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Atomic model of the human cardiac muscle

myosin lament
Hind A. AL-Khayata,1, Robert W. Kenslerb, John M. Squirec, Steven B. Marstona, and Edward P. Morrisd
a
National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London W12 0NN, United Kingdom; bDepartment of Anatomy and
Neurobiology, University of Puerto Rico Medical School, San Juan, Puerto Rico 00936-5067; cSchool of Physiology and Pharmacology, University of Bristol,
Bristol BS8 1TD, United Kingdom; and dDivision of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB,
United Kingdom

Edited by J. G. Seidman, Harvard Medical School, Boston, MA, and approved November 19, 2012 (received for review August 2, 2012)

Of all the myosin laments in muscle, the most important in terms (Ig) and bronectin 3 (Fn3) domains connected by short linkers.
of human health, and so far the least studied, are those in the Within the C zone, the titin domains are distributed in a pattern
human heart. Here we report a 3D single-particle analysis of corresponding to an 11-domain superrepeat every 429 . The
electron micrograph images of negatively stained myosin la- cardiac isoform of MyBP-C (cMyBP-C) has a mass of 140 kDa
ments isolated from human cardiac muscle in the normal (undis- and is made up of a linear array of 11 Ig and Fn3 domains.
eased) relaxed state. The resulting 28- resolution 3D reconstruction Myosin lament structure has been investigated both by
shows axial and azimuthal (no radial) myosin head perturbations electron microscopy and by X-ray ber diffraction to explore
within the 429- axial repeat, with rotations between successive the different organizations and properties of myosin laments
132 -, 148 -, and 149 -spaced crowns of heads close to 60, 35, from a variety of organisms and tissues (7, 8). The most detailed
and 25 (all would be 40 in an unperturbed three-stranded helix). structural description of a myosin lament (to date) derives from
We have dened the myosin head atomic arrangements within helical 3D reconstruction of cryoelectron microscope images of
the three crown levels and have modeled the organization of my- tarantula myosin laments at a resolution of 25 (9, 10). In the
osin subfragment 2 and the possible locations of the 39 -spaced tarantula myosin lament, pairs of myosin heads were identied
domains of titin and the cardiac isoform of myosin-binding pro- forming a similar interaction to that found in the off state of
tein-C on the surface of the myosin lament backbone. Best ts vertebrate smooth muscle myosin (11). Vertebrate skeletal and
were obtained with head conformations on all crowns close to cardiac muscle myosin laments differ from those in inverte-
the structure of the two-headed myosin molecule of vertebrate brates such as tarantula in that the myosin heads depart from an
chicken smooth muscle in the dephosphorylated relaxed state. In- exact helical arrangement. Hence, for vertebrate skeletal and
dividual crowns show differences in head-pair tilts and subfragment cardiac myosin laments, helical reconstruction is not a valid
2 orientations, which, together with the observed perturbations, approach for the analysis of electron microscope data. Working
result in different intercrown head interactions, including one on a variety of myosin laments, we have developed an alter-
not reported before. Analysis of the interactions between the my- native method to determine their 3D structures from electron
osin heads, the cardiac isoform of myosin-binding protein-C, and microscope data based on single-particle analysis (12). This ap-
titin will aid in understanding of the structural effects of muta- proach is well-suited to the analysis of quasihelical myosin la-
tions in these proteins known to be associated with human ments. We have applied single-particle analysis to myosin
cardiomyopathies. laments from a variety of muscle types including vertebrate
skeletal and cardiac muscles (13, 14) and invertebrate striated
thick laments 3D structure | human heart muscle | electron microscopy | muscle (15). A similar approach has been used by others in the
image processing analysis of mouse cardiac myosin lament structure (16), where
myosin heads were also found to adopt a paired conformation
similar to that in tarantula myosin laments.
M uscle contraction involves the interaction between actin
and myosin laments that leads to force production me-
diated by the hydrolysis of ATP. Myosin laments are assemblies
Mutations in human cardiac muscle myosin and its associated
proteins, cMyBP-C and titin, are known to cause a number of
of myosin molecules and accessory proteins. Myosin molecules human cardiomyopathies including familial hypertrophic car-
consist of two heavy chains arranged to form two globular do- diomyopathy and dilated cardiomyopathy (17, 18). A detailed
mains [two subfragment-1s (S1s)] at the N-terminal end and knowledge of the structure of human cardiac myosin laments in
a long rod-like -helical coiled-coil domain (the myosin tail). the normal (undiseased) relaxed state is likely to be important in
Each of the globular domains with its associated essential and understanding how the mutations give rise to these cardiomy-
regulatory light chains forms a myosin head containing the opathies. To address this issue, we have successfully developed
ATPase activity necessary for contraction (1). This is linked to a laboratory method to isolate myosin laments from human
the N-terminal part of the tail known as the subfragment-2 (S2) cardiac muscle that preserves the highly ordered pseudohelical
region. Myosin laments are generally bipolar, with the myosin structure of the relaxed laments, thus making them amenable to
tails packing together in an almost cylindrical backbone and the
heads projecting out laterally in a helical or quasihelical fashion
at intervals of 143 . In the myosin laments of vertebrate Author contributions: H.A.A.-K. designed research; H.A.A.-K. performed research; R.W.K.,
striated muscles, the myosin heads are arranged in a three- J.M.S., S.B.M., and E.P.M. contributed new reagents/analytic tools; H.A.A.-K. and E.P.M.
stranded quasihelical array on the lament surface (2) with the analyzed data; and H.A.A.-K., R.W.K., J.M.S., S.B.M., and E.P.M. wrote the paper.
accessory proteins titin (3, 4), myosin-binding protein-C (MyBP- The authors declare no conict of interest.
C) (5), and possibly the MyBP-C analog, X protein (6), located This article is a PNAS Direct Submission.
on the surface of the backbone. MyBP-C is located within the C Freely available online through the PNAS open access option.
zones of the myosin lament, which are regions 3,500 long Data deposition: The map of the human cardiac muscle myosin lament has been de-
centrally located in the two halves of the bipolar lament. Titin is posited in the EMDataBank (accession code EMD-2240).
an unusually large elongated protein (3.8 MDa) that runs par- 1
To whom correspondence should be addressed. E-mail: h.al-khayat@imperial.ac.uk.
allel to the actin and myosin laments and spans half the length of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
a sarcomere. It is composed of a linear array of immunoglobullin 1073/pnas.1212708110/-/DCSupplemental.

