J. Mol. Biol. (1998) 276, 1±6

COMMUNICATION

Evaluating Atomic Models of F-actin with anUndecagold-tagged Phalloidin Derivative

Michel O. Steinmetz1, Daniel Stoffler1, Shirley A. MuÈ ller1, Werner Jahn3

Bettina Wolpensinger1, Kenneth N. Goldie1,2, Andreas Engel1

Heinz Faulstich3 and Ueli Aebi1*

1M.E. MuÈ ller Institute forMicroscopy, BiozentrumKlingelbergstrasse 70University of BaselCH-4056 Basel, Switzerland2School of Biological SciencesUniversity of AucklandPrivate Bag 92019, AucklandNew Zealand3Max-Planck-Institut fuÈ rMedizinische ForschungD-6900 Heidelberg, Germany

Abbreviations used: ADF, annuundecagold; F-actin, ®lamentous amonomeric actin; MPL, mass-per-photoactivatable phalloidin derivaSTEM, scanning transmission elec

0022±2836/98/060001±06 $25.00/0/m

We have prepared an undecagold-tagged phalloidin derivative to deter-mine this mushroom toxin's binding site and orientation within the F-actin ®lament by scanning transmission electron microscopy (STEM) and3-D helical reconstruction. Remarkably, when stoichiometrically bound toF-actin, the undecagold moiety of the derivative could be directly visual-ized by STEM along the two half-staggered long-pitch helical strands ofsingle ®laments. Most importantly, the structural data obtained whencombined with various biochemical constraints enabled us to criticallyevaluate two distinct atomic models of the F-actin ®lament (i.e. theHolmes±Lorenz versus the Schutt±Lindberg model). Taken together, ourdata are in excellent agreement with the Holmes±Lorenz model.

# 1998 Academic Press Limited

Keywords: F-actin; phalloidin; scanning transmission electron microscopy;image processing; 3-D reconstructions

*Corresponding author

Actin ®laments (F-actin) are found in mosteukaryotic cells as constituents of the cytoskeleton.They play a central role in various types of motility(including muscle contraction) and transport pro-cesses. A major step towards understanding howactin can ful®ll its various functions was the deter-mination of the atomic structure of monomericactin (G-actin) in complex with accessory proteinssuch as DNase I (Kabsch et al., 1990), gelsolinsegment 1 (McLaughlin et al., 1993) and pro®lin(Schutt et al., 1993). Combining the atomic struc-ture of G-actin with ®ber diffraction patternsobtained from oriented gels of actin ®lamentsenabled Holmes and co-workers to construct anatomic model of the F-actin ®lament, i.e., theHolmes-Lorenz model (Holmes et al., 1990; Lorenzet al., 1993). This ®lament model has provided astructural framework for an increasing number ofbiochemical, cell biological and mechanical investi-gations on actin (for a review see, e.g., Steinmetzet al., 1997b), including attempts to map the bind-

lar dark-®eld; Au11,ctin; G-actin,

length; PAL,tive; PHD, phalloidin;tron microscopy.

b971529

ing sites of interacting proteins and drugs at atom-ic scale (e.g. see Lorenz et al., 1993, 1995; Raymentet al., 1993; Owen & DeRosier, 1993; Schmid et al.,1994; McGough et al., 1994, 1997). More recently,Schutt and colleagues have constructed an alterna-tive atomic model of F-actin which they derivedfrom the structural analysis of bovine pro®lin±b-actin co-crystals, i.e. the Schutt±Lindberg model(Schutt et al., 1995a,b, 1997). Their ribbon-based®lament model is substantially different from theHolmes-Lorenz model, although it is corroboratedby the same structural constraints as were used tobuild and re®ne the former (Schutt et al., 1995b).Moreover, Schutt and co-workers have employedtheir F-actin model as a key element in a recenthypothesis on the mechanism of muscle contrac-tion (Schutt & Lindberg, 1992; Schutt et al., 1995b).Hence, new experiments are now necessary tomore critically evaluate the two models and toarrive at a consensus structure for the F-actin ®la-ment at atomic scale. A powerful approachtowards this goal is to visualize site-speci®cmarkers within the F-actin polymer by electronmicroscopy, and then assess the mapped positionsof these hallmarks in terms of available structuraland biochemical data.

