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Muscle proteins: actin
Muscle proteins: actin Kenneth C. Holmes and Wolfgang Kabsch Max-Planck-lnstitut f6r medizinische Forschung, Heidelberg, FRG In the past year, structural knowledge of actin has advanced to atomic detail. A synthesis of X-ray fibre-diffraction data and electron-microscopy observations of actin filaments with the atomic model has been achieved. Current Opinion in Structural Biology 1991, 1:270-280
Introduction Ever since its discovery in muscle tissue almost 50 years ago [1], actin has been the subject of intensive biochem ical and structural studies. Actin has been found in ev ci~ eukaryotic cell studied so far and participates in var i,)u:, fonns of ccllula~ motilit-y ~md the structure of tl~c ~3~oplasmic matrix. Recently, actin-like sequences have also been found in some prokaryotic cells [2]. In mus cle cells, actin - - together with the regulatory proteins tropomyosin and troponin - - forms the thin filament, ~hich ~Tclically interacts ~vith the thick tllyosiIi filamcl~t t(~ produce a mutual sliding that is the basis of muscle ,'~mtracti~m (see [3"], for the most recent review of thc cross bridge cycle). Actin can exist in a monomeric form, G-actin, or as a heheal poMner, F-actin, that possesses a rise per monomer of 27,5~ and a rotation angle of - 166.2 ° around the fil :m~ent axis Thus, the genetic helix, which is generated by the sTmmetry operators, is left handed but because the rotation per monomer is close to 180 °, the structure has the appearance of two right-handed long-pitch helices that are slowly twisting around each other (see Fig. 4} The diameter of the F actin helix is close to 100~ G actin consists of a single 375-residue polypeptide chain, of which the amino acid sequence and biochemical properties are highly conserved throughout evolution. It binds one molecule of ATP or ADP, and has a single high aff]ni U' and several low aft]nit} binding sites for diva lent cations. In vivo, actin is believed to bind Mg2 +, but this can be replaced by Ca 2 + in vitro with some effects on the kinetic properties of actin. It has been shown that the tightly bound cation is directly associated with the phosphates of the nucleotide [4"]. In the ab sence of divalent cations, actin is unstable and denatures rapidly. Nucleotide exchange for actin is relatively slow and monomeric actin is a slow ATPase. The normal assembly of actin into a filament begins with ATP-actin, and may be triggered by increasing the ionic strength. The rate of addition of subunits to the two ends of the growing filament is different: the ends of the filament are referred to as 'barbed' and 'pointed' according to the polarity of the arrow-head-like structure that is generated upon the binding of actin to myosin
270
S1 or heavy meromyosin (see Fig. 4). ATP-actin binds faster to the barbed end, whereupon bound ATP is hydrotyzed to ADP. However, ADP-actin dissociates faster than ATP-actin from the pointed end. This behaviour leads to the phenomenon of treadmilling [5]. Formation of F-actin stimulates actin ATPase; however, the release of phosphate from the newly4brmed filament proceeds more slowly than the formation of the filament, so that the growing filament has a 'cap' of ATP-actin at its barbed end followed by a zone containing ADP and phosphate. The mature filament contains just ADP-actin. ADP-actin may also be induced to form polymers by increasing the ionic strength. It has recently been proposed, however, that such ADP-F-actin polymers are different in structure from normal F-actin [6.]. The physiological importance of the putative ADP-F-actin has yet to be established. A number of excellent reviews have recently appeared that cover the biochemistry, polymerization and dynamics of actin ]7",8"] as well as the large number of actinbinding proteins [9",10]. In the past year, major progress in our understanding of the structure of actin has been achieved. The atomic structure of monomeric actin has been determined by X-ray analysis of crystals of rabbit skeletal muscle actin in complex with bovine pancreatic deoxyribonuclease I (DNase I) [11.]. Also, the atomic model of the F-actin helix has been derived from the crystal structure of monomeric actin and the fibre diffraction pattern from orientated actin gels [12.]. Furthermore, ice-embedded electron micrographs of actin filaments and decorated actin have been obtained at a resolution of 25-30A which show important facets of the binding of myosin and tropomyosin to actin [13"]. The two approaches, X-ray fibre diffraction and electron microscopy, have independently arrived at substantially the same structure for the actin filament. As the fibrediffraction data have presented an atomic model for the actin filament, it will now be possible to achieve a synthesis of the results of electron microscopy with the atomic structure. The following account, therefore, is mostly concerned with structure, and we largely omit mention of the earlier electron-microscopy studies on the F-actin structure of individual filaments or paracrystals consisting of arrays of filaments, which have been reviewed by Egelman [ 14 ]. The variability of images of single filaments
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Muscle proteins: actin Holmes and Kabsch produced by negative staining has shifted attention to the study of actin filaments embedded in vitreous ice [13"], an approach that is discussed below.
Crystal structure of actin in complex with DNase I The strong tendency of G actin to polymerize prevents the formation of crystals suitable for X-ray analysis. Many other proteins bind to actin and some of them - like profilin and DNase I - - form stable 1:1 complexes, ensur ing that actin cannot polymerize. Both the actin:profilin [15,16"] and the actin:DNase I [17-19] complexes have been crystallized and analyzed by X-ray diffraction. On the following pages, we describe the atomic structures of rabbit skeletal muscle actin in complex with bovine pan creatic DNase I in both the ATP- and ADP-linked forms, which have been solved at resolutions of 2.8~ and 3,0A, respectively [11.]. The two structures are very similar.
Domain structure The actin molecule (.Fig .1) fits into a square of side 55A and thickness 35& Actin consists of two domains, which, for historical reasons, are referred to as large and small, although they are now known to be not very different in size. The small domain comprises residues 1-144 and 338-375, the large domain residues 145-337. Each domain can be further subdivided into two subdomains; thus, subdomain 1 (l-32,70-144 and 338-375) and subdomain 2 (334;9) together constitute the small domain, and subdomain 3 (145-180 and 270-337) and s u b d o main 4 (181-269) the large domain. It appears likely that subdomain 2 is an insertion into subdomain 1 and that subdomain 4 is an insertion into subdomain 3. The motif of a five-stranded [3-sheet, consisting of a [3meander and a right-handed [3a[3 unit, is found in both subdomains 1 and 3, suggesting that gene duplication may have oc curred. 75 pairs of corresponding residues can be super imposed by an approximate twofold rotation with a root mean square deviation of 2.8A. The same chain topology is found in hexokinase [20] and the 44kD ATPase fragment of the heat-shock protein HSC70 [21. ] (see below). Both the amino- and carboxymrminal of actin are 1o cated in the small domain (Fig, 1), the polypeptide chain crossing over into the large domain at residue 140 and crossing back at residue 338. The C~ atoms of these two residues are only 4.0A apart, suggesting the possibility of a hinge in this region. The position of the adenine nucleotide between the two domains is of central importance for the stability of the actin molecule. Notably, all of the interactions that are responsible for the stable association of the large and small domains are mediated by salt bridges or hydrogen bonds involving the adenine phosphates. The experimental binding of DNase I to two regions on actin adds further to the stability of the domains. The minor contact region involves residues Thr203 and
Glu207 of subdomain 4, and Glu13 and His44 of DNase I. The major contact involves residues Arg39-Gly46 and Lys61-Ile64 of subdomain 2. The binding energy between the two proteins derives from hydrogen bonds as well as electrostatic and hydrophobic interactions. Residues Gly42, Va143 and Met44 of subdomain 2 are localized to one strand of a parallel [3-sheet, the other strand of which involves Tyr65, Va166 and Va167 of DNase I. The tight binding results in a structural distortion of the DNase I from its native form by as much as 1.8~_ in the C a positions of residues 65-67. It is likely that the structure of actin is also disturbed at the major contact region in subdomain 2. In particular, we expect that the loop structure adopted by residues 40-49 in the crystal structure will be different from the corresponding structure of the native Gactin conformation. The X-ray analysis of crystals of the actin-profilin complex is in progress [16"], and is expected to provide an undistorted structure of subdomain 2, as the contact region between these two proteins is close to Glu364 in subdomain 1 [22]. Interestingly, the crystal structure of HSC70 - - which is undisturbed by DNase I - - is very similar to that of actin (see below). A structure corresponding to the loop His40-Gln49 is not present in HSCY0, but the three strands of the 18-sheet and the a-helix that connects the second with the third strand in subdomain 2 of actin have moved only slightly with respect to the equivalent residues in HSC70. Importantly, the environments of the phosphate groups are essentially identical: the relative orientation of domains 1 and 3 is the same in both proteins. Therefore, it seems reasonable to assume that the structure of actin determined in complex with DNAse I is very like the structure of G-actin. Moreover, our studies [12"] (see below) have shown that the movement of domains necessary to account for the fibre diffraction pattern were small except for those of the loops 40-59 and 260-270. This comparison leads us to believe that the differences between F- and G-actin are subtle.