318323 | PNAS | January 2, 2013 | vol. 110 | no. 1 www.pnas.org/cgi/doi/10.1073/pnas.1212708110


analysis by electron microscopy and single-particle image anal- The location of the crowns within the C zone matches that de-
ysis. From such samples, we have produced a 3D reconstruction scribed in earlier studies of vertebrate skeletal and cardiac
of the C zone of the myosin lament at 28- resolution, which muscle (13, 14), and the crowns are labeled 1, 2, and 3 following
allows the detailed docking of myosin heads and the modeling of the same convention. Here crown 1 is the most massive, with its
the constituent domains of titin and cMyBP-C. extra mass arising from cMyBP-C [n.b. this is a different num-
bering scheme from the unconventional one used by Zoghbi
Results et al. (16) in their analysis of mouse cardiac myosin lament,
Three-Dimensional Structure of the Human Cardiac Myosin Filament. where the equivalent crowns are labeled 1, 3, and 2, respectively].
Electron microscope images were recorded from negatively Each crown is made up of three pairs of myosin heads. Direct
stained preparations of myosin laments isolated from human inspection of the density associated with each crown allows the
ventricular muscle. From these images, well-preserved examples recognition of myosin head pairs that form triangular motifs
of myosin laments were selected in which the C zone could be (Fig. 1 C and D) very similar in appearance to the conformation
clearly identied (Fig. S1 A and B). From 237 myosin lament C of myosin head pairs in the off state of vertebrate smooth muscle
zones, a total of 1,559 segments were extracted, each segment myosin (Fig. 1E) (11). Similar shapes were seen in the tarantula
corresponding to two 429- repeats. An initial reference 3D myosin lament reconstruction (9), where the base of each tri-
structure was calculated using C3 symmetry (threefold rotational angle oriented towards the M band corresponds to the two motor
symmetry) from a single class average of the segments. Starting domains and the two sides correspond to the lever arms.
from this reference, a rened 3D structure was calculated from
the individual segments by successive rounds of forward pro- Fitting the Crown Densities with Myosin Molecular Models. Because
jection, multireference alignment, and 3D reconstruction. Dur- we identied motifs in each of the crowns with close resemblance
ing the renement, the number of segments was reduced to 285 to the myosin head pairs in tarantula myosin laments, we pro-
by selecting only those that agreed visually most strongly with ceeded to dock the corresponding atomic model into the crown
densities. For this purpose, we used Protein Data Bank (PDB)
forward projections of the 3D structure. Fourier analysis of the
ID code 3DTP (10) [a modied version of the off state of ver-
selected segments (Fig. S2A) shows good recovery of detail, with
tebrate smooth muscle myosin (11), which also includes part of
the characteristic layer-line pattern arising from the quasihelical
the coiled-coil S2 structure]. We found that the best t within the
myosin head distribution together with meridional reections
density map could be obtained by uncoupling and separately
extending to 36 (the 12th order of the 429- repeat). Fur-
docking the two S1 domains, resulting in minor changes in the
thermore, these segments provide an even distribution of pro- mutual interaction of the motor domains and minor reor-
jection angles (Fig. S3). The nal 3D reconstruction (Fig. 1), ientations of the lever arms. Nevertheless, in each case, the
which has an estimated resolution of 28 (Fig. S4), is charac- overall structure of the docked head pair is not very different
terized by a wealth of detail, and Fourier analysis of repro- from the original structure (11) and had similar intramolecular
jections (Fig. S2B) shows good agreement with the original interactions between the motor domains of the blocked and free
segments. The visual selection is supported by quantitative heads as described previously (11). We also found it necessary to
comparison (Fig. S5) and Fourier analysis (Figs. S2A and S6A) of reorient the S2. Fig. 2 A and B shows a similar view of the map
the selected and rejected segments. Moreover, 3D recon- shown in Fig. 1B along with our best-t structure within the map.
structions of the rejected segments are characterized by signi- The map is also shown as a movie (Movie S1). Overall, the tted
cantly lower levels of detail (Fig. S7) and correspondingly lower coordinates correspond closely to the density while revealing
resolution (Figs. S6 and S8). Within the 429- repeating unit of characteristic features of the individual crowns. In crown 3, there
the nal 3D reconstruction, there are three distinct sets of is little density unaccounted for within the head-pair motif, and
densities on the outer surface of the lament (Fig. 1 A and B) almost all of the coordinates are contained within the density.
that correspond to the three crowns or levels of myosin heads. However, in the case of crown 1 there are minor excursions as-
sociated with the regulatory light chain (RLC) of the blocked
head and the essential light chain (ELC) of the free head,
whereas for both crowns 1 and 3, the N-terminal SH3 domains
A B C D E (residues 3378) of both the blocked and free heads extend
outside the density. This latter effect may be associated with the
1
conformational variability observed in this region of myosin (19).
On the other hand, in the case of crown 2, although the overall
3 shape of the head pair conforms quite well to the tted coor-
dinates, there are regions where the coordinates lie outside the
density more substantially, particularly at the N-terminal SH3
2 domains of the two heads and in the regions of the lever arms.
The least density is recovered for the head domains in crown 2.
MEDICAL SCIENCES