# 1998 Academic Press Limited

Figure 1. Structure of the Leu7 amino acid derivative ofPHD linked to an Au11 cluster by an �1.7 nm longspacer moiety. A threefold molar excess of bis-(4-nitro-phenyl) adipate was added to 9 mg of d-aminophalloi-din (Wieland et al., 1983) in 0.1 ml dimethylformamideand 0.005 ml triethylamine, and allowed to react forten minutes at room temperature. The product wasprecipitated with diethylether, dissolved in 0.15 ml ofdimethylformamide and 0.01 ml triethylamine, and trea-ted with the amino derivate of the Au11 cluster preparedas described by Jahn (1989). The product was isolatedby precipitation with acetone. In the actin binding assaydescribed by Faulstich et al. (1988), Au11-PHD had a�100-fold lower af®nity for F-actin than PHD. Theresulting 7308 Da Au11-PHD derivative bound stoichio-metrically to F-actin. The undecagold cluster isrepresented schematically by a 1 nm diameter goldensphere. The atomic coordinates of PHD were kindly pro-vided by Dr N. Kobayashi, University of Tsukuba,Japan.

2 Orientation of Undecagold-tagged Phalloidin

For this purpose, we conjugated the modi®edside-chain of (OH)2-Leu7 of the bicyclic heptapep-tide phalloidin (PHD) to an undecagold cluster(Au11) via an �1.7 nm long organic linker. Theorganometallic compound Au11-PHD (Figure 1)containing 11 gold atoms within a cluster of0.8 nm diameter (Bartlett et al., 1978) bound speci®-cally to F-actin and hence served as a structuralhallmark. Remarkably, scanning transmission elec-tron microscopy (STEM) dark-®eld images ofunstained, freeze-dried specimens revealed Au11 aselectron-dense particles directly, without compu-tational averaging of the images (Figure 2c and d).The gold particles were spaced every 5.5 nm (i.e.the axial extent of the actin subunit) along the twohalf-staggered (i.e. by 2.75 nm) long-pitch helicalstrands of single F-actin ®laments (Figure 2d).Determination of the mass-per-length (MPL) bySTEM (cf. MuÈ ller et al., 1992; Engel & Colliex, 1993)of native PHD (Figure 2b) and Au11-PHD(Figure 2c)-stabilized F-actin ®laments yieldedvalues of 15.4(�1.4) kDa/nm and 19.8(�1.6) kDa/nm (Figure 2e), respectively, consistent with themass of the actin:PHD (predicted MPL of15.6 kDa/nm) and the actin:Au11-PHD complex(predicted MPL of 19.3 kDa/nm). Together, thesedata indicate a 1:1 binding stoichiometry ofAu11-PHD to F-actin protomer.

To determine more accurately the Au11-PHDbinding site within F-actin, we recorded STEM

dark-®eld images of negatively stained nativeF-actin ®laments, as well as PHD and Au11-PHD-stabilized F-actin ®laments (Figure 2a), and sub-jected them to 3-D helical reconstruction. Figure 2fdisplays corresponding re®ned and averaged 3-Dreconstructions at a nominal resolution of 2.5 nm.Visual inspection of these reconstructions (i.e.native (1), �PHD (2), and �Au11-PHD (3)) revealsa distinct site (see arrowheads) of increasing massdensity at larger ®lament radii between the twolong-pitch helical strands (see also Bremer et al.,1991; Steinmetz et al., 1997a), whereas the overall®lament morphology and F-actin subunit confor-mation remain conserved. Subtracting reconstruc-tion (2) (i.e. �PHD) from reconstruction (3) (i.e.�Au11-PHD) produced a single positive differencedensity peak per actin subunit at a ®lament radiusof approximately 3 nm, whose statistical signi®-cance yielded a level of con®dence >99.9% (i.e. bysubjecting the difference map to a t-test). Thedifference map revealed the position of the Au11

cluster to a resolution of better than 1 nm (Figure 2f(4 bottom)).

Using a directed mutation algorithm togetherwith X-ray ®ber diffraction data from orientednative and PHD-stabilized F-actin ®lament gels,Lorenz et al. (1993) have determined the PHD bind-ing site within F-actin to �1 nm resolution. Toobtain an atomic model of PHD:F-actin, they usedthe subunit orientation within the ®lament asdetermined by Holmes et al. (1990), together withchemical crosslinking data (Vandekerckhove et al.,1985) and stereochemical considerations as con-straints. However, as stated by these authors, theorientation of PHD was only tentative due to lackof resolution. As illustrated in Figure 3A, to testtheir proposed position and orientation of PHD,we overlaid our Au11-PHD:F-actin 3-D reconstruc-tion (Figure 2f (3)) with the atomic model ofPHD:F-actin (Lorenz et al., 1993) and replaced thePHD molecule within the atomic model with ourAu11-PHD derivative (Figure 1), so that the goldcluster coincided with the difference peak asmapped in Figure 2f (4). Lorenz's orientation of thetoxin molecule caused the attached gold cluster tocollide sterically with an adjacent actin monomer(Figure 3B). In contrast, rotation of the PHD moietyby �180� about an axis roughly parallel to the ®la-ment axis (Figure 3C) yielded close coincidence ofthe gold cluster with the difference peak(Figure 3A).