tigand binding ATP (or ADP) resides in the cleft between the two domains in the company of a Ca 2 + ion that is bound to the 13- and y , or [3-phosphates, respectively (Fig. 2). The observed geometry of the Ca 2 + ion with respect to the phosphate groups agrees with biochemical evidence that the tightly bound divalent metal ion interacts with the [3and y-phosphates of ATP and shows a high specificity for the A isomer [4"]. The environment of the divalent cation is a hydrophilic pocket that is closed off from the top by the di- or tri- phosphate moiety of the adenine. Other charged residues also found in this pocket include Aspll, Asp154 and Lysl8, and also Gln137 in the helix linking the two domains. Thus, the Ca2+ ion is shielded from the bulk solvent, which may explain its tight protein binding. In the case of ATP, the [3-phosphate is bound into the 18bend formed by residues Ser14, Gly15 and Leu16, and the y-phosphate is bound into the [3-bend formed by Asp157, Gly158 and Vail59. These two phosphate-bind-
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M a c r o m o l e c u l a r assemblages
L65
T203
P38
Subdomain 4 T194 Subdomain 2 1250 K68 K238
N92 F223
L1
A29
1274
Ell , F21
A131 L320
I282
G1
C
8348
L
N " " $350
Subdomain 3
Subdomain 1
D288
Fig. 1. Schematic representation of the structure of actin. ATP and Ca2+ are located between the small (right) and large (left) domains. Published with permission [11 ° ]
ing loops are equivalent with respect to the approximate twofold rotation that relates subdomains 1 to subdomain 3. In each case, the amide groups of the 13bends are orientated such that they hydrogen bond to the phosphate oxygens. In the case of ADP, only the [3-phosphate is bonded and, in both the ADP and ATP forms, Lys18 forms a bidentate complex with the a- and [3-phosphate oxygens. The adenine binds into a hydrophobic pocket formed between domains 3 and 4. The floor of this pocket comprises one turn of a 310-helix (302-308) connecting the second strand of the [3-sheet to the a-helix 309-320. In particular, the residue Gly302 is hydrogen bonded to the a-phosphate group. There is no space for a side chain between this residue and the ribose-phosphate moiety so that Gly302 is essential for nucleotide binding. The back of the pocket (see Fig. 1) is formed by the aromatic ring of Tyr306. The main components of the upper part of
the pocket are the hydrophobic chain of Glu214 (which is held in place by a hydrogen bond to the ribose 2'OH group and a salt bridge to Arg210) and Lys213 (which forms a hydrogen bond to the carbonyl group of 182). The N6 side of the adenine group points towards the bulk solvent and is thus available for chemical modification.
Comparison with HSCT0
The atomic structure of the 44 kD ATPase fragment of the bovine heat-shock protein HSC70 has recently been determined [21.]. Its striking structural similarity with actin is rather unexpected as there is hardly any sequence homology between the two proteins. The Ca positions of 241 pairs of equivalent amino acid residues in the two structures can be superimposed with a root mean square distance of 2.3A. In addition, the conformations of the
Muscle proteins: adin H o l m e s and Kabsch
Fig. 2. Stereo-plot of the environment of Ca 2+ and ATP in actin, l h e Ca 2+ ion is in the center of the picture. The atoms belonging to ATP are connected by thick lines. An approximate twofold axis (vertical in the figure) passes through the Ca 2 + ion and relates the two phospate-binding loops and associated [3-sheets. Published with permission [11"].
adenine nucleotides and their environments are very sire liar in both proteins. Subdomains 1~i of actin roughly correspond to HSC70 domains IA, IB, IIA and liB, as defined by Flaherty et aL [21"]. The structural differences between the two molecules mainly occur in those loop regions of actin known to be involved in interactions with other monomers in the actin filament or in binding myosin or tropomyosin.
Comparison with hexokinase In order to draw this comparison, the structure of hexokinase b [20] has to be deformed so as to overlay actin. The I3-sheet of the large domain is first brought into co incidence with the [3-sheet of subdomain 3 of actin, then the small domain (63-188) is rotated so as to bring its [3-sheet into coincidence with subdomain 1 of actin. The amino-terminal extension is not fitted to actin as it has no counterpart in the actin or HSP70 structure. For the above procedure, the rotation is large (37 °) and the translational component along the rotation axis is small (2.5A), so that the movement is practically a pure hinge motion. This rotation is greater than that necessary to bring hexokinase b into coincidence with hexokinase a [23], which suggests that even hexokinase a is not completely shut. After appropriately rotating domains, the structures of hexokinase b and actin in the [3sheets of subdomains 2 and 4 are very similar. One area of discrepancy is the bend adjoining strand 4 in the large domain, a bend which together with the adjoining 310 helix and a-helix forms the floor of the adenine-binding pocket (390-408). This structure has the same shape as the corresponding bend in actin, but protrudes by about 3~, into the putative adenine-binding site in the hexo-
Mnase b structure, thereby occluding the binding site. If these residues are moved by about 3~, so that they correspond to the positions occupied in actin or HSP70, the ATP coordinates taken from actin fit readily into the hexokinase pocket.
Hypothesis of the mechanism of ATP hydrolysis All three proteins may share a common mechanism for ATP hydrolysis. If we assume that the ATP binding found in actin is typical for all three proteins, the I3-phosphate is hydrogen bonded by three amide groups from the [3bend in the amino-terminal end of the small domain, and the 7 phosphate is hydrogen bonded by three amide groups of the equivalent 13-bend in the amino-terminal region of the large domain. The amide groups each carry a positive fractional charge which polarizes the phosphate oxygens. The 6- and y-phosphates form a tight bidentate complex with the divalent cation which further polarizes the phosphate oxygens. One expects an in-line mechanism and, therefore, seeks an attacking group situated on the far side of the T-phosphate group in line with the bond joining the O3'7-oxygen and the y-phosphorus. For all three proteins, candidates for the attacking group can be provided. For hexokinase, as expected, it is the glucose 0 6 group which is very well situated for inline attack. To be effective, the group must be polarized. As Steitz et al. [22] have pointed out, the group able to do this could be Asp189, which is situated in the crossover between the domains and, therefore, could also be sensitive to domain closure. In the case of HSP70, an ordered water molecule has been specified by Flaherty et aL [21.] which - - we surmise - - could be an attacking water group. It lies 3.6~, from the y-phosphorus and is
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Macromolecular assemblages ideally positioned for an in-line attack. Moreover, it forms a hydrogen bond to Thr204 and is polarized by Asp206. For actin, the situation is less clear. In place of the polarizing group Asp206 in HSC70 we have His161 in actin. To be effective as a base, this histidine would need to be part of a charge-relay system.
Domain
movement
The comparison of actin with hexokinase strongly suggests that actin may exist in an open form, wherein the large and small domains swing apart as in hexokinase. Moreover, the fact that nucleotide exchange in the closed form (i.e. bound to actin in F-actin or bound to DNase I) is very slow suggests that opening may assist nucleotide and divalent cation exchange. Both of these ideas are substantiated by an inspection of the actin structure: the packing around the phosphates is dense, the envi ronment of the cation is an occluded hydrophilic hole formed between the two domains. The open form is, however, not a long-lived species, as low-angle scatter ing measurements of G-actin report values for the radius of gyration of 22A [24] or 18.5,~ [25], which are the same or smaller than the corresponding calculated values for ATP-actin deduced from the crystal structure of this com plex.
The structure of the actin filament The atomic model fired to X-ray fibre diagrams F actin can be orientated in capillary tubes [26] such that the resulting gel yields X-ray fibre patterns that diffract to better than 8 A. An atomic model of the actin filament has been constructed [ 12"] from transformed coordinates of the actin monomer [11"]. The set of four free parameters for this transformation that lead to the best agree ment between the observed and calculated fibre diffraction patterns is unique. The resulting atomic model of the filament is shown in Fig. 3.
The maximum diameter of the actin filament is 90-95A. The large domain (comprising subdomains 3 and 4) is at a small radius and the small domain (subdomains 1 and 2) is at a large radius from the filament axis. The large domain is about 55A long and fits along the longpitch helix. The interactions between molecules along this helix are extensive, and are formed between residues 322-325 and 243-245,286-289 and 202-204, and between 166-169, 375 and 41-45. The hydrophobic loop (41-50), which binds DNase I in the crystal, makes contact with the large domain of the molecule above it. In the middle of the fibre, packing density is low. Contacts along the left-handed genetic helix are made between residues 110-112 and 195-197. In addition, a loop from the opposite strand (264-273) appears to insert into the hydrophobic pocket formed by residues Tyr166, Ala169, Leu171, His173, Cys285, Ile289, Gly63, Ile64 and residues 40-45. The loop (264-273) can be rebuilt as an antiparallel [B-sheet [12.] in the middle of which lies a [Bbend, thereby generating a finger-like structure which ex-
tends across the helix axis. This extension positions the sequence Phe266-Ile-Gly-Met in the [B-bend in such an orientation that these hydrophobic residues can intercollate between the molecules of the opposing strand to form a hydrophobic 'plug'. Some structural differences between monomeric actin and F-actin are to be expected. A preliminary investigation of the possible magnitude of subdomain movement has been carried out. The structure was divided into the four domains described above. Upon refinement against fibre diffraction data, a marked improvement in the fit to the fibre diagram was obtained with small domain movements (1-2A.) for all subdomains except for subdomain 2, which had to be moved approximately 6A. The model presented above agrees with many biochemical observations (see [12.]. Moreover, it was possible to establish the polarity of the filament; the barbed end is at the bottom in Fig. 3; this agrees with electron micrograph image reconstructions [13°,27]. By comparing the association constants at the ends of the filament with the very high association constant for annealing broken filaments, Erickson [28] showed that the longitudinal bonds in actin were three times stronger than the transverse bonds. On the basis of the number of interacting residues found in the model, this result seems quite plausible.
