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Shape of the myosin head in the rigor complex
J. Mol. Riol. (1988) 204, 639-652
Shape of the Myosin Head in the Rigor Complex Three-dimensional Image Reconstruction of the ActinTropomyosin-Heavy Meromyosin Complex Hitomi Kajiyama Department of Physics, Faculty of Science The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113, Japan (Received 14 September 1986, and in revised form, 22 June 1988) The structure of the actin-tropomyosin-heavy meromyosin rigor complex was studied by image analysis of electron micrographs. The arrowhead of the rigor complex has a whiskerlike structure with a dense turning point at the “barb” of the arrowhead. The neck region of the myosin head in the reconstructed three-dimensional image is present in the area corresponding to the dense point. It is concluded that at least one extra-thin area contributes to the neck region, and that the two heads in the heavy meromyosin molecule join a double helical rope beyond the end of the large head (G in this study). (This is different from previous interpretations.) It is also concluded that the heavy meromyosin has a short bent part near the head/rod junction in the rigor complex.
sional reconstruction is better for studies on the molecular morphology of a single filament. Moore et al. (1970) first reconstructed the threedimensional image of the thin filament decorated with myosin Sl. Since then, improvements in electron microscopy (low dose imaging) and the use of other specimens (chymotryptic Sl from vertebrate muscle and Sl from scallop myosin), have resulted in more detailed information on the structure (Wakabayashi & Toyoshima, 1981; Taylor & Amos, 1981; Amos et al., 1982; Vibert & Craig, 1982; Toyoshima & Wakabayashi, 1985a,b). However, it is important to study the structure of the decorated thin filament in preparations in which myosin heads are as intact as possible. The three-dimensional structure of the AC-HMM complex in the rigor state has been reconstructed by Katayama & Wakabayashi (1981) and Seymour & O’Brien (1985). Although it was found that the Ac-HMM complex had regions (F and G) in addition to those of AC-Sl, the reconstructed images showed only a part of the HMM. However, one of these studies (Katayama & Wakabayashi. 1981) used only a few short filaments that were put on a carbon microgrid and the other (Seymour & O’Brien, 1985) used only a single filament on a carbon-coated copper grid. To determine the overall structure, I examined the structure of AC-TM-HMM with an improved method: the use of thin filaments that contained tropomyosin to obtain straight filaments, averaging
1. Introduction Muscle is composed of two kinds of filament, thick filaments that consist mainly of two-headed myosin molecules (480,000 M,) and thin filaments that’ consist of actin, tropomyosin and troponin (T+I+C). In the rigor state, the myosin binds to the actin filament at a ratio of one myosin head to one actin monomer. In the force-generating, i.e. activated, state, myosin heads (Sit) interact with the actin filament cyclically (Huxley & Brown, 1967; Pepe, 1971). Harrington , 1969; 1967; Huxley, Crossbridges move out towards the actin filaments by an amount depending on the sarcomere length (Huxley & Brown, 1967). The hinge regions and S-2 part. enable myosin heads to move towards the actin filament. Thus, studies on the hinge region and S-2, as well as on the shape and configuration of the two heads, are important. X-ray analysis is useful for studies on muscle fibres, but electron microscopy with three-diment Abbreviations used: Sl, subfragment-l; S-2, rod part of heavy meromyosin; AC, actin; HMM, heavy meromyosin; TM, tropomyosin; DTNB light chain, the light chain that is dissociated from myosin with 5,5-dithiobis-(2. nitrobenzoate); LC2. light chain-2 (i.e. DTNB light chain); LCl, light chain-l (i.e. alkali light chain 1); LC3, light chain-3 (i.e. alkali light chain-2); LMM, light meromyosin; HMM/LMM hinge, rod part that divides myosin into HMM and LMM.
639 002%2836/88/230639
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II. Kajiyama
of dat’a on more filaments and careful handling of higher layer-lines (21 to 27). This made it possible to obt’ain details of the structure around the neck region of the myosin head and the region near the head/rod junction beyond the thin end of the large myosin head (G in this study). The position of the DTNH light chain proposed by Katayama & Wakabayashi (1981), Vibert & Craig (1982) and Seymour & O’Brien (1985) was confirmed. Characteristic regions of density besides that of the large head were observed for the first, time, and “bent” part was seen near the head/rod junction.
2. Materials and Methods (a) Preparation
of protein rigor complex
and
Actin was prepared by the method of Spudich & Watt, (1971) from rabbit skeletal muscle. Myosin was extracted in Guba&+raub (0.3 .M-K(‘1. 0.15 M-potassium phosphate (pH 6.4)) solution and purified by the procedure outlined by Kzent,-Gyorgyi (1951). Heavy meromyosin was obtained by digesting myosin with chymotrypsin (Yagi & Otani, 1974) in the presence of 1 mM-MgCl,, 0.6 M-EaCl? 10 ma-sodium phosphate buffer (pH 7.0) and 1 mM-dithiothreitol (Weeds & Taylor. 1975). Tropomyosin was extracted with 0.4 M-LiCl from acetone powder from which actin had been extracted, and was purified by 3 cycles of isoelectric precipitation and fractionation with 60 to 7076 saturated ammonium sulphate in the presence of 10 mM-p-merraptoethanol. The concentration of actin was determined by the method of Bradford (1976) and those of myosin, heavy meromyosin and tropomyosin were determined by the Biuret method. (b) Preparation
of the rigor complex
Ac-TM solution (0.48 to 0.96 mg actin/ml and 0.3 mg TM/ml in the presence of 50 mM-KC], 1 mM-MgCl, 10 mM-potassium phosphate (pH 7.5) and 1 mMdithiothreitol) was mixed with an equal volume of HMM solution (3.0 to 3.78 mg/ml in 10 mM-imidazole. HCl (pH 7.0). 50 mM-KC1 and 1 mnr-dithiothreitol). allowed to stand for 18 h at 4°C and then centrifuged at 36,000 revs/min for 2 h. The pellet used for electron microscopy (Figs 1 and 2(a)) was washed twice without suspension, and then resuspended in its initial volume of AC-TM buffer. The final preparation was obtained by diluting this solution with AC-TM buffer. Most 3-dimensional images were reconstructed using filaments prepared from the mixture without centrifugation (Fig. 2(b) and (c)). For electrophoresis, a portion of the sample was dialysed on a membrane filter against distilled water or AC-TM buffer in which sodium was substituted for potassium; it was then freeze-dried. (c) Preparation of specimen for electron microscopy
Carbon microgrids were used. They were obtained from plastic microgrids (Fukami & Adachi, 1965) by removal of the Triafol film after carbon deposition. The carbon microgrids were covered with a heavy deposit of carbon before use so that actual holes might be distinguished from pseudoholes.
The specimen solution was put’ on the carbon microgrid and allowed to enter the holes in the carbon film; it was
negatively st,ained with 1 q/o or 216 (w/v) uranyl acetate. Excess stain was removed with filter paper and the grid was dried under a tungsten lamp. Most of the specimens analysed werr (Lmbeddetl in stain sheets, which were reinforced by thin “unbroken” in an elect ran carbon backing before examination microscope (Craig et al., 1980; Taylor & Amos, 1981). (d) Electron
microscopy and anulyais of electron ‘micrographs
Specimens were examined with a .JEOL 100~(‘5 electron microscope at an accelerating voltage of 80 k\ and a magnification of about 50,000 x The electron microscope was operated with an anti-contamination device and objective aperture of 40 pm. Fuji FQ film was used. The magnification was calibrated assuming t,hat t,he axial distance of the left-handed generic helix of the F-a&in helix was l/59 A (1 A =O.l nm). The optical diffraction of filaments that were straight over a region of 4 arrowheads were examined. (e) Digitization
of the image
Densitometry of electron micrographs for computer analysis was done with a Joyce-Loebl microdensitometer (MDM-6) controlled by a PDP-1 l/44. An effective sampling interval of about 9 A and a slit size of 5 A x 5 .h were selected. These conditions were chosen t,o include images containing nearly 6 crossovers into an array size of 256 along the helix. Optical densities in the range 0.1 to 3.0 were digitized in 1024 grey levels. (r) Fourier
transform
The optical densit,y data were displayed on a line printer in 10 grey levels. Data were processed with a HITAC M-200/280 H computer at the Computer Centre of the IJniversity of Tokyo. The selection rule was determined wit,h reference to: (1) the spacing corresponding to the axial repeat distance in the left-handed generic helix of F-actin, which was assumed to be l/59 A; and (2) the true pitch of the F-actin filament, which varied depending on the selection rule. The actin helix is a left-handed helix of 6 t,urnx per true pitch of about 380 8, when the selection rule is I= -6nf 13m. In this case. both (I = 3. /I = -7) and (1= 3, n = 6) helices contribute t)o the 3rd layer-line. Tn t,he layer-line data studied here, the J-, and J6 terms could be separated depending upon their distances from the meridian. (g) Kejnement of thr position of the helix axis and averaging of the $laments
The shift of phase origin (s) and the tilt. a,ngle (w) of t,hr helix axis out of t’he plane normal to the direction of view the intraparticlr phase were refined by minimizing residual (&. defined by DeRosier & Moore (1970)). The layer-line data of .rn values of high order were examined in view of the characteristic features of Bessel function, that is J,(r) gives the maximum value at, about x = n + 1: The position of the nearest peak to the meridian in the Fourier transform was examined to determine whether it was larger than (n+ 1)/27cr,,,. Almost, all the peaks selected were found to be satisfactory.