To probe for the PHD binding site of F-actin,Vandekerckhove et al. (1985) conjugated the modi-®ed side-chain of (OH)2-Leu7 of PHD with aphotolabile carbene-generating organic linker, simi-lar in length (i.e. �2 nm) to our Au11-complexedorganic linker (i.e. �1.7 nm). Stoichiometricamounts of this photoactivatable PHD derivative(called PAL) were expected to react fast andspeci®cally with nucleophilic amino acid residuesthat are close to the PHD binding site. By doing so,Glu117 of actin was identi®ed as the modi®edamino acid residue (Vandekerckhove et al., 1985).

Figure 2. Structural analysis of native, PHD, and Au11-PHD-stabilized rabbit muscle F-actin ®laments. For all samplespolymerization was initiated at room temperature by adding 2 mM MgCl2 and 50 mM KCl to 24 mM G-actin in2.5 mM imidazole, 0.2 mM CaCl2, 0.2 mM ATP, 0.005% NaN3 (pH 7.4), in the absence or presence of a 2:1 molarexcess of the respective toxin over actin. a, STEM annular dark-®eld (ADF) micrograph of a negatively stained (i.e.with 0.75% uranyl formate, pH 4.25) PHD-stabilized F-actin ®lament stretch. b, As a, but unstained and freeze-dried.c, Freeze-dried and unstained Au11-PHD-stabilized F-actin ®lament. d, As c but contrast adjusted (i.e. by top/bottomslicing) to display the highest intensities only, which correspond to single gold clusters (diameter �1 nm). The imagereveals single gold clusters approximately every 5.5 nm along the two long-pitch helical strands which are staggeredby 2.75 nm (see yellow lines). Scale bar represents 20 nm (a to d). (e) Histogram with pooled mass data from twoexperiments with Gaussian peaks ®tted: peak b from unstained, PHD-stabilized F-actin ®laments (shown in b); peakc from ®laments when Au11-PHD was employed (shown in c). Expected experimental value for 1:1 stoichiometricbinding of Au11-PHD to F-actin: 19.3 kDa/nm. Specimen preparation for STEM was according to the method ofSteinmetz et al. (1997a), and data acquisition and mass analysis were performed as described by MuÈ ller et al. (1992).f, Re®ned and averaged 3-D helical reconstructions of negatively stained native, PHD stabilized, and Au11-PHDstabilized F-actin ®laments. In each case ten two-crossover-repeat long F-actin ®lament stretches (26 subunits each)were 3-D helically reconstructed and re®ned, aligned and averaged as described by Steinmetz et al. (1997a) based ona processing protocol established by Bremer et al. (1994). They are displayed surface-rendered to include 100% of thenominal molecular volume. The bottom panels of reconstructions 1 to 3 show single sections normal to the ®lamentaxis and along the line drawn above them. Contours including 100, 75 and 50%, respectively, of the total mass aresuperimposed on the grey level representations. The white arrowheads point to the interface where the two long-pitch helical strands come to lie side by side, thereby de®ning a distinct ``groove''. In reconstruction 4, the differencebetween 3 and 2 (in yellow) is superimposed on reconstruction 3, revealing spatially unambiguously the position ofthe gold cluster at a ®lament radius of �3 nm. The bottom panel of 4 depicts the t-test evaluated difference map (inyellow) between 3 and 2, which has a >99.9% con®dence level. Scale bar represents 2.75 nm.

Orientation of Undecagold-tagged Phalloidin 3

Figure 3. Orientation of PHD within its F-actin binding site. A, Alignment and overlay of an atomic PHD:F-actin tri-mer (yellow ribbon; data from Lorenz et al., 1993) on the Au11-PHD:F-actin 3-D reconstruction displayed in Figure 2f(3). The PHD molecule was rotated by �180� about an axis roughly parallel to the ®lament axis and then replaced byour Au11-PHD derivative (violet CPK with a golden sphere). The experimentally determined Au11 density peak(Figure 2f (4)) is marked by the red broken contour. B, Top view of the atomic Au11-PHD:F-actin trimer as displayedin A but with the PHD moiety oriented as proposed by Lorenz et al. (1993). In this orientation the Au11 cluster col-lides with an actin monomer near the ®lament axis (red cross). C, Same as B but this time with the PHD oriented asshown in A. All modeling work was performed using Insight II (Biosym/Molecular Dynamics, Inc., San Diego).