Cryo-electron microscopic structure of F-actin The structures of F-actin and actm decorated with myosin $1 have been obtained by Milligan et al. [13"] by cryoelectron microscopy in vitreous ice. These results pro vide important new information about the shape of F actin and the location of the binding sites for myosin and tropomyosin. The structure of the actin filament so deduced is substantially the same structure as that deduced by Holmes et al. [12"]. A comparison of the structures is shown in Fig. 4. The atomic model shown in Fig. 4a was calculated from the atomic coordinates of the actin helix by convoluting each atom with a Gauss function to produce an electron-density map with the effective resolution of 25~.. Two cut-off levels of resolution are shown. 20% and 80% of the maximum density. Figures 4b and 4c are taken from [13"]. The two structures of F-actin are very similar, excepting some differences in contrast; these differences can be attributed to the way in which the electron micrographs were processed, as the correction for the contrast transfer function arising from u n d e r focus was neglected. This leads to a substantial underestimation of the low-resolution terms in the Fourier synthesis and to an overestimation of detail. Although this error is not important in comparing two electron micrograph images taken under similar conditions, it does affect the kind of absolute comparison being made here. MJlligan et al. [13 °] were also able to localize the
carboxy-terminus by labeling Cys374 with an undecagold derivative. The radius they obtained (27.~.) agrees well with the value obtained by Holmes et al. [12"].
Muscle proteins: actin
H o l m e s and Kabsch
Fig. 3. Atomic model of F-actin as a stereo pair. The barbed end of the filament is at the bottom. The C a positions of eight monomers, a,DP and Ca 2+ are shown. A few amino acid sequence numbers are given for the actin monomer at the bottom of the figure. Strong contacts are made along the long-pitch helices. Published with permission [12"].
Location of surface binding sites on F-actin The myosin-binding site The myosin head interacts with the actin filament in a tangential manner, obscuring the front face of the actin monomer which, by comparison with the atomic model, can be identified as one face of subdomain 1 in the
small domain. This situation agrees with the identification made by Holmes et al. [12.] based on the positions of known myosin-binding residues (Aspl, Glu2, Glu4, Asp24, Asp25, Arg28, Glu93, Arg95 and Lys336, see discussion in [11"]). Moreover, the reconstruction shows that the rigor myosin-binding site spans two subunits. Be-
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Macromolecular assemblages
Fig.4. Ca>Atomic model of a(tin reduced to 25,~ resolution, shown at 70o,/0 and 20% of the maximum density. (b) Cryoelectron microscopic image reconstruction of F-actin at high and low density. (c) Cryo-electron mmroscopic ~mage reconstruction of F actin decorated with $1 at high and Iov, density. A bridge of density connects the second actinbinding site of $1 to the neighbouring actin molecule on the long-pitch helix. Adapted from [13"]. low the major site of interaction, there is an additional thin bridge of density extending between the main body of S1 and the top of the outer domain in the adjacent long-pitch actin monomer (Fig. 4c). This bridge lies at a filament radius of 40~. Inspection of the atomic structure suggests that residues Glu93 and Arg95 participate in this interaction. The idea of two distinct myosin-binding sites on actin may be of significance for theories of muscle contraction (,see Fig. 6 in [29]). Moreover, it has been shown b.v crosslinking of $1 to F-actin using short cross lifters that one S1 links to t w o different actin monomers
[3o].
The tropomyosin-binding site Tropomyosin follows the long-pitch helix at a filament radius of 38~ [13"]. Comparison of Fig. 4b of [13"] with the atomic model (Fig. 3) suggests that tropomyosin runs in the groove between subdomains 3 and 4 with contacts around Lys215 and Pro307. The helix Asp222 Ser233 may form part of this contact. This situation conforms with the observation that the sequence of Tetrahymena thermophila actin [32,33], which does not bind muscle tropomyosin [34"], differs by eight amino acids in the region 222-233 from the rabbit muscle sequence. It is also interesting to note that the region Cys217-Leu236 in actin must have an actin-specific function, a~s it is replaced by the short stretch Ser275-Thr278 in HSC70.
The light-chain-binding site By comparing actins decorated with SI(A1) or with SI CA2) ($1 containing A1 or A2 light chains, respective~0, Milligan et al. [13"] located the extra density" corresponding to the 41 amino-terminal residues that are present in the A1 light chain but are missing from the A2 light chain. The amino-terminal region of the A1 light chain binds to residues 360-363 in actin [31]. The position of the binding site determined by Milligan el al. is on the un dersurface of the actin monomer; this is consistent with the position found by Holmes et al. [12. ] for residues 360-363.
Comparison of the atomic model with previous results In comparison with previous results obtained in vitreous ice [27,35], the actin filament shows a stronger connectivity along the long-pitch helix. Mthough the latest structure incorporates better resolution, it agrees well with the earlier image reconstructions obtained from negatively stained Mg-paracrystals of F-actin [36], particu-
Muscle proteins: actin Holmes and Kabsch 277 larly with respect to the localization of tropomyosin and the presence of strong contacts along the long-pitch helix. Hence, reservations about results obtained from paracrystals seem to have been unnecessarily stressed. A discussion of the difficulties inherent in working with paracrystals has been presented by Francis and DeRosier [37]. The strength of the contact along the long-pitch helix has been stressed by Aebi eta/. ([38], see below). The structure obtained for Sl-decorated actin shows important detail (e.g. the second myosin-binding site) that could not be seen in the earlier results of Milligan and Flicker [27]. Moreover, it bears a welcome family resemblance to the original reconstructions of decorated actin [39].
try. However, supported actin does not exhibit such behavior: neither decorated actin nor paracrystals of actin show any marked variability in pitch. In muscle in rigor, it is the actin symmetry which impresses itself on the myosin heads, and not vice versa.
Disorder in the filaments
Millonig et al. [41 °] have classified individual negatively stained actin filaments into three classes according to their apparent radius, and have established the threedimensional structures for each class by image reconstruction. Of these classes, the one showing maximal lateral slipping (i.e. the largest diameter) agrees qualitatively in respect of shape and radius with the results of Holmes et al. [12.] and Milligan et al. [13"] discussed above. On the basis of this comparison, it appears to us that the classes showing more overlap, which were thought by MAllonig et al. [41 °] to represent native actin, are prob ably being distorted by compression during specimen preparation.
Electron micrographs of single actin filaments obtained using negative staining show considerable variability in the pitch of the long-pitch two-start helix. Moreover, the apparent radius of the filament is variable. These two phenomena have been studied by Egelman et al. [40] as 'cumulative angular disorder' and by Millonig et al. [41.] as the 'lateral slipping' model. However, as the effects seem much less obvious in vitreous ice (see Fig. l l c in [42] and [13"]), the onus still remains with electron microscopists to demonstrate that the polymorphisms seen in negatively stained images of single filaments are real effects and not artefacts produced by binding to the carbon film or artefacts of staining.
Cumulative angular disorder An explanation of the variable pitch has been provided by Egelman et aL [40], who advanced the theory that the azimuthal angular position of each subunit with respect to its neighbor is subject to a random error (standard deviation, a). Moreover, as one proceeds along the filament, these errors propagate as a one-dimensional random walk. They termed this effect cumulative angular disorder. This theory has been carefully tested by Stokes and DeRosier [42] who established a value of c~ = 12 ° for negatively stained images. The magnitude of c~ has been the subject of discussion. Erickson [28] criticized the value of cr obtained by Stokes and DeRosier on the grounds that protein structures are precise. Indeed, from the model reported above, it is not easy to see where one would accommodate such large angular variations. The main contacts are 15-20A from the axis. Moreover, the stronger contacts are made between next nearest neighbours along the long-pitch helix, for which the expected standard error in position is ~ x ,/2. The standard deviation in displacement between neighboring molecules becomes 6h, which is ditficult to reconcile with the known precision of protein-protein interactions. Stokes and DeRosier [42] take the view that this flexibility is an intrinsic property of actin which can be used to explain the structure of many actin filament bundles which often express a symmetry inconsistent with actin symme-
Lateral slipping The lateral slipping model [41o] addresses the problem of variable pitch together with the problem of varying diameter. Aebi et al. [38] stressed the importance of strong contacts along the long-pitch helicies, whereas contacts along the genetic helix are weaker. The actin filament may therefore behave as two independent strands, which may slip past each other in an approximately radial direction to give rise to filaments of variable diameter.
Variable lateral slipping produces changes in the apparent distance between crossovers, and could account for some of the phenomena explained by cumulative angular disorder. A critical appraisal of the relationship between the two phenomena has yet to be made.
The ribbon hypothesis Schutt et al. [16o] have published a low-resolution electron-density map of the crystalline complex between G-actin and profilin. The structure shows actin as having two domains with the nucleotide sitting at the top of the cleft between the two. The X-ray crystallographic analysis proved very difficult as the crystals displayed extensive polymorphism: the c-axis was subject to considerable variation. The authors note also that the b-axis showed a superlattice at low pH, which causes dissociation of profilin. The crystals were obtained from high salt concentrations in the presence of EDTA, which is likely to remove the essential divalent action. Moreover, a high salt concentration will weaken the salt bridges that hold the two domains together. Under these conditions, one might expect that the two domains could swing apart, as in hexokinase. A hinge motion might explain the bizarre changes in cell constants observed. Further, the authors have presented a theory as to how changes in the structure of the actin filament may be in-
votved in muscle contraction. The theory is based upon the hypothesis that the packing of molecules seen along the b-axis, a twofold screw axis with a rise per monomer of 36~. along the axis of syn'unetry and a twist per monomer of 180 ° (the 'ribbon' structure), can transform into the normal actin helix, which has a rise of 27.5A and a twist of 166 °.