Fig. 1. (AD) Surface views of the nal 3D reconstruction of the human This may arise because the heads in crown 2 are less well ordered
cardiac myosin lament obtained by single-particle EM analysis and dis- than in crowns 1 and 3.
played using PyMOL showing a length of a full 429- repeat. The re-
construction is shown in two views (A and C; B and D) related by a 25 Myosin Head Arrangement in Crowns 1, 2, and 3. The tted positions
rotation about the lament axis, such that in A and C, a myosin head pair on of the myosin heads within the cardiac myosin lament (Fig. 2)
level 1 is facing the viewer, and in B and D, a myosin head pair on level 3 is can be used to characterize the pseudohelical head arrangement
facing the viewer. Note that the two views in A and C and in B and D are of the lament as summarized in Fig. S9 and Table S1. This gives
quite distinct because of the different perturbations in the crowns. The tri- the axial and angular displacements between successive crowns
angular-shaped congurations for the three head pairs on each crown level
measured both between the centers of each head pair and be-
are shown in yellow, blue, and pink for levels 2, 3, and 1, respectively, in C
and D. The bare zone is at the bottom of the map in all of the four views. (E)
tween the ends of the lever arm at the C-terminal position of the
Crystal structure for the head pair (11). The blocked and free heads are color- S1 domains, where they connect to S2. The azimuthal displace-
coded as in refs. 911. Blocked head: motor domain, green; essential light ment represents the rotation about the lament axis between
chain, orange; regulatory light chain, yellow. Free head: motor domain, head pairs on successive crowns (Fig. S9B). Comparison of these
cyan; essential light chain, pink; regulatory light chain, beige. parameters with the corresponding values for a regular three-