Shape of the Myosin
The relative orientation and radial scaling factor of each image were refined: the values that minimized the interparticle phase residuals (P, defined by Wakabayashi
et al. (1975) or Rmin,defined by Amos & Klug (1975)) were procedure was done using layerlines with numbers of up to 23(1/33.5 A). selected. All the fitting
After all the 3-dimensional images, which were reconstructed from each side, had been put in identical positions, all the sets of extracted layer-line data were averaged (Wakabayashi et al., 1975) to show the common feature of Ac-TM-HMM images. Final Fourier synthesis was executed with and without layer-lines higher than the 21st (l/36.7 A). Two-sides averaged data for filament F (see Table 2) were analysed and reconstructed using 13 layer-lines (1=0 to 12 (1/29.5A)) including both (1=3, n=6) and (E=3, n = -7) peaks throughout the process. (h) Model reconstruction
Images were reconstructed using 10, 9, 4 and l-sides averaged data from the filaments whose selection rule was 1= - 13%+ 28m and using 2-sides averaged data from the filament whose selection rule was I = - 6n + 13m. A solid model of the rigor complex was built to the radius of 130 A with styrene foam, the thickness of which corresponded to 9 A. Transparent density maps were
made to the radius of 1708, with sections at 9 A intervals to show the continuation from the myosin head to the outer part. The cut-off densities in both the solid model and transparent density-maps were such that the volume of the AC-TM-Sl( +LC2) was 87% (shaded area in Fig. 5) of the expected volume. The theoretical volume was calculated, assuming that the molecular weight of a myosin head that retains LC2 is 186,000. The density of
protein (mass relative to that of a hydrogen atom) was assumed to be 0,8/A’ (Mikhailov & Vainshtein, 1971). The contour levels were graduated at constant intervals on a transparent sheet.
3. Results (a) Attachment of heavy meromyosin the thin jllament
to
Figures 1 and 2(a) are electron micrographs of suspensions of washed pellets from AC-TM-HMM mixtures containing small (20%) and large (200%) excess amounts of HMM, respectively. The former of three species: suspension (Fig. 1) consists apparently complete rigor complex, undecorated thin filaments, and intermediate ones (1 -sidedly contrasted images along a double-stranded helix, Fig. 1(c)). The latter suspension (Fig. 2(a)) consists of fully decorated thin filaments. This suggests that HMM binds to, or detaches from, the thin filaments co-operatively. A pellet of the latter suspension was used for three-dimensional image analysis. The cooperative tendency was more apparent in the AcTM-HMM complex than in the AC-TM-S1 complex (data not shown). (b) Whisker-like structure and density barb of the arrowhead structure
641
Head in the Rigor Complex
at the
Figure 2 shows typical rigor complexes obtained from the pellet (Fig. 2(a)) and the mixture (Fig. 2(b)
and (c)). The HMM used in this study contained DTNB light chains (LC2, 18,000 M,) on each head in addition to either the alkali light chain, LCl or LC3 (25,000 or 14,000 M,, respectively). It also retained S-2 rod. Electron microscopic images of AC-TM-HMM complexes (Figs 2(a), (b)? (c) and 4(f)) show a whisker-like structure (Katayama 6 Wakabayashi, 1981) in addition to the arrowhead structure. The arrowhead is a common feature of rigor complexes such as AC-Sl and AC-TM-Sl. The barb of the arrowhead is denser and is different from that of AC-TM-S1 (Fig. 2(d)), as described for the complex with the fragment from scallop myosin by Craig et al. (1980). In some cases, the whisker is packed against the filament giving a “closed” whisker pattern (Fig. 4(f)). It turns towards the arrowhead that is in the direction of the M-line, the centre of the sarcomere in situ. (c) Image analysis
of the AC-TM-HMM
complex
(i) Optical and computed transforms A total of 100 straight images were examined with an optical diffractometer. Six filaments that showed clear diffraction patterns were subjected to computer processing. Their enlarged views and the lineprinter outputs of their digitized images are shown in Figure 3. Five filaments showed a helical symmetry of 28 units per 13 turns and the other showed 13 units per 6 turns. The width of the box used in transformation was 340 A. The computed transforms consist of two groups of layer-lines (Fig. 4(e)). The first group shows a typical X-shaped pattern crossing over the meridian around l/54 A, which is a feature shared by AC-TM-Sl. The second one shows another X-shaped pattern that crosses over the meridian around l/l08 A reciprocal spacing (Fig. 4(b) and (d)). These meridional reflections (l/108 A) were not due to the spikes from the box used for transformation. Sometimes additional X-shaped patterns were seen around the 22nd (l/34.9 A) layer-line (Fig. 4(d)). Images were reconstructed with and without higher layer-lines (from the 21st to 27th). Four of six transforms also showed the 28th (l/27.5 A) meridional reflections. (ii) ReJinement of the position
of the helix axis and
the average of the Jilaments
Values of the intraparticle phase residual, &, were found to be satisfactory, together with the number of peaks examined are jisted in Table 1. The phase correlation of the peaks across the meridian on the higher layer-lines (1221) were carefully examined. Most peaks were satisfactory and these are contoured in Figure 4(d). Most of the processed images gave a reasonable interparticle phase residual (P value) after four rounds of fitting, as shown in Table 2, which indicates that the selection rules (l= - 6n + 13m and I= - 13n+ 28m) adopted for these electron micrographs were suitable. The P value (84.0) for which
642
II. h’ajiyama
Figure 1. Electron micrographs of the HMM and TM in small excess (20% and are seen. (c) Filaments with one-sidedly pattern can be seen. The bars represent
suspension of the washed pellet, from the AC-TM-HMM mixture thnt contained 50%, respectively). (a) and (b) Both undecorated and fully decorated filaments contrasted images along double-stranded helices in which the oblique banding 1000 A.
E is large, but the values of the set of parameters were clearly defined.
filament
(d) Three-dimensional
structure
the AC-TM-HMM
com$ex
of
The final images were obtained from lo- and O-sides averaged data using 26 layer-lines. At the cut-off level described in Materials and Methods, the course of density from the high-density moiety
(A + K+ D+E area) to the outer circumference (I, and I( areas) can be more or less followed as shown in Figure 5(a) and (d). The thin end (G) of the large head nearest to the head/rod junction can be seen. together with two extra densities F and f’. After the end of the large head (G), there are regions of low density on the bent line -1-J (or - I-K). The radius of the whole AC-TM-S1 (+1X2) complex is IIOA.
Shape of the Myosin Head in the Rigor Complex
643
Figure 2. Electron micrographs of typical rigor complexes. (a) Suspension of washed pellet from an AC-TM-HMM mixture. mixture containing TM and HMM in large excess (140% and 200%, respectively). (b) and (c) AC-TM-HMM (d) AC-TM-S1 complex for comparison. Arrowheads indicate whiskers. Double arrowheads indicate the density at the barb of the arrowhead structure. The bar represents 1000 A.
(e) Sl moiety and the neck region of the myosin head Figure 5(a) shows the end-on view reconstructed image, where the two-start F-actin is untwisted to become parallel.
of the helix of A high-
density region including the A, D and E areas (at radii of about 20, 40 to 70 and 80 A, respectively) looks like an Archimedian spiral. The view also shows three circles consisting of E-G-I (r= lOO+lO A), L-(J)-K (130+10 A) and the
H. Kajiyanaa
644
(a
(b)
Id)
(e)
Figure 3. Enlarged images of single filaments which were used in the 3-dimensional reconst,ruction. Their digitized images with boxed areas are beside them. The selection rules are I = - 13n + 28m and I= - 6%+ 13m for filaments (a) to (e) and (f), respectively. The bar represents 500 A. Single arrowheads indicate whiskers. Double arrowheads indicate the density at the barb of the arrowhead structure. The density at the barb is seen between 60 and 144 A from filament axis.
Shape of the Myosin
Head in the Rigor Complex
645
-l/59 -l/108
lilO8 ,v 59
(a)
lb)
(d)
(0,28)---
-
(e) Figure 4. Transforms and the whisker-like structure. (a) Filament shown in Fig. 3(a). (b) Optical diffraction pattern of (a). (c) Digitized image. (d) Computed Fourier transform of the boxed area in the image (c). (e) Diagram of transforms from the filaments with a selection rule of I= -13n+%n. (f) Closed whisker pattern. Left, an electron micrograph; right, drawing of the micrograph. The bar represents 500 A. edge of the density region (r=170A), which is not labelled. At these distances (x) from the filament axis in electron micrographs (Fig. 3), one can see the centre of the dense barb (100 A
whisker (x=li'OA).