The use of quantitative cryo-electron microscopy reported by Milligan et al. [13"] represents a quantum leap forward. The successful localization of undeca-gold and determination of the difference between the A1 and A2 light chains demonstrates the potential of this method. Putting aside possible difficulties arising from spec:men preservation, these results could not have been obtained by negative staining. However, cryo-electron microscopy has not yet been fully exploited: the present resolution limit of 25 A arises to a large extent from the high degree of underfocus presently employed in order to obtain sufficient contrast. The situation can be improved dramati cally by filtering out inelasticalty scattered electrons [44] and may be further improved by the use of methods developed by Henderson et aL [45], such as microscopy at liquid helium temperatures and spot scanning. Cryo-electron microscopy, therefore, will undoubtedly be used further to establish the structure of macromolecular assemblies to higher resolutions. A resolution of 10--15A should be attainable in most situations and much better resolutions will be obtainable in a few hardy specimens. In any case, the resolution will be adequate to allow a determi nation of the orientation of an atomic structure obtained from protein crystallography within the macromolecular assemb~, with even some resolution left over with which to estimate distortions. The combination of X-ray crystallography with high-resolution cryo-electron microscopy seems to be the general way forward for detenmning the fine structure of a large class of macromolecular assem blages.
Unfortunately, the packing in the ribbon form and the packing now established for the monomer in F actin [11.,12,] are quite different in several respects. Firstly, in the ribbon structure, the large domain would be at high radius it is actually at small radius~ In support of their identification, the authors cited that the carboxT terminus is located at large radius according to fluores cence energy transfer measurements [43], mad that the carboxy-terminus lies in the large domain, which turns out to be wrong. Secondly, the contacts between do mains in the ribbon model constitute a tenuous zigzag, which is thought to transform into contacts along the lefthanded genetic helix upon transformation into the helical structure. There are no contacts along the long-pitch he !ix In the recentb~ determined ~tructure major contact~ are made involving the large domain along the long-pitch helix, and these contacts prox4de the backbone for the whole filament This structure has no counterpart in the ribbon model. By renaming the actin domains, i.e posttflating that larg~: and small have been incorrectly assigned, and rotating the monomers by approximately 30 °, it may be possible to establish a correspondence with the present known structure. The rotation, however, leads to expression of a completely new set of contacts, which are quite different from those present in the crystal. Therefore, we think that it is unlikely that the ribbon form has any relationship to [.-actin. In this context, it may be worth mentioning how actin packs in the orthorhombic fore1 of actin-DNase I crystals: the actin-actin contacts occur in the large domain near a crystallographic twofold screw-axis along b. This results in a zig zag helix with a rise per monomer of 28.3,tk and a rotation angle of 180 ° along the crystallographic helix axis. Despite the close resemblance of these parameters with the true F-actin helical parameters, the contacts expressed in the crystal are quite different from those found in the filament.
3.
Macromolecular assemblages: outlook
4. .
In the case of actin, a synthesis of X-ray crystallography, X-ray fibre diffraction and electron microscopy has been achieved to yield a consistent, near-atomic picture of the structure of the actin filament. In each case, progress has depended upon technoloDcal advances: accurate data collection from crystals of varying cell parameters; successful phase extension using the known structure of DNase I as a starting model; and, progress in cryo-electron microscopy. Solving the structure of the actin filament by combining fibre diffraction with protein crystallography couM be a paradigm for other helical structures such as microtubules or Rec-A filaments.
References and recommended reading Papers of special interest, published within the annual peri(xt of re'clew. have been highlighted as: • of interest •• of outstanding interest 1
STRAt~FB: Actin. Studies, UniversRy of Szeged 1942, 1I:3-15.
2.
GARC~-CUELLARC, TENOPaO V, CISNEROS B, MONTATNEZ C, HERNANDEZ JM, ALVAm~Z J, DE LA G~ZZA M: Actin-like seq u e n c e s in Escherichia coli and Salmonella typhimurium [abstract]. J Cell Biol 1990, I ! l:31a.
COOKER: Force G e n e r a t i o n in Muscle. Curt Opin Cell Btol 1990, 2:62~o6. ~he most recent review of the crossbridge cycle. VALENTIN-RANC TC, CARLmRM F: Evidence for the Direct Interaction b e t w e e n Tightly Bound Divalent Metal I o n and ATP on Actin. J Biol Chem 1989, 264:20871 20880. Biochemical evidence that the tightly bound divalent metal ion interacts with the [~-and ?-phosphates of ATP and shows a high specificity for the A isomers. SELVE N, WEGNERA= Rate o f Treadmilliug of Actin Filaments in vitro. J Mol Biol 1986, 187:627q531. JANMEYPA, HVIDT S, OSTER G, LAMBJ, STOSSELT, HARTW1G J: Effect of ATP o n Actin Filament Stiffness. Nature 1990, 347:95-99. Actin filaments made from ATP-containing monomers are stiffer than those prepared from ADP-monomers. As both filaments mainly contain ADP, it is suggested that the normal F-actin stores extra internal energy, which is utilized by actin-binding proteins. 6. .
Muscle proteins: actin Holmes and Kabsch 279 7.
POLLARDT D: Actin. Curr Opin Cell Biol 1990, 2:33-40.
22.
VANDEKERCKHOVE JS, KAISERDA, POLLARDTD: A c a n t h a m o e b a Actin and Profilin can be Cross-linked Between Glutamic Acid 364 of Actin and Lysine 115 of Profilin. J Cell Biol 1989, 109:619-626.
23.
STEITZTA, SHOHAMM, BENNETt WS: Structural Dynamics of Yeast Hexokinase During Catalysis. Phil Trans R Soc Lond [Biol] 1981, 293:43-52.
24.
CURMIPM, STONE DB, SCHNEIDERDK, SPUDICnJA, MENDELSON R& Comparison of the Structure of Myosin Subfragment 1 Bound to Actin and Free in Solution. J Mol Biol 1988, 203:781-798.
25.
GODDETIEDW, UBERBACHEREC, BUNICK GJ, FR1EDENC: Formation of Actin Dimers as Studied by Small Angle Neutron Scattering. J Biol Chem 1986, 261:2605-2609.
26
PoPP D, LEDNEV W, JAHN W: Methods of Preparing WellOrientated Sols of F-actin Containing Filaments Suitable for X-ray Diffraction. J Mol Biol 1987, 197:679-684.
27.
MALUGANRA, FUCKER PF: Structural Relationships of Actin, Myosin, and Tropomyosin Revealed by Cryo-Electron Microscopy. J Cell Biol 1987, 105:29-39.
28.
ERICKSONHP: Cooperativity in Protein-Protein Association. J Mol Biol 1989, 206:465470.
20
(~OODYIL~. ttOLMES KC: Cross-Bridges and the Mechanism of Muscle Contraction. Biochim Biophvs Acta 1983, 726:13 39.
30
MORNET D, BETR&ND R, PANTEL P, AUDEMARD E, KASSAB R: Structure of the Actin-Myosin Interface. Nature 1981, 292:301-306.
31
SUTOHK: Identification of Myosin Binding Sites on the Actin Sequence. Biochemist~. 1982, 21:3654 3661.
~2
HIRONOM. ENDOH H, OKADA N, NUMATA O, WATANABE Y: Cloning and Sequencing of the Tetrahymena Actin Gene and Identification of its Gene Product. J Mol Biol 1987, 194:181-192.
33.
CupPlzSCG, PEARINIANRE: Isolation and Characterization of the Actin Gene From T e t r a h y m e n a thermophila. Proc Natl Acad Sci ~.~et 1986, 83:5160-51(34.
review of the structure, biochemistry, polymerization and cellular dy namics of actin. CARLIERM-F: Role of Nucleotide Hydrolysis in the Dynamics of Actin Filaments and Microtubules. lnt Rev Cytol 1989, 115:139-170. Authoritative review of actin kinetics. 8.
•
9. VANDEKERCKHOVE J: Actin-hinding proteins. Curr Opin Cell • Biol 1990, 2:41-50. An up-to-date review of actin-binding proteins excluding myosin. 10.
KORNED, HAMMERJA: Myosins of non-muscle cells. A n n u Rev Biophys Biophys CJaem 1988, 17:23-45.
KABSCHW, MANNHERZ HG, SUCK D, PAl EF, tiOLMES KC: . Atomic Structure of the Actin:DNase I Complex. Nature 1990, 347:37-44. The first report of the use of X ray crystallography to describe the atomic structure of actin. 11. •
HOLMESKC, POPP D, GEBHARD W, KABSCH W: Atomic Model • of the Actin Filament. Nature 1990, 347:44~9. Construction of the F-actin filament from the atomic structure of the actin monomer that fits X ray fibre data obtained from orientated gels of F actin. 12.
MIUJGANRA, WHFIq'AKER M, SAFER D: Molecular Structure ot • F-Actin and Location of Surface Binding Sites. Nature 1990, 348:217 221. Cryo-electron microscopy of muscle thin fdanmnts reveals the structure of F actin, the binding sites of tropomyosin, the myosin head and the myosin AI light chain of the filament at a resolution of 25-30A. 13.