AL-Khayat et al. PNAS | January 2, 2013 | vol. 110 | no. 1 | 319


intramolecular arrangement for the head pairs in which the
A B intramolecular interactions observed in previous studies of
smooth muscle myosin (11) and tarantula muscle (9, 10) are
1 substantially conserved. However, intermolecular interactions
between the head pairs on adjacent crowns vary much more
substantially (Fig. 3A). As noted above, the head pairs in the
2,3,1 crown sequence are quite closely connected, whereas there
3
is a distinct gap between crowns 1 and 2, where the head pairs
appear not to be connected (Fig. 3D). In contrast, the head pairs
in crowns 2 and 3 are connected through an interaction between
the ELC of the blocked head on crown 2 and the motor domain
of the free head on crown 3 (Fig. 3C). A similar interaction was
2 observed between heads in successive crowns in the purely he-
lical tarantula myosin lament (9, 10). This structural similarity
can be understood because the head pairs on crowns 2 and 3
have the same tilt angles (Table S1 and Fig. S9C). Moreover, the
Fig. 2. (A) Three-dimensional map of the view in Fig. 1B showing a full 429- angular rise between crowns 2 and 3 (35) in the human cardiac
repeat together with tting of the atomic model of myosin heads in the myosin lament is quite similar to the angular separation of 30
off state to the head motifs in crowns 2, 3, and 1 after splitting and tting
separately the two heads from Alamo et al. (10). The majority of the atomic
in tarantula, and the axial spacing of 148 is also similar to that in
structures for the head pairs is well-contained within the density map on tarantula (145 ). A notably different type of intermolecular
crowns 3 and 1 but less on crown 2, especially for the two lever-arm parts. connection is observed between crowns 3 and 1. This is mediated
The S2 is also tted, as described in the text. (B) Same view as in A, but
shown without the back part of the map to illustrate the tting of the heads
and S2 within the densities on levels 2, 3, and 1. S2 is shown in red and the
bare-zone direction is toward the bottom. A B
1 1
start helix with a repeat of 429 allows the perturbations from
an exact helical structure to be accurately dened. For the axial
displacements (Fig. S9A), the values are very similar whether
measured between the centers of the head pairs or between the
ends of the lever arms, and it is apparent that crowns 1 and 2 are 3 3
closer together (axial displacement 133 ) than either crowns 2
and 3 or crowns 3 and 1 (axial displacement 148 ). In com-
parison, an unperturbed helix would have a uniform axial dis-
placement of 143 . On the other hand, the angular displacement C
between crowns 1 and 2 (60 measured between the centers of the 2 3
head pairs, and 58 between the ends of the lever arms) is very
much greater than the equivalent measurements between crowns
2 and 3 (35 and 30, respectively) and between crowns 3 and 1
(25 and 32, respectively). These values can be compared with an 1
unperturbed helix, which would have a constant azimuthal dis-
2
placement of 40. Hence, the head arrangement in the cardiac
myosin lament is characterized by a sequence of three crowns
(2,3,1) with similar axial displacements (148 ) and approxi-
mately similar angular displacements (30). This is followed by D
a discontinuity in which the transition to the next crown has
a reduced axial displacement (133 ) but a substantially in- 2
creased angular separation (60). This gives rise to the phe-
nomenon in which the head pairs in the 2,3,1 crown sequence are
quite closely connected along a pseudohelical track, but due to
the angular discontinuity, the connection with the next 2,3,1 se- 1
quence is not maintained. In addition to the variations in axial
and angular displacements between adjacent crowns, the head
pairs also exhibit a variation in their tilt angles. This can be most
readily appreciated by comparing the orientation (or tilt angle) of
the extended density arising from the two motor domains of each Fig. 3. (A) Fitted crystal structures showing head pairs and S2 on successive
head pair (Fig. S9C). Here it is apparent that crowns 2 and 3 have crowns along one strand of the myosin lament. Head pairs in the 2,3,1
very similar orientations, whereas crown 1 is rotated by 19 in crown sequence are quite closely connected, whereas there is a distinct gap
a clockwise direction as viewed from outside the lament. Despite between crowns 1 and 2. (B) Head pairs on crowns 3 and 1 are connected
through an interaction between the RLC of the blocked head on crown 3
these perturbations in axial displacement, azimuthal displace-
and the motor domain of the free head on crown 1. The red arrowed star
ment, and tilt, the radial perturbation of myosin heads is minor in indicates the predicted position of serine 15 in human myosin RLC. (C) A
the human cardiac myosin lament, in agreement with previous different type of intermolecular connection is observed between crowns 3
observations for both sh and rabbit myosin laments (13, 14). and 2 that is mediated by an interaction between the ELC of the blocked
head on crown 2 and the motor domain of the free head on crown 3. (D) No
Interactions Between the Myosin Heads. Independent docking of intermolecular interaction is seen between the head pairs in crowns 1 and 2.
the heads within the head pairs in each crown leads to a similar S2 is shown in red, and the bare zone is oriented vertically down in all views.

320 | www.pnas.org/cgi/doi/10.1073/pnas.1212708110 AL-Khayat et al.