(x= 135 A) and the frame of the box The first circle explains the densities
around and after the neck region of the myosin head (Fig. 5(a)). Around the first circle (r= lOOk 10 A), the G area shows the highest density and looks globular (Fig. 6(b)). It has been assigned to the part of the DTNB light chain (Katayama & Wakabayashi, 1981; Seymour & O’Brien, 1985) or the regulatory
H. Kajiyama
646
Table 1 Intraparticle
Filament A 8 c D E Ft t Filament
phase correlation
Number of peaks examined
Q (deg.1
x (‘Q
UJ (deg.)
6 4 5 5 5 4
35 36 16 31 4 9
- 10.4 -4.0 0.3 0.8 0.0 0.0
-84 8.0 4.4 8-O 04 6.0
with a selection rule of 1= - 6n + 13n. Notation
light chain (Vibert & Craig, 1982). Above this, the area F is observed as in the previous model of t,he AC-HMM complex (Katayama & Wakabayashi, 1981). In this study, a new thin area, f, which runs down from the G area, was observed inside the Sl spiral (Figs 5(a) and 6(c)). The views of the superimposed cylindrical sections (Fig. 5(b)) and the solid model (Fig. 6(b)) show that the neck region of the myosin head includes these two protrusions (or thin areas), F and f, above and below G, respectively. Thus, the curved contour length of the large myosin head in the rigor complex is roughly estimated to be 180 to 190 A, when measured along the centre of the density course with the view of the untwisted horizontal (Fig. 5(a)) and cylindrical sections (Fig. 5(b), see also Fig. 7(b)), assuming that the large head begins at the position marked with the double arrowhead (Fig. 5(a), r=20 A) and ends at the end of the G area marked with the single arrowhead (Fig. 5(a)). The longest chord of the large head, a straight line from the edge of I) to the end of G, measured with the solid model (Fig. 6(d), see also Fig. 7(b)) was about 150 to 160 A long. In this study, the thin part of the large head appeared
is as for Fig. 3.
to be about 20 A wide, but area G was 35 A x 27 A wide. (f) The bent part near the head/rod juraction
4. Discussion and computed transforms expected for the AC-TM-HMM complex
(a) Optical
The AC-TM-HMM complex should give information about the spatial relationship between the two
phase correlation
Filament
~‘nli” (deg.)
A0 (deg.)
A near far B near far C near far D near7 far E near far FJ near far
59.6 57.8 43.0 57.6 61.1 64.6 65.6 67.3 84.0 70.2 52.7 62-3
196.5 191.1 27.0 28.0 6.8 15.5 195.8 122.7 120.0 122.1 04 10.0
the
As shown in Figures 5(a) and (b) and 6(c), a density line from the G area goes down, bends back at a radius of 110 A and goes up to the J area forming a “short bent line” G-I-J (or G-I-K) that is 120 to 150 A long in the top view (Fig. 5(a)). Figure 5(a) shows that the outer peaks nearest to the I area are K (r= 135A) and J-L (r=125 to 130 A). J goes up to L (Fig. 6 (c)), and L goes down to the outer circle (r= 165 A) as indicated by a curved line with an arrowhead (Fig. 5(d)), whereas K goes up along the circle at a radius of 135 A (Fig. 5(c)). Peaks K and J-L have higher density than G and I.
Table 2 Interparticle
and
part beyond the bent part (90 A < r < 135 ‘4)
Radial scaling factor 30.9 29.1 14.0 8.6 13.3 17.5 31.3 0.0 0.0 04 1.o 3.0
140 0.95 I.00 0.75 0.98 I .O3 1.14 I 40 1.21 1.oo 140 0.89
t The reconstructed image of this side is fuzzy and was not included in the g-sides averaged data. The P-values tend to increase when the full data (for filament E or D) were fitted to the averaged data that contain the dummy data. The more layer-lines were used (from 1=22 to 27), the larger the P-values became (data not shown). 1 Filament with a selection rule of 1= - 6n + 13m.
Shape of the Myosin Head in the Rigor Complex
647
Figure 5. Horizontal and cylindrical sections (piled up at 9 a intervals) of the image reconstructed from g-sides averaged data using 26 layer-lines. Areas D and E indicate the main body of SI and its tail, respectively. The shaded area corresponds to the cut-off density of the solid model (see Materials and Methods). The radius of the circle is 130 A. This radius is equal to that of the cylinder that contains the solid model. (a) End-on view of untwisted horizontal sections. A “bent chain” (C&I-J) is shown as a broken line. L continues to the outer circumference as indicated by a curved line. Scales are graduated at 10 A intervals. The double arrowhead indicates the beginning of the large head. The single arrowhead indicates the end of it. (b) Superimposed cylindrical sections (63 A
heads
of
meridional for
the
the
HMM
reflections first
time.
In this study, l/110 L%were found
molecule.
around Some
of
these
meridional
reflections had a clear X-shaped pattern around them, indicating the presence of another helical symmetry besides that’ of the actin helix (Fig. 4(b)
and (d) to (e)). There are layer-lines with spacing corresponding to a structure of doubled true-pitch by subsidiary repeat (380 X 4 A), accompanied peaks; these were not included in the image analysis. This indicates that the units, which are arranged at about 108 A (54 x 2 8) vertical spacing,
648
H. Kajiyama
Figure 6. Solid model of the A+TM-HMM complex reconstructed from g-sides averaged data with 26 layer-lines. The thin sheets of styrene foam on the bent course corresponds to the unshaded area (99% of the expected volume) in Fig. 5. (a) End-on view. Some areas on course G-I are 18 to 45 if wide in vertical direction. (b), (c) and (d) Oblique views showing areas G, F, f and the short-bent chain that follows area G. The bars represent 100 A. A short-bent chain is indicated by a broken line. Labels are the same as for Fig. 5.
Shape of the Myosin
Head in the Rigor Complex
649
(b)
(a)
j* ,___-_--
id)
(e)
Figure 7. (a) Schematic drawing of the HMM part of the reconstructed image in the radial projectSion (see legend to Fig. 5(b)). The HMM spiral is uncoiled in a plane. F and f are shown by dotted areas. F is outside, and f is inside the neck region. (b) Hypothetical interpretation of (a). It was assumed that the end of each large head is identical. Two neck regions of one HMM join up to somewhere on the bent line G-I-J-L (or G-I-K). The curved contour and the longest chord of the large head are indicated by a curved line and a straight line, respectively. (c) Drawing of the HMM molecule. The dotted area indicates the contribution of LCZ. (d) and (e) Hypothetical models of binding of the HMM/LMM hinge to another HMM molecule. The HMM/LMM hinge is indicated by a broken line. The HMM/LMM hinge binds to (d) the lower part (near the head/rod junction) of the upper HMM or to (e) the middle of t’he upper S-2. Lengths estimat,ed by Sutoh et al. (1978) are shown.
sometimes make a helix with a pitch of 380 x 4 A (Z= -6n+ 13m). Figure 8(a) explains this phenomenon. When the selection rule for the actin helix is I= -6n+ 13m (Fig. 8(a)), the true repeat corresponds to the length of one arrowhead. Actin units are arranged with 54 A vertical spacing along one strand of double helix. When HMM binds to the actin filament, adjacent actin molecules are decorated with leading (open circles) and trailing (filled circles) heads of one HMM molecule: HMM units are arranged with 54 x 2= 108 A vertical spacing, making a new true repeat that corresponds to four arrowhead repeats. Here, filament F whose selection rule was 1= - 6n+ 13m did not provide layer-lines to show the correct selection rule. HMM binding to the act’in filament whose selection rule is I= - 13n + 28m (Fig. 8(b)) will also
change the vertical spacing of the repeating unit from 54 to 108 A, but will leave the true repeat as two arrowhead repeats. In this study, layer-lines from I=0 to 27 (except E= 1 and 14) were used (Fig. 9). The layer-line data include peaks that arose from the pairing of the two heads, which shows a new selection rule (see Fig. 8(b)). However, the selection rule used here (2= - 13n + 28m) assumes that the heads are not paired. So, the P values in this study are good to t,he extent that the image reconstructed with 28/13 symmetry and put in an identical position was shifted by an actin monomer to obtain these values. (b) Neck region of the myosh head in the rigor complex image (Fig. 5(a)) shows The reconstructed apparent continuation from the Sl moiety to the G
H. MO _.--
h*ajtyanrcl
_
e___._-c_ -c__
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I----=l . . . . . . . . . . . . . . .._
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(a) Figure 8.
lb) lattice (,I’ (a) 13 IIIII~S 11rr 6-Iurn heIt\ per l$tutcc h&s. Open and fikd k&s and trailing head3 of HMM. wyectivel:. indicatrs thr one-stat? helix getterated by
Surt’ace
artd (b) 28 unttx jndlcatc leading The broken line
area. and extra prutrusions F and f III thr neck region (Figs Z(b) and 6(b). see ak.o Fig. Y(B)). The curved contnur length of the large head (180 to 1% 1%) A: Fig. 7(h)) . . oonsisknt. with the siztt of t.hr head it\ an isolated ntyosin tnolwule (front mhhit skeletal mus&) detrrmiwd bv the shadow-csasting method (Slavtrr e; Lowry. 1967: E:l\iot.t & Offw. 1978) by this method. thP heat1 mrusures 1.90 A in
. ..‘._ I-t.13,
. .