14.
EGELMANE: The Structure of F-Actin. J Muscle Res 1985, 6:129-151.
15.
CARkSSON L, NYSTROEM 1,-E, IaNDBERG U, KANNAN KK, CID DRESDNER H, LOEVGREN S, JOERNVALLH: Crystallization of a Non-Muscle Actin. J Mol Biol 1976, 105:353-366.
16. .
SCHUTTCE, LINDBERGU, MYSIJKJ, STRAUSSN: Molecular Packing in Profilin~ctin Crystals and its Implications. J Mol Biol 1989, 209:735-746. The authors present a low-resolution structure of the actin-profilin complex. A ribbon structure for F actin is proposed on the basis of crystalline packing data. This structure is not compatible with the atomic model of F-actin [12•]. 17.
LINDBERG U: Crystallization from Calf Spleen of Two lnhibitors of Deoxyribonuclease I. J Biol Chem 1966, 241:1246-1248.
18.
MANNHERZHG, KABSCH W, LEBERMAN R: Crystals of Skeletal Muscle Actin:Pancreatic DNase I Complex. FEBS Lett 1977, 73:141-143.
19.
SUGINO H, SAKABE N, SAKABE K, HATANO S, OOSAWA F, MIKAWAT, EBASHIS: Crystallization and Preliminary Crystallographic Data of Chicken Gizzard G-Actin:DNase I Complex and P h y s a r u m G-Actin:DNase I Complex. J Biochem 1979, 86:257 260.
20.
ANDERSONCA, STENKAMPRE, STEITZT& Sequencing a Protein by X-Ray Crystallography. II. Refinement of Yeast Hexokinase B Co-ordinates and Sequence at 2.1Jk. J Mol Biol 1978, 123:15-33.
FLAHERTYKM, DELUCA-FIAHERTYC, MCKAY D: Three Dimensional Structure of the ATPase Fragment of a 70K HeatShock Cognate Protein. Nature 1990, 347:623~28. The atomic structure of the 44 kD ATPase fragment of the heat-shock protein HSC70 is reported. The observation that this structure is very similar to actin came as rather a surprise, as there is no significant sequence homology between the two proteins. This provokes interesting questions about the evolutionary relationship between actin and the heat-shock proteins. 21.
HIRONOM, TANAKA R, WATANABE Y: T e t r a h y m e n a Actin: Copolymerization with Skeletal Muscle Actin and Interactions with Muscle Actin-Binding Proteins. J Biochem 1990, 107:3206. Tetrahymena actin is very unusual as it binds neither DNase I, nor phalloidin, :~-actinin nor muscle tropomyosin. Its primary' structure has only 75% homology with rabbit skeletal muscle actin. 34. •
35.
TPaVaCKJ, COOPER J, SEYMOUR J, EGEtMAN EH: Cryo-electron Microscopy and Three-Dimensional Reconstruction of Actin Filaments. J Microsc 1986, 141:349-360.
36.
O'BRIENEJ, COUCH J, JOHNSON GRP, MORRIS EP: Structure of Actin and the Thin Filament. In Actin: Its Structure a n d Function in Muscle a n d Non-Muscle Cells edited by dos Remedios CG, Barden SA. London: Academic Press, 1983, pp 3-16.
37.
FRANCISNR, DEROSIERDJ: A Polymorphism Peculiar to Bipolar Actin Bundles. Biophys J 1990, 58:771-776.
38.
AliBI U, MILLON1GR, SALVO H, ENGEL A: The Three-Dimensional Structure of the Actin Filament Revisited. A n n N Y Acad Sci 1986, 483:100-119.
39.
MOOREPB, HUXLEYHE, DEROSIERD: Three-Dimensional Reconstructions of F-Actin, Thin Filaments and Decorated Thin Filaments. J Mol Biol 1970, 50:279-295.
40.
EGEtMANEN, FRANCISN, DEROSm~ DJ: F-Actin is a Helix with Random Variable Twist. Nature 1982, 298:131-135.
41. •
MILLONIGR, SOTrERL~ R, EN6EL A, POLLARDTD, AEBI U: The 'Lateral Slipping' Model of F-actin Filaments. In: Springer Series in Biophysics Vol 3, Cytoskeletal a n d Extracellular Pr~
•
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Macromolecular assemblages teins edited by Aebi U, Engel A. Heidelberg: Springer-Verlag, 1989, pp 51-53. Negatively stained images of F-actin: are classified according to their diameter. The hypothesis is advanced that these images are related to each other by the lateral slipping of the tightly bonded long-pitch he. lices with respect to each other.
42.
STOKESDL, DEROSlER DJ: The Variable Twist of Actin and its Modulation by Actin-Binding Proteins. J Cell Biol 1987, 104:1005-1017.
43.
TAYLORDL, REIDLER K, SPUDICH JA, STRYER L: Detection of Actin Assembly by Fluorescence Energy Transfer. J Cell Biol 1981 89:362 367.
44
SCHROEDERRR, HOFMANN W, MENETRET JF: Zero-Loss Energy Filtering as Improved Imaging Mode in Cryoelectron-
Microscopy of Frozen-Hydrated Specimens. J Struct Biol 1990, 105. 45.
HENDERSON R, BALDWINJM, CESKATA, ZEMLINF, BECKMANNE, DOWNING KH: Model for the Structure of Bacteriorhodopsin Based on High-Resolution Electron Cryomicroscopy. J Mol Biol 1990, 213:899-929.
KC Holmes and W Kabsch, Max-Planck-Institut far medizinische Forschung, Abteilung Biophysik, Jahnstral~e 29, W-6900, Heidelberg, FRG.
Introduction Ever since its discovery in muscle tissue almost 50 years ago [1], actin has been the subject of intensive biochem ical and structural studies. Actin has been found in ev ci~ eukaryotic cell studied so far and participates in var i,)u:, fonns of ccllula~ motilit-y ~md the structure of tl~c ~3~oplasmic matrix. Recently, actin-like sequences have also been found in some prokaryotic cells [2]. In mus cle cells, actin - - together with the regulatory proteins tropomyosin and troponin - - forms the thin filament, ~hich ~Tclically interacts ~vith the thick tllyosiIi filamcl~t t(~ produce a mutual sliding that is the basis of muscle ,'~mtracti~m (see [3"], for the most recent review of thc cross bridge cycle). Actin can exist in a monomeric form, G-actin, or as a heheal poMner, F-actin, that possesses a rise per monomer of 27,5~ and a rotation angle of - 166.2 ° around the fil :m~ent axis Thus, the genetic helix, which is generated by the sTmmetry operators, is left handed but because the rotation per monomer is close to 180 °, the structure has the appearance of two right-handed long-pitch helices that are slowly twisting around each other (see Fig. 4} The diameter of the F actin helix is close to 100~ G actin consists of a single 375-residue polypeptide chain, of which the amino acid sequence and biochemical properties are highly conserved throughout evolution. It binds one molecule of ATP or ADP, and has a single high aff]ni U' and several low aft]nit} binding sites for diva lent cations. In vivo, actin is believed to bind Mg2 +, but this can be replaced by Ca 2 + in vitro with some effects on the kinetic properties of actin. It has been shown that the tightly bound cation is directly associated with the phosphates of the nucleotide [4"]. In the ab sence of divalent cations, actin is unstable and denatures rapidly. Nucleotide exchange for actin is relatively slow and monomeric actin is a slow ATPase. The normal assembly of actin into a filament begins with ATP-actin, and may be triggered by increasing the ionic strength. The rate of addition of subunits to the two ends of the growing filament is different: the ends of the filament are referred to as 'barbed' and 'pointed' according to the polarity of the arrow-head-like structure that is generated upon the binding of actin to myosin
270
S1 or heavy meromyosin (see Fig. 4). ATP-actin binds faster to the barbed end, whereupon bound ATP is hydrotyzed to ADP. However, ADP-actin dissociates faster than ATP-actin from the pointed end. This behaviour leads to the phenomenon of treadmilling [5]. Formation of F-actin stimulates actin ATPase; however, the release of phosphate from the newly4brmed filament proceeds more slowly than the formation of the filament, so that the growing filament has a 'cap' of ATP-actin at its barbed end followed by a zone containing ADP and phosphate. The mature filament contains just ADP-actin. ADP-actin may also be induced to form polymers by increasing the ionic strength. It has recently been proposed, however, that such ADP-F-actin polymers are different in structure from normal F-actin [6.]. The physiological importance of the putative ADP-F-actin has yet to be established. A number of excellent reviews have recently appeared that cover the biochemistry, polymerization and dynamics of actin ]7",8"] as well as the large number of actinbinding proteins [9",10]. In the past year, major progress in our understanding of the structure of actin has been achieved. The atomic structure of monomeric actin has been determined by X-ray analysis of crystals of rabbit skeletal muscle actin in complex with bovine pancreatic deoxyribonuclease I (DNase I) [11.]. Also, the atomic model of the F-actin helix has been derived from the crystal structure of monomeric actin and the fibre diffraction pattern from orientated actin gels [12.]. Furthermore, ice-embedded electron micrographs of actin filaments and decorated actin have been obtained at a resolution of 25-30A which show important facets of the binding of myosin and tropomyosin to actin [13"]. The two approaches, X-ray fibre diffraction and electron microscopy, have independently arrived at substantially the same structure for the actin filament. As the fibrediffraction data have presented an atomic model for the actin filament, it will now be possible to achieve a synthesis of the results of electron microscopy with the atomic structure. The following account, therefore, is mostly concerned with structure, and we largely omit mention of the earlier electron-microscopy studies on the F-actin structure of individual filaments or paracrystals consisting of arrays of filaments, which have been reviewed by Egelman [ 14 ]. The variability of images of single filaments
(~ Current Biology Ltd ISSN 0959-440X
Muscle proteins: actin Holmes and Kabsch produced by negative staining has shifted attention to the study of actin filaments embedded in vitreous ice [13"], an approach that is discussed below.