by the interaction between the RLC of the blocked head on Titin is thought to extend from the Z band (N terminus) to the
crown 3 and the motor domain of the free head on crown 1 (Fig. M band (C terminus), with six titin strands per half thick lament
3B). The different mode of interaction compared with that be- (20, 21), and it is likely that the six titins are grouped as three
tween crowns 2 and 3 can be considered as the consequence of strands of titin pairs. Titin Ig and Fn3 domains are arranged in
the clockwise 19 rotation of the head pair on crown 1 and the long-range patterns or superrepeats. Within the myosin lament
reduced angular rise between crowns 3 and 1 (25), both of which bridge region, two types of titin superrepeats are found. There
serve to shift the motor domain of the free head on crown 1 from are 7 consecutive copies of the 7-domain small superrepeat in
the ELC to the RLC of the blocked head on crown 3 (Fig. 3B). the D zone at the distal edge of the A band. These are followed
Upon searching the literature, we could not nd a record of this by 11 copies of the 11-domain large superrepeat. The 11-do-
type of interaction between myosin head pairs in adjacent crowns. main repeats each follow the sequence (N)-Ig-Fn3-Fn3-Ig-Fn3-
Although in their studies of mouse cardiac myosin lament, Fn3-Fn3-Ig-Fn3-Fn3-Fn3-(C). The axial distance between each
Zoghbi et al. (16) presented a docking of myosin head pairs on large superrepeat copy is 429 , and this series ends at the edge
crowns 3 and 1 (they call them crowns 2 and 1) in which the of the bare zone (2225). The 11 copies of the 11-domain large
motor domain of the free head on crown 1 can be seen to make superrepeat of titin cover both the C zone and the P zone (Fig.
contact with the RLC of the blocked head on crown 3, this was S1B). cMyBP-C is a modular structure comprising 11 Fn3 and Ig
not mentioned by the authors. domains (C0C10) (26, 27).
We used the crystal structure of the Fn3 tandem A77A78
Location of S2. S2 was docked into the reconstruction separately domains (22) (PDB ID code 3LPW) from the part of titin in the
from the myosin head pairs starting from the conguration ob- C-zone region to create models of titin and cMyBP-C (Fig. 4 and
served in tarantula myosin laments (9, 10) (Fig. 2). Further- Movie S2). Each Fn3 domain is also homologous to Ig domains,
more, the variation in tilt angle of the head pairs on different and is thus likely to be similar in structure, particularly at the res-
crowns required S2 to be further subdivided into the segment olution of our current analysis (28 ). The titin model consists of
running from the end of the lever arms to the blocked motor a series of 11 appropriately spaced domains: Two parallel copies
domain, the proximal region of S2 (residues 854927 of S2), and are docked into the region alongside the 2,3,1 crown sequence of
the remaining C-terminal segment, the distal region of S2 (res- myosin heads extending 429 along the lament. We considered
idues 928972 of S2). Although not fully resolved from the rest it likely that two strands could be side by side and in contact, and
of the density of the head pair, the proximal region of S2 can be indeed these match the density well (yellow in Fig. 4). We also
accommodated within crown 3, where it is integrated with the used the Fn3 structure to t three further domains that are
density of the lever arms of the blocked heads. A similar location sandwiched between the titin and the myosin head pair on crown
with respect to the myosin heads for the proximal region of S2 1 (pink in Fig. 4). It is likely that these are the C-terminal part of
was obtained in the case of crown 1, although in this case it is not cMyBP-C domains C8C10. With this t, these domains run
fully accommodated in the density. The less-complete recovery parallel to the myosin lament axis in agreement with Squire
of myosin head-pair density on crown 2, noted above, also et al. (28) and not as a collar, as suggested by Moolman-Smook
applies to this segment of S2. For consistency, given the fact that et al. (29). With this arrangement, there is an apparent interaction
the head pairs on level 2 have the same tilt angle as the head pair between the C10 domain of cMyBP-C and the motor domain of
on level 3, the proximal region of S2 is docked in a similar the free head of crown 1.
conformation to that in crown 3 but is not accommodated in the These modeled locations for titin and cMyBP-C are consistent
density. The distal regions of S2 in all of the three crowns are with the earlier proposals of Zoghbi et al. (16) based on their
accommodated in the density and run approximately parallel to structural analysis of native and knockout mouse cardiac myosin
the lament axis close to the surface of the lament backbone. laments. However, unlike the previous study, the additional
The conformation of S2 on each crown is illustrated in Fig. S10, detail achieved in the current analysis allows explicit docking of
which shows that on each level, S2 is in a similar position in the model structures for the individual domains. The assignment of
region between its junction with the C-terminal region of the the three domains to cMyBP-C is also consistent with their loca-
heads (i.e., end of the lever arm) and its interaction with the tion adjacent to the crown 1 myosin heads, whereas the suggestion
blocked head. This conformation is very similar to that observed that these represent C8C10 is consistent with the location of
in tarantula myosin laments (9, 10), suggesting that the intra-
molecular interaction between S2 and the actin-binding domain
of the blocked motor domain may be conserved between taran-
tula and human cardiac myosin laments. Beyond its interaction
with the blocked head, S2 continues in a similar direction on level
1, but on levels 2 and 3 it is hinged to the right. The difference in
orientation of S2 in this region serves to accommodate the dif- 1
ference between the tilt angle of the head pair on level 1 and the
MEDICAL SCIENCES

tilt angle of the head pairs on levels 2 and 3 (19, as documented


in Table S1; see also Fig. S9), while allowing the distal regions of
S2 to remain approximately parallel to the lament axis. 3

Location of Nonmyosin Protein Densities: Titin and cMyBP-C. Having


identied the density in the structure of the human cardiac
myosin lament that can be ascribed to the myosin heads and S2, 2
we could now seek to identify the nonmyosin components.
Alongside the 2,3,1 crown sequence of myosin heads with strong
intermolecular interactions, the myosin lament is characterized
by regions with prominent and well-dened surface features with
a regular modulation every 39 , consistent with the size of the Ig Fig. 4. Final 3D map (Fig. 1) showing a full 429- repeat, shown with my-
and Fn3 domains of titin and cMyBP-C. Accordingly, we sought osin head pairs, S2 (red), titin (yellow), and the three C-terminal domains of
to develop models of titin and cMyBP-C to t these densities. cMyBP-C (C8C10) (magenta). The bare-zone direction is toward the bottom.