4 Orientation of Undecagold-tagged Phalloidin

Remarkably, when PAL was modeled into thePHD binding site of the Holmes±Lorenz modelwith the same orientation as the Au11-PHD(Figure 3C), slight rotation of the derivative placedit closer than 0.4 nm to Glu117 of the nearest actinsubunit (Figure 4A).

Using the same structural and biochemical con-straints (see above), we tested the Schutt±LindbergF-actin model (Schutt et al., 1995a,b, 1997), whichindicates a rather different orientation of the sub-units within the ®lament. Relative to the proposedactin ribbon (Schutt et al., 1993), an F-actin ®lamentstructure was generated by a twist of 13� and ashortening by 0.83 nm per actin subunit (seeFigure 2 of Schutt et al., 1997). The resulting modelreveals subdomain 2 at the lowest and subdomain3 at the highest ®lament radius (with subdomains1 and 4 at medium radii). Moreover, with this sub-unit orientation the hydrophobic loop (i.e. aminoacid residues 262 to 274; Kabsch et al., 1990) isexposed at the ®lament surface rather than beingburied in the major intersubunit contact holdingthe two long-pitch helical strands together (Holmeset al., 1990; Lorenz et al., 1993). Hence, in the

Schutt±Lindberg F-actin model the subunit orien-tation is ``inside-out'' compared to the Holmes±Lorenz model, although the locations of the barbedand pointed ends of the ®lament are in common.First, overlaying the Shutt±Lindberg model withour Au11-PHD:F-actin 3-D reconstruction was lesssatisfactory than with the Holmes±Lorenz model.Second and most signi®cant, according to ourdetermined Au11 site within the Schutt±Lindbergmodel, the closest possible linear distance of PALto Glu117 of the nearest actin subunit was >2 nm(Figure 4B).

In conclusion, we have demonstrated that Au11

can serve as a site-speci®c hallmark on F-actin,which can be directly visualized by STEM andlocated in 3-D by helical reconstruction of nega-tively stained ®laments. This powerful techniqueallowed atomic scale re®nement of the molecularorientation of PHD within its F-actin binding sitefor the ®rst time. Importantly, whereas theHolmes±Lorenz model of the F-actin ®lament pro-duces a close match with our EM-based Au11-PHD:F-actin 3-D reconstruction and crosslinkingdata from Vandekerckhove et al. (1985), both our

Figure 4. Evaluating atomic F-actin models by considering crosslinking data with PAL as constraint (Vandekerckhoveet al., 1985). Ribbon represented F-actin trimers were overlaid with calculated 1 nm resolution electron density maps.A, The Holmes±Lorenz model (Holmes et al., 1990; Lorenz et al., 1993). Here Glu117 (red CPK) of the green actinmonomer is at a distance of �0.4 nm from the reactive group of PAL (tip of the white CPK), placed in the sameorientation as the Au11-PHD derivative in Figure 3, A and C. B, The Schutt±Lindberg model (Schutt et al., 1995a,1997). According to our determined Au11 site within this model, Glu117 is separated by a portion of actin's subdo-main 1 from PAL, making this residue inaccessible for the PHD derivative. For details, see the text. Both ®lamentmodels were aligned with their barbed (�) end at the bottom (white arrow); B, is rotated by �130� about the ®lamentaxis relative to A. A and B are stereo images. All modeling work was performed using Insight II (Biosym/MolecularDynamics, Inc., San Diego).

Orientation of Undecagold-tagged Phalloidin 5

EM and the crosslinking data yielded a rather poor®t with the Schutt±Lindberg model of F-actin.

Acknowledgments

We thank Dr W. Chiu for critical comments and help-ful suggestions. This work was supported by the CantonBasel-Stadt, the M.E. MuÈ ller Foundation of Switzerland,and a research grant from the Swiss National ScienceFoundation (31-39691.93).

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6 Orientation of Undecagold-tagged Phalloidin

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Edited by M. F. Moody

(Received 29 July 1997; received in revised form 3 November 1997; accepted 12 November 1997)