--
AXape of the Myosin Head in the Rigor Complex length and about 45 A at its widest part and tapers to about 20 A at, the thin end, which is close to the head/tail junction. If one considers that the longest chord of the large head (150 to 160 A) in the rigor state corresponds to the head observed in isolated myosin, it would seem to leave an adequate length of neck on the bent line G-I-J (or G-I-K). Anyway, each neck region of one HMM may continue t’o somewhere on the bent line -1-J-L (or -I-K), because some areas at a radius of 90 A are 18 to 45 A wide vertically (Figs 5(b) and 6(a)). Whether the end of each large head is identical or not cannot be determined in this study. The present results do not support the suggestion of the previous studies that the neck region of each head continues from the G area upward or downward depending upon its relative position (Katayama & Wakabayashi, 1981; Seymour & O’Brien? 1985) for the following reasons. Jn the image from a single filament F (Fig. 3(f)), which clearly shows six layer-lines of the actin helical symmetry, a continuation from the G area in the azimuthal direction, and the F area, but no lower protrusion of the G area are visible. Instead, final and images show a “short bent chain (-I-J-L)” areas f and/or F at this radius. Images from single filaments, C and E (Table 1) also showed f. (c) Candidates for the DTNB light chain (areas F and/or f) LC2 is assumed to be long and to contain a globular part, (Alexis & Gratzer, 1978). The regulatory light chain isolated from scallop myosin was concluded to be 100 A long (Stafford & SzentGyorgyi, 1978). In this study, areas F and f are candidates for LC2 together with area G, because they are found in the image of the rigor complex with HMM but not with Sl, in which LC2 on the myosin head is lost (Wakabayashi & Toyoshima, 1981; Taylor & Amos, 1981; Toyoshima & Wakabayashi, 1985a,b). Their assignment to LC2 fits in with other available findings that the regulatory light chain contributes to the barbs on filaments decorated with Sl and HMM (Craig et al., 1980; Vibert & Craig, 1982), and to the visibility in the neck region of the head in the isolated myosin molecule (Flicker et al.. 1983). In this study, certain HMM preparations contain less 18,000 Mr peptide than 14,000 M, peptide when analysed by SDS/polyacrylamide gel electrophoresis (Laemmli, 1970), suggesting that the partially degraded DTNB light chain (LC2) comigrated with LC3: LC2 of HMM is known to be rapidly degraded with chymotrypsin before the SljS-2 junction is cleaved under physiological conditions without actin and/or divalent cation (Weeds & Pope, 1977; Oda et al., 1980). So, area f is not the result from t’he inclusion of inadequate layer-line data. Thus, it is concluded that at least one extra thin area (either or both of F and f) contributes to the neck region.
651
(d) The bent part near the head/rod junction and areas K and L (90 A < r < 1,3.5‘4) Three peaks I, J-L and K were observed in both of two final averaged images. The G-J-L bent chain and peak K are more apparent in a g-sides averaged image, omitting one fuzzy image, than in a IO-sides one. Higher layer-lines also increased the density at the bent chain and area L. Since this shape of the Sl moiety (Fig. 5(a)) is in agreement with the previous work, this cut-off seems to be reasonable. So, an average of about 160 HMM molecules appearing as a short bent chain (GI-J) of about 120 to 150 A long (Figs 5(a), 6(a) and (c), see also Fig. 7(b)) may be the continuation from the end of the large head. (e) The basal part of the whksker and areas K and L Though S2 whiskers in Figures 2 and 3 appear to be lying flat on the surface of the grid, the centre of a dense barb or dense turning point is seen at a distance between 99.5 and 123.8 A from the filament axis. Each turning point is 27 to 60 A wide, suggesting that it contributes to three peaks I, J-L, and K (r=lOO, 125 and 135 A) in the reconstructed image (Fig. 5(a)). The HMM/LMM hinge of the myosin molecule is known somehow to lock the cross-bridge on the surface of the thick filament in the presence of divalent ion, Ca2+ and/or Mg2+ (Mendelson & Cheung, 1976; Sutoh & Harrington, 1977; Sutoh et al., 1978; Borejdo & Werber, 1982; Ueno et al., 1983). The HMM/LMM hinge may bind to the other S-2 rod, directly near the head/rod junction (see Fig. 7(d)) or in the middle part (see Fig. 7(e)). If this kind of binding occurs also in the rigor complex, the binding of the HMM/LMM hinge with the upper molecule shown in Figure 7(d) and (e) may cause the “closed” whisker seen in elect.ron micrographs (Fig. 4(f)); the whisker may open by turning at the bent part.. However, areas I, J: L and K cannot yet be assigned to particular parts of HMM. Figure 7(c) shows a schematic drawing of the HMM molecule proposed from this work. The real head/rod junction, where two single polypeptide chains join to a double helical rope. may differ from the end of the large myosin head (end of G in Fig. 7(a) and (b)). A proteolytic site that produces chymotryptic Sl may be within the dotted area, because LC2 effectively protects myosin from chymotryptic proteolysis at the Sl/S2 junction in 0.12 M-NaC1 without divalent cation (Weeds & Pope, 1977; Oda et al., 1980). A papain Sl site would be somewhere between the end of the large myosin head and the head/rod junction, because an 81 preparation that retains LC2 or the regulatory light chain has been reported (Werber rt al., 1972; Margossian bz Lowey, 1973; Margossian et al., 1975; Bagshow, 1977; Kuwayama & Yagi. 1977, 1980; Yamamoto & Sekine, 1980). I could not determine the exact position of the head/rod junction in this study.
H. Kaji yama -
652
Tn future, information on the “pairing” features may be obtained by carrying out image analysis using the extracted peaks that belong to a correct selection rule and using the filament with a closed whisker (Fig. 4(f)), which gave the clearest X-shaped pattern that crosses over the l/108 a meridian (data not shown). Image analysis of the one-sidedly contrasted specimen shown in Figure 1 and of the data for the “pairing” features should be examined. I am grateful to Professor T. Wakabayashi for suggesting analysis of the AC-TM-HMM complex and t,o Dr. Y. Shirakihara for examining the program used. This work was supported by Grants-in-Aid for Special Project Research and Grants-in-Aid for Scientific Research to T.W. from the Ministry of Education. Science and Culture of ,Japan.
Kuwayama. H. di Yagi, K. (1980). J. Hiocltem. 87. 16031607. Laemmli. I-. K. (1970). ,V&~rp (London). 227. 68W 685. Margossian, S. S. & Lowry. S. (1973). d. ~llo/. Biol. 74. 301-31 I. Margossian. S. S., Lowry, S. & Barshop. 13. (1975). X&trr (London),
.J. Mol. Riol. 50, 279-295. Oriol-Audit. (‘. cyi Rt~islrr. E. (1980). s., Biochmistry, 19, 5614-5618. Pepe. F. A. (1967). .I. Mol. J&Z. 27, 227 -%ci. Seymour. ,J. & O’Brien, E. ,J. (1985). .J. :Mus~‘. Km. (‘c/l Motil. 6. 725 756.
Oda.
Slagter. H. S. & Lowry. S. (1965). i’roc.. i$‘trf. .3&. I..A.A. 58. 161 I-- 1618. Staflord.
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Mendelson. R. A. & Cheung. I’. (1976). Scipncr. 194. l90192. Mikhailov. A. M. & Vainshtein. 1~. K. (19il). Soclirt f’hiys. Prystalloyr. 16. 4~8~~436. Moore. I’. B., Huxley. H. E. & Uetlosirr. I). ,I. (19iO).
\1’.
SC~..
F.
K- Hzent-($orgyi. .\. (:. (I!YiX). 17. 607-614. Sutoh. K. & Harringt,on. iv. F. (1977). Hiochenlistry. 16. 2441 2449. Sutoh. K.. S&oh, K.. Karr. T. Hr Harriugton. \V. E‘. (l!+i8). .J. Mol. H/i/. 126. 1 “L’. Sent-Cyorgyi. A. (1951). (‘hrmist,y of Nuwultr~ ~‘ontraciion. 2nd edit.. Acatlemic~ Press. Inc.. iYe\{ 1.ark. Riochrnri&y.
17. Alexis, M. N. & Gratzer. W. B. (1978). fkJchrvn&ry, 2319-2325. Amos. L. A. & Klug. A. (1975). J. Mol. Biol. 99. 51-64. Amos, I,. A., Huxley, H. E., Holmes. K. C.. Goody. R. S. 299. 467& Taylor. K. A. (1982). AGzturr (London). 469.
Bagshow, C. R. (1977). Biochemistry. 16, 59-67. Borejdo. ,J. & Werber, M. M. (1982). J?iochsmi.stry. 21. 549-555. Bradford, M. M. (1976). Anal. Hiochum. 72, 21X-d.54. Craig, R., Szent-Gyorgyi, A. G., Beese, I,., Flicker, 1’. F.. Vibert, I’. & Cohen. (“. (1980). J. Mol. Hiol. 140. 3556. DeRosier. I). ,J. & Moore. I’. B. (1970). .J. Nol. Hiol. 52, 355-369.