AL-Khayat et al. PNAS | January 2, 2013 | vol. 110 | no. 1 | 321


binding sites to myosin heavy chain (MHC), light meromyosin predicted to be tilting back toward the backbone surface, as ob-
(LMM), and titin within these domains (30). served in our structure, and forming interactions, as proposed by
Stewart and colleagues (3133) for the SRX state.
Discussion
The structure described here for the human cardiac myosin l- Materials and Methods
ament in the normal (undiseased) state is an essential starting Chemicals. Type III lyophilized powder elastase from porcine pancreas (code
point from which to understand the mechanisms of various E0127), N-p-tosyl-L-phenylalanine chloromethyl ketone (code T4376), apro-
cardiac diseases, in particular the effects of mutations in myosin, tinin from bovine lung (code A1153), pepstatin A from microbial source
(code P5318), trypsin inhibitor from Glycine max (soyabean) (code T6522),
C protein, and titin associated with different cardiomyopathies.
PMSF (code P7626), and leupeptin hemisulfate salt from microbial source
The current analysis shows a substantial improvement in the
(code L2884) were obtained from Sigma-Aldrich. The elastase obtained from
resolution and level of detail compared with previous studies of Sigma-Aldrich with code E0127 had an elastase content of 5585%, elastase
vertebrate striated muscle myosin laments and allows detailed enzymatic activity 4% unit/mg, and trypsin activity 200 unit/mg.
modeling of myosin heads and accessory proteins and their
interactions. Using a negative-stain approach, we have achieved Isolation of Myosin Filaments from Human Heart Muscle. Myosin laments
a degree of resolution and detail that approaches that obtained were isolated from human ventricular muscle under relaxing conditions (35).
with frozen-hydrated preparations of highly ordered invertebrate To avoid chloride-induced disorder of myosin heads (36), MgCl2 was replaced
myosin laments (9, 10). We have been able to exploit the high by MgAc in both the relaxing solution and the rinsing solution for EM grids.
level of detail to demonstrate that the myosin head pairs in each Similarly, KCl was replaced by NH4Ac in the EM rinsing solution. Ventricular
crown adopt the off-state conformation previously identied in muscles removed from donor human hearts were frozen and stored in liquid
nitrogen until use. Small muscle pieces, about 80 mg, were dissected and
smooth muscle myosin (11) and tarantula myosin laments (9,
thawed for 30 min in relaxing solution containing the components listed in
10). These observations are consistent with a previous analysis of
SI Text 1. Samples were cut with a razor blade into small pieces and then
mouse cardiac myosin laments at lower resolution (16) that teased with a pair of forceps into ne bundles 0.5 mm or less in diameter on
identied the off-state conformation on crowns 1 and 3. These a microscope slide and stirred for 1 h in 10 mL of fresh relaxing solution to
studies, however, were unsuccessful in recovering head-pair ensure full relaxation (assessed by light microscope as showing long un-
density on crown 2, and it was suggested that this was due to contracted sarcomeres). Myosin laments were released by incubation for 3
higher mobility. The higher resolution in the current analysis min in 0.22 mg/mL elastase and 0.44 mg/mL trypsin inhibitor in a relaxing
allows us to recognize the off-state head-pair conformation on solution containing the components listed in SI Text 2. The reaction was
crown 2 and identify the orientation of the head pair by docking. stopped by transfer into the same buffer as in SI Text 2, but with 1 mM PMSF
It is apparent that recovery of density for the head pair is less- and no elastase. After vigorous shaking by hand for 3 min and then gentle
pipetting, the muscle broke up, yielding a suspension of separated thick and
complete on level 2, consistent with the earlier suggestion of
thin laments. The suspension was centrifuged for 2 min at 735 g using an
increased mobility in this region. Increased mobility on crown 2
Eppendorf Centrifuge 5415C, and EM grids were made using the supernatant.
may be a consequence of the pattern of intermolecular inter- Donor heart muscle tissue samples were collected by C. dos Remedios,
actions between crowns, because crown 2 lacks the interaction of University of Sydney, Australia. Ethical approval was obtained from the
its free-head motor domain with the lever arm of the adjacent Brompton, Hareeld, and NHLI Ethics Committee, London, UK and the
crown 1. The docked locations for titin and cMyBP-C described Australian Red Cross Blood Service and St. Vincents Hospital, Sydney,
here are broadly consistent with previous proposals (16). How- Australia. The investigation conformed to the principles outlined in the
ever, the enhanced resolution of the current study makes explicit Declaration of Helsinki.
docking of both the myosin heads and S2 possible and allows the
development of a molecular model. The locations we identify for Negative Staining and Electron Microscopy. Five microliters of the isolated
the myosin heads are quite close to the backbone, and the myosin lament suspension was applied to 400-mesh copper grids with a thin
carbon lm (57 nm thickness) supported by a holey formvar lm and left
groupings of three crowns show well-dened interactions be-
for 10 s. The grids were rinsed sequentially with eight drops of half-strength
tween myosin head pairs in adjacent crowns. A head confor-
relaxing solution containing the components listed in SI Text 3 and then ve
mation recently identied biochemically has been called the drops of 1% (wt/vol) uranyl acetate and 0.025% glycerol (37, 38). Samples
superrelaxed (SRX) state, in which the myosin ATPase activity is were blotted with lter paper after each drop of uranyl acetate stain except
inhibited (3133). It was concluded that the myosin heads in the for the last drop of stain, which was left on the grid for 30 s. Most of the
SRX state are strongly stabilized on the backbone of the thick stain was then removed with lter paper and the grid was rapidly dried
lament and have the J-like motif (11). It could well be this par- with a hair dryer. Samples were imaged at room temperature with an FEI
ticular structure, with many of the heads making strong (some- Tecnai TF20 electron microscope at an accelerating voltage of 200 kV, in
times blocking) interactions with their neighbors, that we have low-dose mode with an exposure of 100 e/2, a nominal magnication of
now captured in our current cardiac myosin lament preparation. 29,000, and an underfocus chosen to place the rst minimum in the
contrast transfer function at 17 . Images were recorded using a Tietz
In our structure, the interaction between levels 3 and 1 is
F415 (4k 4k) CCD camera and elds were binned by a factor of 2, resulting
between the motor domain of the free head on level 1 and the
in a calibrated sampling of 17 m per pixel (equivalent to 5.86- sampling
RLC of the blocked head on level 3 below it. By comparing in the specimen). Only laments lying over holes were used in the cur-
the sequence of the human cardiac RLC with that of tarantula, rent analysis.
the origin of the myosin head coordinates used here (10), it is
apparent that serine 15, which is the major phosphorylation site Preparation of Filament Segments and 3D Analysis. CCD frames were collected
in the human RLC, is at the base of an -helix that forms and evaluated using MRC (39), IMAGIC (40), and locally developed software.
a prominent contact between the RLC and the free-head motor Regions were selected that contained intact half-laments that were rela-
domain on level 1 (Fig. 3B, red star). Thus, phosphorylation tively straight and minimally overlapped by actin and other myosin la-
might well act to destabilize this interaction in a similar manner ments, with readily identiable bare zones (Fig. S1 A and B). Whole myosin
to the phosphorylation effect proposed by Alamo et al. (10) for laments were cut into two half-laments (each including the whole bare
zone) and half-laments were oriented with their bare zones pointing
the tarantula myosin lament. From these considerations, it seems
vertically downward (Fig. S1B). Fourier transforms of the half-laments were
likely that the relaxed myosin laments studied here are sub-
calculated. The spacing of the sixth order of the 429- repeat, the 71.5-
stantially in the dephosphorylated state. Moreover, X-ray diffrac- meridional reection, was used to calibrate the magnication and to adjust
tion studies of mouse heart muscle (34) have shown an increase in the sampling of each half-lament from all of the different micrographs to
the proximity of the heads to actin and thus a displacement of the be exactly 5.94 /pixel. This sixth-order meridional reection was particularly
heads away from the backbone upon phosphorylation. Conversely, strong in most of the Fourier transforms. From the C zone of these half-
in the dephosphorylated state, the myosin heads would be laments, particles/segments were extracted, each segment being centered