Crystal structure of actin in complex with DNase I The strong tendency of G actin to polymerize prevents the formation of crystals suitable for X-ray analysis. Many other proteins bind to actin and some of them - like profilin and DNase I - - form stable 1:1 complexes, ensur ing that actin cannot polymerize. Both the actin:profilin [15,16"] and the actin:DNase I [17-19] complexes have been crystallized and analyzed by X-ray diffraction. On the following pages, we describe the atomic structures of rabbit skeletal muscle actin in complex with bovine pan creatic DNase I in both the ATP- and ADP-linked forms, which have been solved at resolutions of 2.8~ and 3,0A, respectively [11.]. The two structures are very similar.
Domain structure The actin molecule (.Fig .1) fits into a square of side 55A and thickness 35& Actin consists of two domains, which, for historical reasons, are referred to as large and small, although they are now known to be not very different in size. The small domain comprises residues 1-144 and 338-375, the large domain residues 145-337. Each domain can be further subdivided into two subdomains; thus, subdomain 1 (l-32,70-144 and 338-375) and subdomain 2 (334;9) together constitute the small domain, and subdomain 3 (145-180 and 270-337) and s u b d o main 4 (181-269) the large domain. It appears likely that subdomain 2 is an insertion into subdomain 1 and that subdomain 4 is an insertion into subdomain 3. The motif of a five-stranded [3-sheet, consisting of a [3meander and a right-handed [3a[3 unit, is found in both subdomains 1 and 3, suggesting that gene duplication may have oc curred. 75 pairs of corresponding residues can be super imposed by an approximate twofold rotation with a root mean square deviation of 2.8A. The same chain topology is found in hexokinase [20] and the 44kD ATPase fragment of the heat-shock protein HSC70 [21. ] (see below). Both the amino- and carboxymrminal of actin are 1o cated in the small domain (Fig, 1), the polypeptide chain crossing over into the large domain at residue 140 and crossing back at residue 338. The C~ atoms of these two residues are only 4.0A apart, suggesting the possibility of a hinge in this region. The position of the adenine nucleotide between the two domains is of central importance for the stability of the actin molecule. Notably, all of the interactions that are responsible for the stable association of the large and small domains are mediated by salt bridges or hydrogen bonds involving the adenine phosphates. The experimental binding of DNase I to two regions on actin adds further to the stability of the domains. The minor contact region involves residues Thr203 and
Glu207 of subdomain 4, and Glu13 and His44 of DNase I. The major contact involves residues Arg39-Gly46 and Lys61-Ile64 of subdomain 2. The binding energy between the two proteins derives from hydrogen bonds as well as electrostatic and hydrophobic interactions. Residues Gly42, Va143 and Met44 of subdomain 2 are localized to one strand of a parallel [3-sheet, the other strand of which involves Tyr65, Va166 and Va167 of DNase I. The tight binding results in a structural distortion of the DNase I from its native form by as much as 1.8~_ in the C a positions of residues 65-67. It is likely that the structure of actin is also disturbed at the major contact region in subdomain 2. In particular, we expect that the loop structure adopted by residues 40-49 in the crystal structure will be different from the corresponding structure of the native Gactin conformation. The X-ray analysis of crystals of the actin-profilin complex is in progress [16"], and is expected to provide an undistorted structure of subdomain 2, as the contact region between these two proteins is close to Glu364 in subdomain 1 [22]. Interestingly, the crystal structure of HSC70 - - which is undisturbed by DNase I - - is very similar to that of actin (see below). A structure corresponding to the loop His40-Gln49 is not present in HSCY0, but the three strands of the 18-sheet and the a-helix that connects the second with the third strand in subdomain 2 of actin have moved only slightly with respect to the equivalent residues in HSC70. Importantly, the environments of the phosphate groups are essentially identical: the relative orientation of domains 1 and 3 is the same in both proteins. Therefore, it seems reasonable to assume that the structure of actin determined in complex with DNAse I is very like the structure of G-actin. Moreover, our studies [12"] (see below) have shown that the movement of domains necessary to account for the fibre diffraction pattern were small except for those of the loops 40-59 and 260-270. This comparison leads us to believe that the differences between F- and G-actin are subtle.
tigand binding ATP (or ADP) resides in the cleft between the two domains in the company of a Ca 2 + ion that is bound to the 13- and y , or [3-phosphates, respectively (Fig. 2). The observed geometry of the Ca 2 + ion with respect to the phosphate groups agrees with biochemical evidence that the tightly bound divalent metal ion interacts with the [3and y-phosphates of ATP and shows a high specificity for the A isomer [4"]. The environment of the divalent cation is a hydrophilic pocket that is closed off from the top by the di- or tri- phosphate moiety of the adenine. Other charged residues also found in this pocket include Aspll, Asp154 and Lysl8, and also Gln137 in the helix linking the two domains. Thus, the Ca2+ ion is shielded from the bulk solvent, which may explain its tight protein binding. In the case of ATP, the [3-phosphate is bound into the 18bend formed by residues Ser14, Gly15 and Leu16, and the y-phosphate is bound into the [3-bend formed by Asp157, Gly158 and Vail59. These two phosphate-bind-
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M a c r o m o l e c u l a r assemblages
L65
T203
P38
Subdomain 4 T194 Subdomain 2 1250 K68 K238
N92 F223
L1
A29
1274
Ell , F21
A131 L320
I282
G1
C
8348
L
N " " $350
Subdomain 3
Subdomain 1
D288
Fig. 1. Schematic representation of the structure of actin. ATP and Ca2+ are located between the small (right) and large (left) domains. Published with permission [11 ° ]
ing loops are equivalent with respect to the approximate twofold rotation that relates subdomains 1 to subdomain 3. In each case, the amide groups of the 13bends are orientated such that they hydrogen bond to the phosphate oxygens. In the case of ADP, only the [3-phosphate is bonded and, in both the ADP and ATP forms, Lys18 forms a bidentate complex with the a- and [3-phosphate oxygens. The adenine binds into a hydrophobic pocket formed between domains 3 and 4. The floor of this pocket comprises one turn of a 310-helix (302-308) connecting the second strand of the [3-sheet to the a-helix 309-320. In particular, the residue Gly302 is hydrogen bonded to the a-phosphate group. There is no space for a side chain between this residue and the ribose-phosphate moiety so that Gly302 is essential for nucleotide binding. The back of the pocket (see Fig. 1) is formed by the aromatic ring of Tyr306. The main components of the upper part of
the pocket are the hydrophobic chain of Glu214 (which is held in place by a hydrogen bond to the ribose 2'OH group and a salt bridge to Arg210) and Lys213 (which forms a hydrogen bond to the carbonyl group of 182). The N6 side of the adenine group points towards the bulk solvent and is thus available for chemical modification.
Comparison with HSCT0
The atomic structure of the 44 kD ATPase fragment of the bovine heat-shock protein HSC70 has recently been determined [21.]. Its striking structural similarity with actin is rather unexpected as there is hardly any sequence homology between the two proteins. The Ca positions of 241 pairs of equivalent amino acid residues in the two structures can be superimposed with a root mean square distance of 2.3A. In addition, the conformations of the
Muscle proteins: adin H o l m e s and Kabsch
Fig. 2. Stereo-plot of the environment of Ca 2+ and ATP in actin, l h e Ca 2+ ion is in the center of the picture. The atoms belonging to ATP are connected by thick lines. An approximate twofold axis (vertical in the figure) passes through the Ca 2 + ion and relates the two phospate-binding loops and associated [3-sheets. Published with permission [11"].
adenine nucleotides and their environments are very sire liar in both proteins. Subdomains 1~i of actin roughly correspond to HSC70 domains IA, IB, IIA and liB, as defined by Flaherty et aL [21"]. The structural differences between the two molecules mainly occur in those loop regions of actin known to be involved in interactions with other monomers in the actin filament or in binding myosin or tropomyosin.
Comparison with hexokinase In order to draw this comparison, the structure of hexokinase b [20] has to be deformed so as to overlay actin. The I3-sheet of the large domain is first brought into co incidence with the [3-sheet of subdomain 3 of actin, then the small domain (63-188) is rotated so as to bring its [3-sheet into coincidence with subdomain 1 of actin. The amino-terminal extension is not fitted to actin as it has no counterpart in the actin or HSP70 structure. For the above procedure, the rotation is large (37 °) and the translational component along the rotation axis is small (2.5A), so that the movement is practically a pure hinge motion. This rotation is greater than that necessary to bring hexokinase b into coincidence with hexokinase a [23], which suggests that even hexokinase a is not completely shut. After appropriately rotating domains, the structures of hexokinase b and actin in the [3sheets of subdomains 2 and 4 are very similar. One area of discrepancy is the bend adjoining strand 4 in the large domain, a bend which together with the adjoining 310 helix and a-helix forms the floor of the adenine-binding pocket (390-408). This structure has the same shape as the corresponding bend in actin, but protrudes by about 3~, into the putative adenine-binding site in the hexo-
Mnase b structure, thereby occluding the binding site. If these residues are moved by about 3~, so that they correspond to the positions occupied in actin or HSP70, the ATP coordinates taken from actin fit readily into the hexokinase pocket.