Elliott. A. & Offer. (:. (197X). .J. Mol. Hiol. 123. 505~ 515. Flicker, P. F.. Walliman. T. & Vihert. I’. (1983). J. &fol. Viol. 169, 723-741. Fukami, A. & Adachi, K. (1965). .J. Electron A’iicrosc. 14. 112-118. Harrington, b’. F. (1971). hoc. ,Vat. Acad. Sci.. lT.#.A. 68, 685-689. Huxley. H. E. (1969). Rciancr, 164. 1356-1366. Huxley. H. E. & Brown. W’. (1967). J. Mol. Hiol. 30, 383 438. Katayama, E. & Wakabayashi. T. (1981). J. Hiorhrm. 90. 703-714. Kuwayama, H. &, Magi. K. (1977). .1. ilfpchunochem. (‘ell Motil. 4. 255-274.
Edited
Toyoshima. (‘. & 1Vakabayashi. ‘I’. (I!)H5o ), .I. flioc,hr,/l. 97. 219 243. Topshima, (‘. P: 1Vakabayashi. T. (19X.56). J. I~i~c~hr/~. 97 “45 %7. ITeno. i-1.. &$rrs. II:. M. $ Harringtorl. \V. 17. (1983). .J. Mol. Viol. 168. 207-22H. Vibert). I’. ei Craig. R. (1982). J. Mol. Jliol. 157. dS!j 31!). Wakabayashi. T. CCToyoshima. (‘. (1981). .I. BiochPm. 90. 683 701. \Vakabayashi. ‘I’.. Huxley. H. E.. Amos. I,. .I. & Klug. ,\ (1!175). .J. Mol. Hiol. 93. 177-497. ~\‘rcds. A. G. Br Pope. B. (1977). .I. No/. Uiol. 111. I”9 157.
LVrrbrr.
%I. Sl.. (:aflin,
S. I,. & Oplatka. .\ (IN:!). 1, RI -!G. Magi. K. & Otjani. F. (1974). -1. Hiochrm 76. Xi:{ 3i:3. Yamamoto. K. & Srkine. T. (1980). ,1. Hiochpn. 87. 219 226. .J. :Wr~hanochum.
by S. Urennrr
Ml
Afotil.
Shape of the Myosin Head in the Rigor Complex Three-dimensional Image Reconstruction of the ActinTropomyosin-Heavy Meromyosin Complex Hitomi Kajiyama Department of Physics, Faculty of Science The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113, Japan (Received 14 September 1986, and in revised form, 22 June 1988) The structure of the actin-tropomyosin-heavy meromyosin rigor complex was studied by image analysis of electron micrographs. The arrowhead of the rigor complex has a whiskerlike structure with a dense turning point at the “barb” of the arrowhead. The neck region of the myosin head in the reconstructed three-dimensional image is present in the area corresponding to the dense point. It is concluded that at least one extra-thin area contributes to the neck region, and that the two heads in the heavy meromyosin molecule join a double helical rope beyond the end of the large head (G in this study). (This is different from previous interpretations.) It is also concluded that the heavy meromyosin has a short bent part near the head/rod junction in the rigor complex.
sional reconstruction is better for studies on the molecular morphology of a single filament. Moore et al. (1970) first reconstructed the threedimensional image of the thin filament decorated with myosin Sl. Since then, improvements in electron microscopy (low dose imaging) and the use of other specimens (chymotryptic Sl from vertebrate muscle and Sl from scallop myosin), have resulted in more detailed information on the structure (Wakabayashi & Toyoshima, 1981; Taylor & Amos, 1981; Amos et al., 1982; Vibert & Craig, 1982; Toyoshima & Wakabayashi, 1985a,b). However, it is important to study the structure of the decorated thin filament in preparations in which myosin heads are as intact as possible. The three-dimensional structure of the AC-HMM complex in the rigor state has been reconstructed by Katayama & Wakabayashi (1981) and Seymour & O’Brien (1985). Although it was found that the Ac-HMM complex had regions (F and G) in addition to those of AC-Sl, the reconstructed images showed only a part of the HMM. However, one of these studies (Katayama & Wakabayashi. 1981) used only a few short filaments that were put on a carbon microgrid and the other (Seymour & O’Brien, 1985) used only a single filament on a carbon-coated copper grid. To determine the overall structure, I examined the structure of AC-TM-HMM with an improved method: the use of thin filaments that contained tropomyosin to obtain straight filaments, averaging
1. Introduction Muscle is composed of two kinds of filament, thick filaments that consist mainly of two-headed myosin molecules (480,000 M,) and thin filaments that’ consist of actin, tropomyosin and troponin (T+I+C). In the rigor state, the myosin binds to the actin filament at a ratio of one myosin head to one actin monomer. In the force-generating, i.e. activated, state, myosin heads (Sit) interact with the actin filament cyclically (Huxley & Brown, 1967; Pepe, 1971). Harrington , 1969; 1967; Huxley, Crossbridges move out towards the actin filaments by an amount depending on the sarcomere length (Huxley & Brown, 1967). The hinge regions and S-2 part. enable myosin heads to move towards the actin filament. Thus, studies on the hinge region and S-2, as well as on the shape and configuration of the two heads, are important. X-ray analysis is useful for studies on muscle fibres, but electron microscopy with three-diment Abbreviations used: Sl, subfragment-l; S-2, rod part of heavy meromyosin; AC, actin; HMM, heavy meromyosin; TM, tropomyosin; DTNB light chain, the light chain that is dissociated from myosin with 5,5-dithiobis-(2. nitrobenzoate); LC2. light chain-2 (i.e. DTNB light chain); LCl, light chain-l (i.e. alkali light chain 1); LC3, light chain-3 (i.e. alkali light chain-2); LMM, light meromyosin; HMM/LMM hinge, rod part that divides myosin into HMM and LMM.
639 002%2836/88/230639
14 $03.00/0
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1988
Academic Press Limited
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II. Kajiyama
of dat’a on more filaments and careful handling of higher layer-lines (21 to 27). This made it possible to obt’ain details of the structure around the neck region of the myosin head and the region near the head/rod junction beyond the thin end of the large myosin head (G in this study). The position of the DTNH light chain proposed by Katayama & Wakabayashi (1981), Vibert & Craig (1982) and Seymour & O’Brien (1985) was confirmed. Characteristic regions of density besides that of the large head were observed for the first, time, and “bent” part was seen near the head/rod junction.
2. Materials and Methods (a) Preparation
of protein rigor complex
and
Actin was prepared by the method of Spudich & Watt, (1971) from rabbit skeletal muscle. Myosin was extracted in Guba&+raub (0.3 .M-K(‘1. 0.15 M-potassium phosphate (pH 6.4)) solution and purified by the procedure outlined by Kzent,-Gyorgyi (1951). Heavy meromyosin was obtained by digesting myosin with chymotrypsin (Yagi & Otani, 1974) in the presence of 1 mM-MgCl,, 0.6 M-EaCl? 10 ma-sodium phosphate buffer (pH 7.0) and 1 mM-dithiothreitol (Weeds & Taylor. 1975). Tropomyosin was extracted with 0.4 M-LiCl from acetone powder from which actin had been extracted, and was purified by 3 cycles of isoelectric precipitation and fractionation with 60 to 7076 saturated ammonium sulphate in the presence of 10 mM-p-merraptoethanol. The concentration of actin was determined by the method of Bradford (1976) and those of myosin, heavy meromyosin and tropomyosin were determined by the Biuret method. (b) Preparation
of the rigor complex
Ac-TM solution (0.48 to 0.96 mg actin/ml and 0.3 mg TM/ml in the presence of 50 mM-KC], 1 mM-MgCl, 10 mM-potassium phosphate (pH 7.5) and 1 mMdithiothreitol) was mixed with an equal volume of HMM solution (3.0 to 3.78 mg/ml in 10 mM-imidazole. HCl (pH 7.0). 50 mM-KC1 and 1 mnr-dithiothreitol). allowed to stand for 18 h at 4°C and then centrifuged at 36,000 revs/min for 2 h. The pellet used for electron microscopy (Figs 1 and 2(a)) was washed twice without suspension, and then resuspended in its initial volume of AC-TM buffer. The final preparation was obtained by diluting this solution with AC-TM buffer. Most 3-dimensional images were reconstructed using filaments prepared from the mixture without centrifugation (Fig. 2(b) and (c)). For electrophoresis, a portion of the sample was dialysed on a membrane filter against distilled water or AC-TM buffer in which sodium was substituted for potassium; it was then freeze-dried. (c) Preparation of specimen for electron microscopy
Carbon microgrids were used. They were obtained from plastic microgrids (Fukami & Adachi, 1965) by removal of the Triafol film after carbon deposition. The carbon microgrids were covered with a heavy deposit of carbon before use so that actual holes might be distinguished from pseudoholes.