322 | www.pnas.org/cgi/doi/10.1073/pnas.1212708110 AL-Khayat et al.


on a 429- repeat and being 950 in length (just over 2 429 repeats). computationally using the VEDA (http://mem.ibs.fr/GAEL/index.html) in-
Three-dimensional analysis was conducted using the approach previously terface to URO (42).
described for vertebrate myosin laments (13, 14). During the later stages of
the renement, we used a brute-force multireference alignment program ACKNOWLEDGMENTS. This work was supported by British Heart Foundation
that performs rotational and translational alignment independently. We Fellowship FS/07/017/22951 (to H.A.A.-K.). R.W.K. was supported by Minor-
ities Basic Research Support Grant 5SC1HL96017 from the National Institutes
also used a locally developed Fourier space 3D reconstruction program (41). of Health (NIH) and, in part, by funding from Research Centers in Minority
Docking of the atomic coordinates into the density was done manually with Institutions Award G12RR-03051 from the National Center for Research
PyMOL (Version 1.2r3pre; Schrdinger), and then docking was rened Resources, NIH.

1. Huxley HE (1963) Electron microscope studies on the structure of natural and syn- 23. LeWinter MM, Granzier H (2010) Cardiac titin: A multifunctional giant. Circulation
thetic protein laments from striated muscle. J Mol Biol 7:281308. 121(19):21372145.
2. Squire JM (1972) General model of myosin lament structure. II. Myosin laments and 24. Krger M, Linke WA (2011) The giant protein titin: A regulatory node that integrates
cross-bridge interactions in vertebrate striated and insect ight muscles. J Mol Biol 72 myocyte signaling pathways. J Biol Chem 286(12):99059912.
(1):125138. 25. Tskhovrebova L, Trinick J (2010) Roles of titin in the structure and elasticity of the
3. Labeit S, Kolmerer B (1995) Titins: Giant proteins in charge of muscle ultrastructure sarcomere. J Biomed Biotechnol 2010: Article 612482.
and elasticity. Science 270(5234):293296. 26. Yasuda M, Koshida S, Sato N, Obinata T (1995) Complete primary structure of chicken
4. Trinick J (1996) Titin as a scaffold and spring. Cytoskeleton. Curr Biol 6(3):258260.
cardiac C-protein (MyBP-C) and its expression in developing striated muscles. J Mol
5. Offer G, Moos C, Starr R (1973) A new protein of the thick laments of vertebrate
Cell Cardiol 27(10):22752286.
skeletal myobrils. Extractions, purication and characterization. J Mol Biol 74(4):
27. Gautel M, Zuffardi O, Freiburg A, Labeit S (1995) Phosphorylation switches specic for
653676.
6. Bennett P, Craig R, Starr R, Offer G (1986) The ultrastructural location of C-protein, the cardiac isoform of myosin binding protein-C: A modulator of cardiac contraction?
X-protein and H-protein in rabbit muscle. J Muscle Res Cell Motil 7(6):550567. EMBO J 14(9):19521960.
7. Craig R (2012) Isolation, electron microscopy and 3D reconstruction of invertebrate 28. Squire JM, Luther PK, Knupp C (2003) Structural evidence for the interaction of
muscle myolaments. Methods 56(1):3343. C-protein (MyBP-C) with actin and sequence identication of a possible actin-binding
8. Squire JM (2009) Muscle myosin laments: Cores, crowns and couplings. Biophys Rev domain. J Mol Biol 331(3):713724.
1(3):149160. 29. Moolman-Smook J, et al. (2002) Identication of novel interactions between domains
9. Woodhead JL, et al. (2005) Atomic model of a myosin lament in the relaxed state. of myosin binding protein-C that are modulated by hypertrophic cardiomyopathy
Nature 436(7054):11951199. missense mutations. Circ Res 91(8):704711.
10. Alamo L, et al. (2008) Three-dimensional reconstruction of tarantula myosin laments 30. Oakley CE, Chamoun J, Brown LJ, Hambly BD (2007) Myosin binding protein-
suggests how phosphorylation may regulate myosin activity. J Mol Biol 384(4): C: Enigmatic regulator of cardiac contraction. Int J Biochem Cell Biol 39(12):21612166.
780797. 31. Stewart MA, Franks-Skiba K, Chen S, Cooke R (2010) Myosin ATP turnover rate is