Hypothesis of the mechanism of ATP hydrolysis All three proteins may share a common mechanism for ATP hydrolysis. If we assume that the ATP binding found in actin is typical for all three proteins, the I3-phosphate is hydrogen bonded by three amide groups from the [3bend in the amino-terminal end of the small domain, and the 7 phosphate is hydrogen bonded by three amide groups of the equivalent 13-bend in the amino-terminal region of the large domain. The amide groups each carry a positive fractional charge which polarizes the phosphate oxygens. The 6- and y-phosphates form a tight bidentate complex with the divalent cation which further polarizes the phosphate oxygens. One expects an in-line mechanism and, therefore, seeks an attacking group situated on the far side of the T-phosphate group in line with the bond joining the O3'7-oxygen and the y-phosphorus. For all three proteins, candidates for the attacking group can be provided. For hexokinase, as expected, it is the glucose 0 6 group which is very well situated for inline attack. To be effective, the group must be polarized. As Steitz et al. [22] have pointed out, the group able to do this could be Asp189, which is situated in the crossover between the domains and, therefore, could also be sensitive to domain closure. In the case of HSP70, an ordered water molecule has been specified by Flaherty et aL [21.] which - - we surmise - - could be an attacking water group. It lies 3.6~, from the y-phosphorus and is
273
274
Macromolecular assemblages ideally positioned for an in-line attack. Moreover, it forms a hydrogen bond to Thr204 and is polarized by Asp206. For actin, the situation is less clear. In place of the polarizing group Asp206 in HSC70 we have His161 in actin. To be effective as a base, this histidine would need to be part of a charge-relay system.
Domain
movement
The comparison of actin with hexokinase strongly suggests that actin may exist in an open form, wherein the large and small domains swing apart as in hexokinase. Moreover, the fact that nucleotide exchange in the closed form (i.e. bound to actin in F-actin or bound to DNase I) is very slow suggests that opening may assist nucleotide and divalent cation exchange. Both of these ideas are substantiated by an inspection of the actin structure: the packing around the phosphates is dense, the envi ronment of the cation is an occluded hydrophilic hole formed between the two domains. The open form is, however, not a long-lived species, as low-angle scatter ing measurements of G-actin report values for the radius of gyration of 22A [24] or 18.5,~ [25], which are the same or smaller than the corresponding calculated values for ATP-actin deduced from the crystal structure of this com plex.
The structure of the actin filament The atomic model fired to X-ray fibre diagrams F actin can be orientated in capillary tubes [26] such that the resulting gel yields X-ray fibre patterns that diffract to better than 8 A. An atomic model of the actin filament has been constructed [ 12"] from transformed coordinates of the actin monomer [11"]. The set of four free parameters for this transformation that lead to the best agree ment between the observed and calculated fibre diffraction patterns is unique. The resulting atomic model of the filament is shown in Fig. 3.
The maximum diameter of the actin filament is 90-95A. The large domain (comprising subdomains 3 and 4) is at a small radius and the small domain (subdomains 1 and 2) is at a large radius from the filament axis. The large domain is about 55A long and fits along the longpitch helix. The interactions between molecules along this helix are extensive, and are formed between residues 322-325 and 243-245,286-289 and 202-204, and between 166-169, 375 and 41-45. The hydrophobic loop (41-50), which binds DNase I in the crystal, makes contact with the large domain of the molecule above it. In the middle of the fibre, packing density is low. Contacts along the left-handed genetic helix are made between residues 110-112 and 195-197. In addition, a loop from the opposite strand (264-273) appears to insert into the hydrophobic pocket formed by residues Tyr166, Ala169, Leu171, His173, Cys285, Ile289, Gly63, Ile64 and residues 40-45. The loop (264-273) can be rebuilt as an antiparallel [B-sheet [12.] in the middle of which lies a [Bbend, thereby generating a finger-like structure which ex-
tends across the helix axis. This extension positions the sequence Phe266-Ile-Gly-Met in the [B-bend in such an orientation that these hydrophobic residues can intercollate between the molecules of the opposing strand to form a hydrophobic 'plug'. Some structural differences between monomeric actin and F-actin are to be expected. A preliminary investigation of the possible magnitude of subdomain movement has been carried out. The structure was divided into the four domains described above. Upon refinement against fibre diffraction data, a marked improvement in the fit to the fibre diagram was obtained with small domain movements (1-2A.) for all subdomains except for subdomain 2, which had to be moved approximately 6A. The model presented above agrees with many biochemical observations (see [12.]. Moreover, it was possible to establish the polarity of the filament; the barbed end is at the bottom in Fig. 3; this agrees with electron micrograph image reconstructions [13°,27]. By comparing the association constants at the ends of the filament with the very high association constant for annealing broken filaments, Erickson [28] showed that the longitudinal bonds in actin were three times stronger than the transverse bonds. On the basis of the number of interacting residues found in the model, this result seems quite plausible.
Cryo-electron microscopic structure of F-actin The structures of F-actin and actm decorated with myosin $1 have been obtained by Milligan et al. [13"] by cryoelectron microscopy in vitreous ice. These results pro vide important new information about the shape of F actin and the location of the binding sites for myosin and tropomyosin. The structure of the actin filament so deduced is substantially the same structure as that deduced by Holmes et al. [12"]. A comparison of the structures is shown in Fig. 4. The atomic model shown in Fig. 4a was calculated from the atomic coordinates of the actin helix by convoluting each atom with a Gauss function to produce an electron-density map with the effective resolution of 25~.. Two cut-off levels of resolution are shown. 20% and 80% of the maximum density. Figures 4b and 4c are taken from [13"]. The two structures of F-actin are very similar, excepting some differences in contrast; these differences can be attributed to the way in which the electron micrographs were processed, as the correction for the contrast transfer function arising from u n d e r focus was neglected. This leads to a substantial underestimation of the low-resolution terms in the Fourier synthesis and to an overestimation of detail. Although this error is not important in comparing two electron micrograph images taken under similar conditions, it does affect the kind of absolute comparison being made here. MJlligan et al. [13 °] were also able to localize the
carboxy-terminus by labeling Cys374 with an undecagold derivative. The radius they obtained (27.~.) agrees well with the value obtained by Holmes et al. [12"].
Muscle proteins: actin
H o l m e s and Kabsch
Fig. 3. Atomic model of F-actin as a stereo pair. The barbed end of the filament is at the bottom. The C a positions of eight monomers, a,DP and Ca 2+ are shown. A few amino acid sequence numbers are given for the actin monomer at the bottom of the figure. Strong contacts are made along the long-pitch helices. Published with permission [12"].
Location of surface binding sites on F-actin The myosin-binding site The myosin head interacts with the actin filament in a tangential manner, obscuring the front face of the actin monomer which, by comparison with the atomic model, can be identified as one face of subdomain 1 in the
small domain. This situation agrees with the identification made by Holmes et al. [12.] based on the positions of known myosin-binding residues (Aspl, Glu2, Glu4, Asp24, Asp25, Arg28, Glu93, Arg95 and Lys336, see discussion in [11"]). Moreover, the reconstruction shows that the rigor myosin-binding site spans two subunits. Be-
275
276
Macromolecular assemblages
Fig.4. Ca>Atomic model of a(tin reduced to 25,~ resolution, shown at 70o,/0 and 20% of the maximum density. (b) Cryoelectron microscopic image reconstruction of F-actin at high and low density. (c) Cryo-electron mmroscopic ~mage reconstruction of F actin decorated with $1 at high and Iov, density. A bridge of density connects the second actinbinding site of $1 to the neighbouring actin molecule on the long-pitch helix. Adapted from [13"]. low the major site of interaction, there is an additional thin bridge of density extending between the main body of S1 and the top of the outer domain in the adjacent long-pitch actin monomer (Fig. 4c). This bridge lies at a filament radius of 40~. Inspection of the atomic structure suggests that residues Glu93 and Arg95 participate in this interaction. The idea of two distinct myosin-binding sites on actin may be of significance for theories of muscle contraction (,see Fig. 6 in [29]). Moreover, it has been shown b.v crosslinking of $1 to F-actin using short cross lifters that one S1 links to t w o different actin monomers
[3o].
The tropomyosin-binding site Tropomyosin follows the long-pitch helix at a filament radius of 38~ [13"]. Comparison of Fig. 4b of [13"] with the atomic model (Fig. 3) suggests that tropomyosin runs in the groove between subdomains 3 and 4 with contacts around Lys215 and Pro307. The helix Asp222 Ser233 may form part of this contact. This situation conforms with the observation that the sequence of Tetrahymena thermophila actin [32,33], which does not bind muscle tropomyosin [34"], differs by eight amino acids in the region 222-233 from the rabbit muscle sequence. It is also interesting to note that the region Cys217-Leu236 in actin must have an actin-specific function, a~s it is replaced by the short stretch Ser275-Thr278 in HSC70.