The specimen solution was put’ on the carbon microgrid and allowed to enter the holes in the carbon film; it was
negatively st,ained with 1 q/o or 216 (w/v) uranyl acetate. Excess stain was removed with filter paper and the grid was dried under a tungsten lamp. Most of the specimens analysed werr (Lmbeddetl in stain sheets, which were reinforced by thin “unbroken” in an elect ran carbon backing before examination microscope (Craig et al., 1980; Taylor & Amos, 1981). (d) Electron
microscopy and anulyais of electron ‘micrographs
Specimens were examined with a .JEOL 100~(‘5 electron microscope at an accelerating voltage of 80 k\ and a magnification of about 50,000 x The electron microscope was operated with an anti-contamination device and objective aperture of 40 pm. Fuji FQ film was used. The magnification was calibrated assuming t,hat t,he axial distance of the left-handed generic helix of the F-a&in helix was l/59 A (1 A =O.l nm). The optical diffraction of filaments that were straight over a region of 4 arrowheads were examined. (e) Digitization
of the image
Densitometry of electron micrographs for computer analysis was done with a Joyce-Loebl microdensitometer (MDM-6) controlled by a PDP-1 l/44. An effective sampling interval of about 9 A and a slit size of 5 A x 5 .h were selected. These conditions were chosen t,o include images containing nearly 6 crossovers into an array size of 256 along the helix. Optical densities in the range 0.1 to 3.0 were digitized in 1024 grey levels. (r) Fourier
transform
The optical densit,y data were displayed on a line printer in 10 grey levels. Data were processed with a HITAC M-200/280 H computer at the Computer Centre of the IJniversity of Tokyo. The selection rule was determined wit,h reference to: (1) the spacing corresponding to the axial repeat distance in the left-handed generic helix of F-actin, which was assumed to be l/59 A; and (2) the true pitch of the F-actin filament, which varied depending on the selection rule. The actin helix is a left-handed helix of 6 t,urnx per true pitch of about 380 8, when the selection rule is I= -6nf 13m. In this case. both (I = 3. /I = -7) and (1= 3, n = 6) helices contribute t)o the 3rd layer-line. Tn t,he layer-line data studied here, the J-, and J6 terms could be separated depending upon their distances from the meridian. (g) Kejnement of thr position of the helix axis and averaging of the $laments
The shift of phase origin (s) and the tilt. a,ngle (w) of t,hr helix axis out of t’he plane normal to the direction of view the intraparticlr phase were refined by minimizing residual (&. defined by DeRosier & Moore (1970)). The layer-line data of .rn values of high order were examined in view of the characteristic features of Bessel function, that is J,(r) gives the maximum value at, about x = n + 1: The position of the nearest peak to the meridian in the Fourier transform was examined to determine whether it was larger than (n+ 1)/27cr,,,. Almost, all the peaks selected were found to be satisfactory.
Shape of the Myosin
The relative orientation and radial scaling factor of each image were refined: the values that minimized the interparticle phase residuals (P, defined by Wakabayashi
et al. (1975) or Rmin,defined by Amos & Klug (1975)) were procedure was done using layerlines with numbers of up to 23(1/33.5 A). selected. All the fitting
After all the 3-dimensional images, which were reconstructed from each side, had been put in identical positions, all the sets of extracted layer-line data were averaged (Wakabayashi et al., 1975) to show the common feature of Ac-TM-HMM images. Final Fourier synthesis was executed with and without layer-lines higher than the 21st (l/36.7 A). Two-sides averaged data for filament F (see Table 2) were analysed and reconstructed using 13 layer-lines (1=0 to 12 (1/29.5A)) including both (1=3, n=6) and (E=3, n = -7) peaks throughout the process. (h) Model reconstruction
Images were reconstructed using 10, 9, 4 and l-sides averaged data from the filaments whose selection rule was 1= - 13%+ 28m and using 2-sides averaged data from the filament whose selection rule was I = - 6n + 13m. A solid model of the rigor complex was built to the radius of 130 A with styrene foam, the thickness of which corresponded to 9 A. Transparent density maps were
made to the radius of 1708, with sections at 9 A intervals to show the continuation from the myosin head to the outer part. The cut-off densities in both the solid model and transparent density-maps were such that the volume of the AC-TM-Sl( +LC2) was 87% (shaded area in Fig. 5) of the expected volume. The theoretical volume was calculated, assuming that the molecular weight of a myosin head that retains LC2 is 186,000. The density of
protein (mass relative to that of a hydrogen atom) was assumed to be 0,8/A’ (Mikhailov & Vainshtein, 1971). The contour levels were graduated at constant intervals on a transparent sheet.
3. Results (a) Attachment of heavy meromyosin the thin jllament
to
Figures 1 and 2(a) are electron micrographs of suspensions of washed pellets from AC-TM-HMM mixtures containing small (20%) and large (200%) excess amounts of HMM, respectively. The former of three species: suspension (Fig. 1) consists apparently complete rigor complex, undecorated thin filaments, and intermediate ones (1 -sidedly contrasted images along a double-stranded helix, Fig. 1(c)). The latter suspension (Fig. 2(a)) consists of fully decorated thin filaments. This suggests that HMM binds to, or detaches from, the thin filaments co-operatively. A pellet of the latter suspension was used for three-dimensional image analysis. The cooperative tendency was more apparent in the AcTM-HMM complex than in the AC-TM-S1 complex (data not shown). (b) Whisker-like structure and density barb of the arrowhead structure
641
Head in the Rigor Complex
at the
Figure 2 shows typical rigor complexes obtained from the pellet (Fig. 2(a)) and the mixture (Fig. 2(b)
and (c)). The HMM used in this study contained DTNB light chains (LC2, 18,000 M,) on each head in addition to either the alkali light chain, LCl or LC3 (25,000 or 14,000 M,, respectively). It also retained S-2 rod. Electron microscopic images of AC-TM-HMM complexes (Figs 2(a), (b)? (c) and 4(f)) show a whisker-like structure (Katayama 6 Wakabayashi, 1981) in addition to the arrowhead structure. The arrowhead is a common feature of rigor complexes such as AC-Sl and AC-TM-Sl. The barb of the arrowhead is denser and is different from that of AC-TM-S1 (Fig. 2(d)), as described for the complex with the fragment from scallop myosin by Craig et al. (1980). In some cases, the whisker is packed against the filament giving a “closed” whisker pattern (Fig. 4(f)). It turns towards the arrowhead that is in the direction of the M-line, the centre of the sarcomere in situ. (c) Image analysis
of the AC-TM-HMM
complex
(i) Optical and computed transforms A total of 100 straight images were examined with an optical diffractometer. Six filaments that showed clear diffraction patterns were subjected to computer processing. Their enlarged views and the lineprinter outputs of their digitized images are shown in Figure 3. Five filaments showed a helical symmetry of 28 units per 13 turns and the other showed 13 units per 6 turns. The width of the box used in transformation was 340 A. The computed transforms consist of two groups of layer-lines (Fig. 4(e)). The first group shows a typical X-shaped pattern crossing over the meridian around l/54 A, which is a feature shared by AC-TM-Sl. The second one shows another X-shaped pattern that crosses over the meridian around l/l08 A reciprocal spacing (Fig. 4(b) and (d)). These meridional reflections (l/108 A) were not due to the spikes from the box used for transformation. Sometimes additional X-shaped patterns were seen around the 22nd (l/34.9 A) layer-line (Fig. 4(d)). Images were reconstructed with and without higher layer-lines (from the 21st to 27th). Four of six transforms also showed the 28th (l/27.5 A) meridional reflections. (ii) ReJinement of the position
of the helix axis and
the average of the Jilaments
Values of the intraparticle phase residual, &, were found to be satisfactory, together with the number of peaks examined are jisted in Table 1. The phase correlation of the peaks across the meridian on the higher layer-lines (1221) were carefully examined. Most peaks were satisfactory and these are contoured in Figure 4(d). Most of the processed images gave a reasonable interparticle phase residual (P value) after four rounds of fitting, as shown in Table 2, which indicates that the selection rules (l= - 6n + 13m and I= - 13n+ 28m) adopted for these electron micrographs were suitable. The P value (84.0) for which
642
II. h’ajiyama
Figure 1. Electron micrographs of the HMM and TM in small excess (20% and are seen. (c) Filaments with one-sidedly pattern can be seen. The bars represent
suspension of the washed pellet, from the AC-TM-HMM mixture thnt contained 50%, respectively). (a) and (b) Both undecorated and fully decorated filaments contrasted images along double-stranded helices in which the oblique banding 1000 A.
E is large, but the values of the set of parameters were clearly defined.
filament
(d) Three-dimensional
structure
the AC-TM-HMM
com$ex
of
The final images were obtained from lo- and O-sides averaged data using 26 layer-lines. At the cut-off level described in Materials and Methods, the course of density from the high-density moiety
(A + K+ D+E area) to the outer circumference (I, and I( areas) can be more or less followed as shown in Figure 5(a) and (d). The thin end (G) of the large head nearest to the head/rod junction can be seen. together with two extra densities F and f’. After the end of the large head (G), there are regions of low density on the bent line -1-J (or - I-K). The radius of the whole AC-TM-S1 (+1X2) complex is IIOA.
Shape of the Myosin Head in the Rigor Complex
643
Figure 2. Electron micrographs of typical rigor complexes. (a) Suspension of washed pellet from an AC-TM-HMM mixture. mixture containing TM and HMM in large excess (140% and 200%, respectively). (b) and (c) AC-TM-HMM (d) AC-TM-S1 complex for comparison. Arrowheads indicate whiskers. Double arrowheads indicate the density at the barb of the arrowhead structure. The bar represents 1000 A.