11. Wendt T, Taylor D, Trybus KM, Taylor K (2001) Three-dimensional image re-
a mechanism involved in thermogenesis in resting skeletal muscle bers. Proc Natl
construction of dephosphorylated smooth muscle heavy meromyosin reveals asym-
Acad Sci USA 107(1):430435.
metry in the interaction between myosin heads and placement of subfragment 2.
32. Hooijman P, Stewart MA, Cooke R (2011) A new state of cardiac myosin with very
Proc Natl Acad Sci USA 98(8):43614366.
slow ATP turnover: A potential cardioprotective mechanism in the heart. Biophys J
12. Al-Khayat HA, Morris EP, Squire JM (2004) Single particle analysis: A new approach to
solving the 3D structure of myosin laments. J Muscle Res Cell Motil 25(8):635644. 100(8):19691976.
13. Al-Khayat HA, Morris EP, Kensler RW, Squire JM (2006) 3D structure of relaxed sh 33. Naber N, Cooke R, Pate E (2011) Slow myosin ATP turnover in the super-relaxed state
muscle myosin laments by single particle analysis. J Struct Biol 155(2):202217. in tarantula muscle. J Mol Biol 411(5):943950.
14. Al-Khayat HA, Morris EP, Kensler RW, Squire JM (2008) Myosin lament 3D structure 34. Colson BA, et al. (2010) Differential roles of regulatory light chain and myosin binding
in mammalian cardiac muscle. J Struct Biol 163(2):117126. protein-C phosphorylations in the modulation of cardiac force development. J Physiol
15. Al-Khayat HA, Morris EP, Squire JM (2009) The 7-stranded structure of relaxed scallop 588(Pt 6):981993.
muscle myosin laments: Support for a common head conguration in myosin-reg- 35. Kensler RW, Harris SP (2008) The structure of isolated cardiac myosin thick laments
ulated muscles. J Struct Biol 166(2):183194. from cardiac myosin binding protein-C knockout mice. Biophys J 94(5):17071718.
16. Zoghbi ME, Woodhead JL, Moss RL, Craig R (2008) Three-dimensional structure of 36. Kensler RW, Levine RJ (1982) An electron microscopic and optical diffraction analysis
vertebrate cardiac muscle myosin laments. Proc Natl Acad Sci USA 105(7):23862390. of the structure of Limulus telson muscle thick laments. J Cell Biol 92(2):443451.
17. Seidman CE, Seidman JG (2011) Identifying sarcomere gene mutations in hypertro- 37. Stewart M, Kensler RW (1986) Arrangement of myosin heads in relaxed thick la-
phic cardiomyopathy: A personal history. Circ Res 108(6):743750. ments from frog skeletal muscle. J Mol Biol 192(4):831851.
18. Marston SB (2011) How do mutations in contractile proteins cause the primary fa- 38. Kensler RW, Stewart M (1983) Frog skeletal muscle thick laments are three-stranded.
milial cardiomyopathies? J Cardiovasc Transl Res 4(3):245255.
J Cell Biol 96(6):17971802.
19. Dominguez R, Freyzon Y, Trybus KM, Cohen C (1998) Crystal structure of a vertebrate
39. Crowther RA, Henderson R, Smith JM (1996) MRC image processing programs. J Struct
smooth muscle myosin motor domain and its complex with the essential light chain:
Biol 116(1):916.
Visualization of the pre-power stroke state. Cell 94(5):559571.
40. van Heel M, et al. (2000) Single-particle electron cryo-microscopy: Towards atomic
20. Liversage AD, Holmes D, Knight PJ, Tskhovrebova L, Trinick J (2001) Titin and the
sarcomere symmetry paradox. J Mol Biol 305(3):401409. resolution. Q Rev Biophys 33(4):307369.
21. Knupp C, Luther PK, Squire JM (2002) Titin organisation and the 3D architecture of 41. da Fonseca PCA, He J, Morris EP (2012) Molecular model of the human 26S protea-
the vertebrate-striated muscle I-band. J Mol Biol 322(4):731739. some. Mol Cell 46(1):5466.
22. Bucher RM, Svergun DI, Muhle-Goll C, Mayans O (2010) The structure of the FnIII 42. Navaza J, Lepault J, Rey FA, Alvarez-Rua C, Borge J (2002) On the tting of model
tandem A77-A78 points to a periodically conserved architecture in the myosin- electron densities into EM reconstructions: A reciprocal-space formulation. Acta
binding region of titin. J Mol Biol 401(5):843853. Crystallogr D Biol Crystallogr 58(Pt 10 Pt 2):18201825.

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