-Thr2-Cys3-Trp6-Leu(OH)7. From several studies on syntheses and modification of phallotoxins, Ala1, Thr2, and Leu(OH)7 are not considered to be important for toxicity. Cross-linking studies (Vandekerckhove et al., 1985; Faulstich et al., 1993) suggest that Leu(OH) faces out as does the electron microscopic study of phalloidin conjugated with a gold cluster at Leu7, PHD-Leu7(OH)-NHCO(CH4)CONH-CH2-Au11 (Steinmetz et al., 1998). Our result provides more direct support for these observations.
We searched for positions and orientations of bound phalloidin consistent with the restrictions derived from the atomic models and diffraction data as discussed above. First, we found several candidates for the position and orientation of phalloidin in F-actin with a low binding energy using AutoDock version 3.0 (Goodsell and Olson, 1990; Morris et al., 1996, 1998). Then, we chose the final candidates for the position of bound phalloidin by applying the following criteria and assumptions:
, we did not find any good candidate for the phalloidin position and orientation within the possible region determined by diffraction data (see below), irrespective of the phalloidin conformation, probably because of a wider separation between the two long-pitch strands in the model. For the F-actin model by Tirion et al. (1995), we also could not find any stable candidate with the major population and the low binding energy, because the extended side chain of His73 interferes with phalloidin binding to the binding site. For the F-actin model by Lorenz et al. (1993), we found three good candidates. (The positions and orientations of phalloidin molecule in these candidates, as well as the binding site in F-actin, have not been optimized for the lowest binding energy, because this requires too many assumptions.) One of these plausible models of phalloidin with a type-II ß-turn conformation is shown in Fig. 8, which might be similar to the model by Steinmetz et al. (1998). It is worth noting, however, that the present results did not decrease the R-factor as initially expected. This might be due to the fact that a substantial conformational change of actin that may be induced by the phalloidin binding is not at all taken into account in this analysis. In fact, Lorenz et al. (1993) modified the actin conformation around the phalloidin binding site to improve the fitting of the calculated diffraction data to the observed data. Without introducing such conformational modification, there is no binding pocket in the models by Holmes et al. (1990) and by Tirion et al. (1995) suitable for accommodating phalloidin.
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). According to the atomic model of F-actin, the interactions between actin subunits in the two long-pitch strands are relatively weak. However, subdomain 4 in one strand and subdomain 1 in the opposite strand appear to change their conformations together under different conditions (Belmont et al., 1999; Orlova and Egelman, 1992, 1995), suggesting that these inter-subdomain interactions may play an important role in the stabilization of F-actin. The binding of phalloidin between the two strands would account for its role in stabilizing the F-actin structure. This idea is supported by a report that phalloidin rescues the polymerization activity lost by mutation of the hydrophobic plug (Kuang and Rubenstein, 1996).
Wriggers and Schulten (1999) have proposed based on their steered molecular dynamics study that the charges of methylated-His73 and Arg177 are essential for phosphate release from actin, and the phosphate release pathway involves these residues. Bound phalloidin would plug the exit of the pathway and thereby inhibit the phosphate release, as was observed by Dancker and Hess (1990). However, a recent biochemical study does not provide support for the hypothesis of Pi exit (Nyman et al., 2002), and a different explanation would be necessary for the inhibition of phosphate release. Although the mechanism is not clear, F-actin polymerized by phalloidin would contain actin-ADP-Pi as the main species. Since ADP-Pi F-actin is more stable than the normal ADP F-actin (Carlier and Pantalone, 1988), this might be an additional mechanism for the stabilization of F-actin by phalloidin binding.
Some groups have presented an idea that the stabilization of F-actin arises from conformational changes of F-actin induced by phalloidin binding (Sampath and Pollard, 1991; Orlova et al., 1995). Unfortunately, we cannot discuss this issue, since conformational changes of actin that may induced by the phalloidin binding are not taken into consideration in our analysis.
A new method to determine the position of bound phalloidin in F-actin filament
In the previous work, the position of the phalloidin molecule bound to actin was determined by modeling based only on the diffraction data from the phalloidin F-actin complex (Lorenz et al., 1993). The method was model-dependent and therefore may not be free from bias. In the present approach, we employed the cylindrical Patterson map to determine the radial position of phalloidin directly from experimental fiber-diffraction data, without any modeling. This new method is widely applicable to any small drug-molecule-bound filament structures. The map may be interpreted even when the map is noisy, if the stoichiometry of a bound molecule is known. Once the radial position is determined, the axial and azimuthal position of the bound molecule relative to the subunit can be determined with high confidence. The method has been successfully applied to determine the bound dolastatin 11, another stabilizer of the F-actin structure (Oda et al., 2003), demonstrating its high potential in other applications to accurately determine the binding position and conformation of drugs in the macromolecular fiber structures.
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ACKNOWLEDGEMENTS |
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The work was partially supported by the Special Coordination Funds from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Submitted on June 17, 2004; accepted for publication October 20, 2004.
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REFERENCES |
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) were those expected from a rhodamine model located at r = 17 Å in the helical lattice of F-actin. The map was normalized to have the height at the origin 1000. Some of the high contour levels were omitted near the meridian for clarity. (b) Cross-Patterson map in radial projection between phalloidin-F-actin structural factors with experimental phases and with the conventional model phases. The experimental phases were deduced from the position of bound rhodamine. The model phase was calculated using the F-actin model of Lorenz et al. (1993)