The light-chain-binding site By comparing actins decorated with SI(A1) or with SI CA2) ($1 containing A1 or A2 light chains, respective~0, Milligan et al. [13"] located the extra density" corresponding to the 41 amino-terminal residues that are present in the A1 light chain but are missing from the A2 light chain. The amino-terminal region of the A1 light chain binds to residues 360-363 in actin [31]. The position of the binding site determined by Milligan el al. is on the un dersurface of the actin monomer; this is consistent with the position found by Holmes et al. [12. ] for residues 360-363.
Comparison of the atomic model with previous results In comparison with previous results obtained in vitreous ice [27,35], the actin filament shows a stronger connectivity along the long-pitch helix. Mthough the latest structure incorporates better resolution, it agrees well with the earlier image reconstructions obtained from negatively stained Mg-paracrystals of F-actin [36], particu-
Muscle proteins: actin Holmes and Kabsch 277 larly with respect to the localization of tropomyosin and the presence of strong contacts along the long-pitch helix. Hence, reservations about results obtained from paracrystals seem to have been unnecessarily stressed. A discussion of the difficulties inherent in working with paracrystals has been presented by Francis and DeRosier [37]. The strength of the contact along the long-pitch helix has been stressed by Aebi eta/. ([38], see below). The structure obtained for Sl-decorated actin shows important detail (e.g. the second myosin-binding site) that could not be seen in the earlier results of Milligan and Flicker [27]. Moreover, it bears a welcome family resemblance to the original reconstructions of decorated actin [39].
try. However, supported actin does not exhibit such behavior: neither decorated actin nor paracrystals of actin show any marked variability in pitch. In muscle in rigor, it is the actin symmetry which impresses itself on the myosin heads, and not vice versa.
Disorder in the filaments
Millonig et al. [41 °] have classified individual negatively stained actin filaments into three classes according to their apparent radius, and have established the threedimensional structures for each class by image reconstruction. Of these classes, the one showing maximal lateral slipping (i.e. the largest diameter) agrees qualitatively in respect of shape and radius with the results of Holmes et al. [12.] and Milligan et al. [13"] discussed above. On the basis of this comparison, it appears to us that the classes showing more overlap, which were thought by MAllonig et al. [41 °] to represent native actin, are prob ably being distorted by compression during specimen preparation.
Electron micrographs of single actin filaments obtained using negative staining show considerable variability in the pitch of the long-pitch two-start helix. Moreover, the apparent radius of the filament is variable. These two phenomena have been studied by Egelman et al. [40] as 'cumulative angular disorder' and by Millonig et al. [41.] as the 'lateral slipping' model. However, as the effects seem much less obvious in vitreous ice (see Fig. l l c in [42] and [13"]), the onus still remains with electron microscopists to demonstrate that the polymorphisms seen in negatively stained images of single filaments are real effects and not artefacts produced by binding to the carbon film or artefacts of staining.
Cumulative angular disorder An explanation of the variable pitch has been provided by Egelman et aL [40], who advanced the theory that the azimuthal angular position of each subunit with respect to its neighbor is subject to a random error (standard deviation, a). Moreover, as one proceeds along the filament, these errors propagate as a one-dimensional random walk. They termed this effect cumulative angular disorder. This theory has been carefully tested by Stokes and DeRosier [42] who established a value of c~ = 12 ° for negatively stained images. The magnitude of c~ has been the subject of discussion. Erickson [28] criticized the value of cr obtained by Stokes and DeRosier on the grounds that protein structures are precise. Indeed, from the model reported above, it is not easy to see where one would accommodate such large angular variations. The main contacts are 15-20A from the axis. Moreover, the stronger contacts are made between next nearest neighbours along the long-pitch helix, for which the expected standard error in position is ~ x ,/2. The standard deviation in displacement between neighboring molecules becomes 6h, which is ditficult to reconcile with the known precision of protein-protein interactions. Stokes and DeRosier [42] take the view that this flexibility is an intrinsic property of actin which can be used to explain the structure of many actin filament bundles which often express a symmetry inconsistent with actin symme-
Lateral slipping The lateral slipping model [41o] addresses the problem of variable pitch together with the problem of varying diameter. Aebi et al. [38] stressed the importance of strong contacts along the long-pitch helicies, whereas contacts along the genetic helix are weaker. The actin filament may therefore behave as two independent strands, which may slip past each other in an approximately radial direction to give rise to filaments of variable diameter.
Variable lateral slipping produces changes in the apparent distance between crossovers, and could account for some of the phenomena explained by cumulative angular disorder. A critical appraisal of the relationship between the two phenomena has yet to be made.
The ribbon hypothesis Schutt et al. [16o] have published a low-resolution electron-density map of the crystalline complex between G-actin and profilin. The structure shows actin as having two domains with the nucleotide sitting at the top of the cleft between the two. The X-ray crystallographic analysis proved very difficult as the crystals displayed extensive polymorphism: the c-axis was subject to considerable variation. The authors note also that the b-axis showed a superlattice at low pH, which causes dissociation of profilin. The crystals were obtained from high salt concentrations in the presence of EDTA, which is likely to remove the essential divalent action. Moreover, a high salt concentration will weaken the salt bridges that hold the two domains together. Under these conditions, one might expect that the two domains could swing apart, as in hexokinase. A hinge motion might explain the bizarre changes in cell constants observed. Further, the authors have presented a theory as to how changes in the structure of the actin filament may be in-
votved in muscle contraction. The theory is based upon the hypothesis that the packing of molecules seen along the b-axis, a twofold screw axis with a rise per monomer of 36~. along the axis of syn'unetry and a twist per monomer of 180 ° (the 'ribbon' structure), can transform into the normal actin helix, which has a rise of 27.5A and a twist of 166 °.
The use of quantitative cryo-electron microscopy reported by Milligan et al. [13"] represents a quantum leap forward. The successful localization of undeca-gold and determination of the difference between the A1 and A2 light chains demonstrates the potential of this method. Putting aside possible difficulties arising from spec:men preservation, these results could not have been obtained by negative staining. However, cryo-electron microscopy has not yet been fully exploited: the present resolution limit of 25 A arises to a large extent from the high degree of underfocus presently employed in order to obtain sufficient contrast. The situation can be improved dramati cally by filtering out inelasticalty scattered electrons [44] and may be further improved by the use of methods developed by Henderson et aL [45], such as microscopy at liquid helium temperatures and spot scanning. Cryo-electron microscopy, therefore, will undoubtedly be used further to establish the structure of macromolecular assemblies to higher resolutions. A resolution of 10--15A should be attainable in most situations and much better resolutions will be obtainable in a few hardy specimens. In any case, the resolution will be adequate to allow a determi nation of the orientation of an atomic structure obtained from protein crystallography within the macromolecular assemb~, with even some resolution left over with which to estimate distortions. The combination of X-ray crystallography with high-resolution cryo-electron microscopy seems to be the general way forward for detenmning the fine structure of a large class of macromolecular assem blages.
Unfortunately, the packing in the ribbon form and the packing now established for the monomer in F actin [11.,12,] are quite different in several respects. Firstly, in the ribbon structure, the large domain would be at high radius it is actually at small radius~ In support of their identification, the authors cited that the carboxT terminus is located at large radius according to fluores cence energy transfer measurements [43], mad that the carboxy-terminus lies in the large domain, which turns out to be wrong. Secondly, the contacts between do mains in the ribbon model constitute a tenuous zigzag, which is thought to transform into contacts along the lefthanded genetic helix upon transformation into the helical structure. There are no contacts along the long-pitch he !ix In the recentb~ determined ~tructure major contact~ are made involving the large domain along the long-pitch helix, and these contacts prox4de the backbone for the whole filament This structure has no counterpart in the ribbon model. By renaming the actin domains, i.e posttflating that larg~: and small have been incorrectly assigned, and rotating the monomers by approximately 30 °, it may be possible to establish a correspondence with the present known structure. The rotation, however, leads to expression of a completely new set of contacts, which are quite different from those present in the crystal. Therefore, we think that it is unlikely that the ribbon form has any relationship to [.-actin. In this context, it may be worth mentioning how actin packs in the orthorhombic fore1 of actin-DNase I crystals: the actin-actin contacts occur in the large domain near a crystallographic twofold screw-axis along b. This results in a zig zag helix with a rise per monomer of 28.3,tk and a rotation angle of 180 ° along the crystallographic helix axis. Despite the close resemblance of these parameters with the true F-actin helical parameters, the contacts expressed in the crystal are quite different from those found in the filament.
3.
Macromolecular assemblages: outlook
4. .
In the case of actin, a synthesis of X-ray crystallography, X-ray fibre diffraction and electron microscopy has been achieved to yield a consistent, near-atomic picture of the structure of the actin filament. In each case, progress has depended upon technoloDcal advances: accurate data collection from crystals of varying cell parameters; successful phase extension using the known structure of DNase I as a starting model; and, progress in cryo-electron microscopy. Solving the structure of the actin filament by combining fibre diffraction with protein crystallography couM be a paradigm for other helical structures such as microtubules or Rec-A filaments.
References and recommended reading Papers of special interest, published within the annual peri(xt of re'clew. have been highlighted as: • of interest •• of outstanding interest 1
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2.
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~2
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KC Holmes and W Kabsch, Max-Planck-Institut far medizinische Forschung, Abteilung Biophysik, Jahnstral~e 29, W-6900, Heidelberg, FRG.