(e) Sl moiety and the neck region of the myosin head Figure 5(a) shows the end-on view reconstructed image, where the two-start F-actin is untwisted to become parallel.
of the helix of A high-
density region including the A, D and E areas (at radii of about 20, 40 to 70 and 80 A, respectively) looks like an Archimedian spiral. The view also shows three circles consisting of E-G-I (r= lOO+lO A), L-(J)-K (130+10 A) and the
H. Kajiyanaa
644
(a
(b)
Id)
(e)
Figure 3. Enlarged images of single filaments which were used in the 3-dimensional reconst,ruction. Their digitized images with boxed areas are beside them. The selection rules are I = - 13n + 28m and I= - 6%+ 13m for filaments (a) to (e) and (f), respectively. The bar represents 500 A. Single arrowheads indicate whiskers. Double arrowheads indicate the density at the barb of the arrowhead structure. The density at the barb is seen between 60 and 144 A from filament axis.
Shape of the Myosin
Head in the Rigor Complex
645
-l/59 -l/108
lilO8 ,v 59
(a)
lb)
(d)
(0,28)---
-
(e) Figure 4. Transforms and the whisker-like structure. (a) Filament shown in Fig. 3(a). (b) Optical diffraction pattern of (a). (c) Digitized image. (d) Computed Fourier transform of the boxed area in the image (c). (e) Diagram of transforms from the filaments with a selection rule of I= -13n+%n. (f) Closed whisker pattern. Left, an electron micrograph; right, drawing of the micrograph. The bar represents 500 A. edge of the density region (r=170A), which is not labelled. At these distances (x) from the filament axis in electron micrographs (Fig. 3), one can see the centre of the dense barb (100 A
whisker (x=li'OA).
(x= 135 A) and the frame of the box The first circle explains the densities
around and after the neck region of the myosin head (Fig. 5(a)). Around the first circle (r= lOOk 10 A), the G area shows the highest density and looks globular (Fig. 6(b)). It has been assigned to the part of the DTNB light chain (Katayama & Wakabayashi, 1981; Seymour & O’Brien, 1985) or the regulatory
H. Kajiyama
646
Table 1 Intraparticle
Filament A 8 c D E Ft t Filament
phase correlation
Number of peaks examined
Q (deg.1
x (‘Q
UJ (deg.)
6 4 5 5 5 4
35 36 16 31 4 9
- 10.4 -4.0 0.3 0.8 0.0 0.0
-84 8.0 4.4 8-O 04 6.0
with a selection rule of 1= - 6n + 13n. Notation
light chain (Vibert & Craig, 1982). Above this, the area F is observed as in the previous model of t,he AC-HMM complex (Katayama & Wakabayashi, 1981). In this study, a new thin area, f, which runs down from the G area, was observed inside the Sl spiral (Figs 5(a) and 6(c)). The views of the superimposed cylindrical sections (Fig. 5(b)) and the solid model (Fig. 6(b)) show that the neck region of the myosin head includes these two protrusions (or thin areas), F and f, above and below G, respectively. Thus, the curved contour length of the large myosin head in the rigor complex is roughly estimated to be 180 to 190 A, when measured along the centre of the density course with the view of the untwisted horizontal (Fig. 5(a)) and cylindrical sections (Fig. 5(b), see also Fig. 7(b)), assuming that the large head begins at the position marked with the double arrowhead (Fig. 5(a), r=20 A) and ends at the end of the G area marked with the single arrowhead (Fig. 5(a)). The longest chord of the large head, a straight line from the edge of I) to the end of G, measured with the solid model (Fig. 6(d), see also Fig. 7(b)) was about 150 to 160 A long. In this study, the thin part of the large head appeared
is as for Fig. 3.
to be about 20 A wide, but area G was 35 A x 27 A wide. (f) The bent part near the head/rod juraction
4. Discussion and computed transforms expected for the AC-TM-HMM complex
(a) Optical
The AC-TM-HMM complex should give information about the spatial relationship between the two
phase correlation
Filament
~‘nli” (deg.)
A0 (deg.)
A near far B near far C near far D near7 far E near far FJ near far
59.6 57.8 43.0 57.6 61.1 64.6 65.6 67.3 84.0 70.2 52.7 62-3
196.5 191.1 27.0 28.0 6.8 15.5 195.8 122.7 120.0 122.1 04 10.0
the
As shown in Figures 5(a) and (b) and 6(c), a density line from the G area goes down, bends back at a radius of 110 A and goes up to the J area forming a “short bent line” G-I-J (or G-I-K) that is 120 to 150 A long in the top view (Fig. 5(a)). Figure 5(a) shows that the outer peaks nearest to the I area are K (r= 135A) and J-L (r=125 to 130 A). J goes up to L (Fig. 6 (c)), and L goes down to the outer circle (r= 165 A) as indicated by a curved line with an arrowhead (Fig. 5(d)), whereas K goes up along the circle at a radius of 135 A (Fig. 5(c)). Peaks K and J-L have higher density than G and I.
Table 2 Interparticle
and
part beyond the bent part (90 A < r < 135 ‘4)
Radial scaling factor 30.9 29.1 14.0 8.6 13.3 17.5 31.3 0.0 0.0 04 1.o 3.0
140 0.95 I.00 0.75 0.98 I .O3 1.14 I 40 1.21 1.oo 140 0.89
t The reconstructed image of this side is fuzzy and was not included in the g-sides averaged data. The P-values tend to increase when the full data (for filament E or D) were fitted to the averaged data that contain the dummy data. The more layer-lines were used (from 1=22 to 27), the larger the P-values became (data not shown). 1 Filament with a selection rule of 1= - 6n + 13m.
Shape of the Myosin Head in the Rigor Complex
647
Figure 5. Horizontal and cylindrical sections (piled up at 9 a intervals) of the image reconstructed from g-sides averaged data using 26 layer-lines. Areas D and E indicate the main body of SI and its tail, respectively. The shaded area corresponds to the cut-off density of the solid model (see Materials and Methods). The radius of the circle is 130 A. This radius is equal to that of the cylinder that contains the solid model. (a) End-on view of untwisted horizontal sections. A “bent chain” (C&I-J) is shown as a broken line. L continues to the outer circumference as indicated by a curved line. Scales are graduated at 10 A intervals. The double arrowhead indicates the beginning of the large head. The single arrowhead indicates the end of it. (b) Superimposed cylindrical sections (63 A
heads
of
meridional for
the
the
HMM
reflections first
time.
In this study, l/110 L%were found
molecule.
around Some
of
these
meridional
reflections had a clear X-shaped pattern around them, indicating the presence of another helical symmetry besides that’ of the actin helix (Fig. 4(b)
and (d) to (e)). There are layer-lines with spacing corresponding to a structure of doubled true-pitch by subsidiary repeat (380 X 4 A), accompanied peaks; these were not included in the image analysis. This indicates that the units, which are arranged at about 108 A (54 x 2 8) vertical spacing,
648
H. Kajiyama
Figure 6. Solid model of the A+TM-HMM complex reconstructed from g-sides averaged data with 26 layer-lines. The thin sheets of styrene foam on the bent course corresponds to the unshaded area (99% of the expected volume) in Fig. 5. (a) End-on view. Some areas on course G-I are 18 to 45 if wide in vertical direction. (b), (c) and (d) Oblique views showing areas G, F, f and the short-bent chain that follows area G. The bars represent 100 A. A short-bent chain is indicated by a broken line. Labels are the same as for Fig. 5.
Shape of the Myosin
Head in the Rigor Complex
649
(b)
(a)
j* ,___-_--
id)
(e)
Figure 7. (a) Schematic drawing of the HMM part of the reconstructed image in the radial projectSion (see legend to Fig. 5(b)). The HMM spiral is uncoiled in a plane. F and f are shown by dotted areas. F is outside, and f is inside the neck region. (b) Hypothetical interpretation of (a). It was assumed that the end of each large head is identical. Two neck regions of one HMM join up to somewhere on the bent line G-I-J-L (or G-I-K). The curved contour and the longest chord of the large head are indicated by a curved line and a straight line, respectively. (c) Drawing of the HMM molecule. The dotted area indicates the contribution of LCZ. (d) and (e) Hypothetical models of binding of the HMM/LMM hinge to another HMM molecule. The HMM/LMM hinge is indicated by a broken line. The HMM/LMM hinge binds to (d) the lower part (near the head/rod junction) of the upper HMM or to (e) the middle of t’he upper S-2. Lengths estimat,ed by Sutoh et al. (1978) are shown.
sometimes make a helix with a pitch of 380 x 4 A (Z= -6n+ 13m). Figure 8(a) explains this phenomenon. When the selection rule for the actin helix is I= -6n+ 13m (Fig. 8(a)), the true repeat corresponds to the length of one arrowhead. Actin units are arranged with 54 A vertical spacing along one strand of double helix. When HMM binds to the actin filament, adjacent actin molecules are decorated with leading (open circles) and trailing (filled circles) heads of one HMM molecule: HMM units are arranged with 54 x 2= 108 A vertical spacing, making a new true repeat that corresponds to four arrowhead repeats. Here, filament F whose selection rule was 1= - 6n+ 13m did not provide layer-lines to show the correct selection rule. HMM binding to the act’in filament whose selection rule is I= - 13n + 28m (Fig. 8(b)) will also
change the vertical spacing of the repeating unit from 54 to 108 A, but will leave the true repeat as two arrowhead repeats. In this study, layer-lines from I=0 to 27 (except E= 1 and 14) were used (Fig. 9). The layer-line data include peaks that arose from the pairing of the two heads, which shows a new selection rule (see Fig. 8(b)). However, the selection rule used here (2= - 13n + 28m) assumes that the heads are not paired. So, the P values in this study are good to t,he extent that the image reconstructed with 28/13 symmetry and put in an identical position was shifted by an actin monomer to obtain these values. (b) Neck region of the myosh head in the rigor complex image (Fig. 5(a)) shows The reconstructed apparent continuation from the Sl moiety to the G
H. MO _.--
h*ajtyanrcl
_
e___._-c_ -c__
__.___.__ --
I----=l . . . . . . . . . . . . . . .._
I .“.
._-___ I13.?7;1
.-
l-2.26)
142.16)
.
.... .. .. .
.
.*.._
. . . -*.
. . . . .
(a) Figure 8.
lb) lattice (,I’ (a) 13 IIIII~S 11rr 6-Iurn heIt\ per l$tutcc h&s. Open and fikd k&s and trailing head3 of HMM. wyectivel:. indicatrs thr one-stat? helix getterated by
Surt’ace
artd (b) 28 unttx jndlcatc leading The broken line
area. and extra prutrusions F and f III thr neck region (Figs Z(b) and 6(b). see ak.o Fig. Y(B)). The curved contnur length of the large head (180 to 1% 1%) A: Fig. 7(h)) . . oonsisknt. with the siztt of t.hr head it\ an isolated ntyosin tnolwule (front mhhit skeletal mus&) detrrmiwd bv the shadow-csasting method (Slavtrr e; Lowry. 1967: E:l\iot.t & Offw. 1978) by this method. thP heat1 mrusures 1.90 A in
. ..‘._ I-t.13,
. .
--
AXape of the Myosin Head in the Rigor Complex length and about 45 A at its widest part and tapers to about 20 A at, the thin end, which is close to the head/tail junction. If one considers that the longest chord of the large head (150 to 160 A) in the rigor state corresponds to the head observed in isolated myosin, it would seem to leave an adequate length of neck on the bent line G-I-J (or G-I-K). Anyway, each neck region of one HMM may continue t’o somewhere on the bent line -1-J-L (or -I-K), because some areas at a radius of 90 A are 18 to 45 A wide vertically (Figs 5(b) and 6(a)). Whether the end of each large head is identical or not cannot be determined in this study. The present results do not support the suggestion of the previous studies that the neck region of each head continues from the G area upward or downward depending upon its relative position (Katayama & Wakabayashi, 1981; Seymour & O’Brien? 1985) for the following reasons. Jn the image from a single filament F (Fig. 3(f)), which clearly shows six layer-lines of the actin helical symmetry, a continuation from the G area in the azimuthal direction, and the F area, but no lower protrusion of the G area are visible. Instead, final and images show a “short bent chain (-I-J-L)” areas f and/or F at this radius. Images from single filaments, C and E (Table 1) also showed f. (c) Candidates for the DTNB light chain (areas F and/or f) LC2 is assumed to be long and to contain a globular part, (Alexis & Gratzer, 1978). The regulatory light chain isolated from scallop myosin was concluded to be 100 A long (Stafford & SzentGyorgyi, 1978). In this study, areas F and f are candidates for LC2 together with area G, because they are found in the image of the rigor complex with HMM but not with Sl, in which LC2 on the myosin head is lost (Wakabayashi & Toyoshima, 1981; Taylor & Amos, 1981; Toyoshima & Wakabayashi, 1985a,b). Their assignment to LC2 fits in with other available findings that the regulatory light chain contributes to the barbs on filaments decorated with Sl and HMM (Craig et al., 1980; Vibert & Craig, 1982), and to the visibility in the neck region of the head in the isolated myosin molecule (Flicker et al.. 1983). In this study, certain HMM preparations contain less 18,000 Mr peptide than 14,000 M, peptide when analysed by SDS/polyacrylamide gel electrophoresis (Laemmli, 1970), suggesting that the partially degraded DTNB light chain (LC2) comigrated with LC3: LC2 of HMM is known to be rapidly degraded with chymotrypsin before the SljS-2 junction is cleaved under physiological conditions without actin and/or divalent cation (Weeds & Pope, 1977; Oda et al., 1980). So, area f is not the result from t’he inclusion of inadequate layer-line data. Thus, it is concluded that at least one extra thin area (either or both of F and f) contributes to the neck region.
651
(d) The bent part near the head/rod junction and areas K and L (90 A < r < 1,3.5‘4) Three peaks I, J-L and K were observed in both of two final averaged images. The G-J-L bent chain and peak K are more apparent in a g-sides averaged image, omitting one fuzzy image, than in a IO-sides one. Higher layer-lines also increased the density at the bent chain and area L. Since this shape of the Sl moiety (Fig. 5(a)) is in agreement with the previous work, this cut-off seems to be reasonable. So, an average of about 160 HMM molecules appearing as a short bent chain (GI-J) of about 120 to 150 A long (Figs 5(a), 6(a) and (c), see also Fig. 7(b)) may be the continuation from the end of the large head. (e) The basal part of the whksker and areas K and L Though S2 whiskers in Figures 2 and 3 appear to be lying flat on the surface of the grid, the centre of a dense barb or dense turning point is seen at a distance between 99.5 and 123.8 A from the filament axis. Each turning point is 27 to 60 A wide, suggesting that it contributes to three peaks I, J-L, and K (r=lOO, 125 and 135 A) in the reconstructed image (Fig. 5(a)). The HMM/LMM hinge of the myosin molecule is known somehow to lock the cross-bridge on the surface of the thick filament in the presence of divalent ion, Ca2+ and/or Mg2+ (Mendelson & Cheung, 1976; Sutoh & Harrington, 1977; Sutoh et al., 1978; Borejdo & Werber, 1982; Ueno et al., 1983). The HMM/LMM hinge may bind to the other S-2 rod, directly near the head/rod junction (see Fig. 7(d)) or in the middle part (see Fig. 7(e)). If this kind of binding occurs also in the rigor complex, the binding of the HMM/LMM hinge with the upper molecule shown in Figure 7(d) and (e) may cause the “closed” whisker seen in elect.ron micrographs (Fig. 4(f)); the whisker may open by turning at the bent part.. However, areas I, J: L and K cannot yet be assigned to particular parts of HMM. Figure 7(c) shows a schematic drawing of the HMM molecule proposed from this work. The real head/rod junction, where two single polypeptide chains join to a double helical rope. may differ from the end of the large myosin head (end of G in Fig. 7(a) and (b)). A proteolytic site that produces chymotryptic Sl may be within the dotted area, because LC2 effectively protects myosin from chymotryptic proteolysis at the Sl/S2 junction in 0.12 M-NaC1 without divalent cation (Weeds & Pope, 1977; Oda et al., 1980). A papain Sl site would be somewhere between the end of the large myosin head and the head/rod junction, because an 81 preparation that retains LC2 or the regulatory light chain has been reported (Werber rt al., 1972; Margossian bz Lowey, 1973; Margossian et al., 1975; Bagshow, 1977; Kuwayama & Yagi. 1977, 1980; Yamamoto & Sekine, 1980). I could not determine the exact position of the head/rod junction in this study.
H. Kaji yama -
652
Tn future, information on the “pairing” features may be obtained by carrying out image analysis using the extracted peaks that belong to a correct selection rule and using the filament with a closed whisker (Fig. 4(f)), which gave the clearest X-shaped pattern that crosses over the l/108 a meridian (data not shown). Image analysis of the one-sidedly contrasted specimen shown in Figure 1 and of the data for the “pairing” features should be examined. I am grateful to Professor T. Wakabayashi for suggesting analysis of the AC-TM-HMM complex and t,o Dr. Y. Shirakihara for examining the program used. This work was supported by Grants-in-Aid for Special Project Research and Grants-in-Aid for Scientific Research to T.W. from the Ministry of Education. Science and Culture of ,Japan.
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.J. Mol. Riol. 50, 279-295. Oriol-Audit. (‘. cyi Rt~islrr. E. (1980). s., Biochmistry, 19, 5614-5618. Pepe. F. A. (1967). .I. Mol. J&Z. 27, 227 -%ci. Seymour. ,J. & O’Brien, E. ,J. (1985). .J. :Mus~‘. Km. (‘c/l Motil. 6. 725 756.
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Slagter. H. S. & Lowry. S. (1965). i’roc.. i$‘trf. .3&. I..A.A. 58. 161 I-- 1618. Staflord.
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LVrrbrr.
%I. Sl.. (:aflin,
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by S. Urennrr
Ml
Afotil.