The concept that MyBP-C might bridge between thick and thin filaments and thus modulate muscle contraction has existed ever since it was first shown that this myosin-binding protein could also bind to F-actin23
. This possibility has been strengthened by subsequent in vitro
studies showing binding to regulated thin filaments24, 26
, and the location of the interaction site has been narrowed to the N-terminal region, primarily the C1 and M domains25, 27, 28
). In vitro
motility studies demonstrate that cMyBP-C slows actin-myosin sliding, an effect that is reduced by cMyBP-C phosphorylation27, 33-36
. A simple interpretation of these results is that cMyBP-C acts as a phosphorylation-dependent tether between the two filaments, modulating their sliding. Direct observation of MyBP-C links between thick and thin filaments in intact muscle suggests that actin interaction is not an in vitro
artifact, but occurs in vivo30
. Our study of the binding of N-terminal fragments of MyBP-C to F-actin was designed to provide new insights into the nature of the actin end of this putatively crucial interaction.
Our EM images and reconstructions directly support the view that it is the N-terminal domains of cMyBP-C that bind to actin25, 27, 28
. First, even the shortest, most N-terminal fragments, C0C1 and C0C1f, appear to bind to actin. Second, we find that the diameter of the decorated filaments increases with fragment length (). All the fragments have C0 and C1 in common, and elongate in the C-terminal direction when additional domains are added (). Assuming that the attachment site to actin is similar in all cases, the increase in diameter is consistent with the addition of C2 and C3 distal to the binding site and above the filament surface, rather than being directly attached to actin. The reconstructions support this view. C0C1f-decorated filaments show a small protrusion on SD1 of actin. With the addition of C2 and then C3, the attached density increases in length and extends further above the surface of the actin filament core in the direction of the pointed end. However, the actin attachment site itself appears similar in each case, covering a region of SD1 centered over the N-terminus of actin. While there is substantial coincidence between the distal, C-terminal regions of the C0C2 and C0C3 fragments in the two reconstructions, there appeared to be some variation in their precise positions. This may reflect some flexibility in the molecule (e.g. due to flexible linkers between the globular domains). In addition, as each domain is added, the fragments could adopt different conformations in their C-termini made possible by new potential interactions between the added domains. An example is shown with C0C3, where the C-terminal domains of one molecule come close to the more N-terminal domains of the next MyBP-C up (, Supplementary Movie 2
). While interaction between domains of neighboring MyBP-C molecules may be possible in this synthetic structure, it would not occur in intact muscle, being precluded by the low MyBP-C:myosin stoichiometry and large (43 nm) axial distance between neighboring MyBP-Cs on the thick filament (see later).
Best-fit position of two C0C3 fragments in C0C3 reconstruction (), superimposed on F-actin reconstruction and shown in relation to tropomyosin and myosin head positions
While most studies suggest that binding of MyBP-C's N-terminal region to actin is specific25, 27, 32, 36
, this view is not universal, and it has been suggested that specific binding to actin might actually involve the C-terminal half of the molecule29
. Our observation that N-terminal fragments bind in a regular way to the actin helix, as demonstrated by Fourier transforms of decorated filaments (not shown) supports the specificity of the interaction. The same conclusion was reached based on the appearance of filtered images and of actin layer lines in Fourier transforms of filaments decorated with C0C2, also under these salt conditions38
. Specific binding is also indicated by our 3D reconstructions (also implied by44
). Our use of helical reconstruction methods ensures that only features having the same helical symmetry as F-actin appear in the final density map; the presence in the reconstructions of substantial mass attached to actin with the actin helical symmetry implies a regular and specific interaction. Finally, while our experiments were carried out at three salt levels, all producing similar looking decoration, the highest was 180 mM which would minimize non-specific electrostatic binding; it was this level that was used for our reconstructions. We conclude that there is substantial regularity and hence specificity to the interaction with actin.
To understand whether and how MyBP-C might modulate filament interaction and contraction in muscle, we have compared its location on actin to that of tropomyosin and myosin heads. Fitting atomic models of thin filaments in high and low Ca2+
to the reconstruction of C0C3-decorated filaments suggests that MyBP-C would not significantly interfere with tropomyosin in the high Ca2+
position (the “closed” state), but that there may be some interference in the low Ca2+
position (the “blocked” state; , Supplementary Movies 3
). (As tropomyosin is located even further away from SD1 in the myosin-induced (“open”) state45, 46
, any clash with cMyBP-C is even less likely than in the closed state; Supplementary Fig. 2
.) However, there is a clear steric interference between the binding sites of MyBP-C and strongly bound myosin heads (rigor or end-of-power-stroke heads) on actin SD1, and this would prevent both from binding simultaneously (, Supplementary Movie 5
These structural results are in good agreement with biochemical and other observations. Several studies have shown that MyBP-C (and N-terminal fragments) can bind to thin filaments at high Ca2+ 24-26
and to actin-tropomyosin without troponin23
: in both cases tropomyosin is in the closed position, which does not appear to clash with cMyBP-C in our reconstruction. In the low Ca2+
state (blocked position) binding is also found, although it may be weaker25, 26
, consistent with the apparent partial overlap of tropomyosin and cMyBP-C binding positions seen in the reconstruction (, Supplementary Movies 3
). Electron tomography of muscle sections in the relaxed (low Ca2+
) state shows that MyBP-C extends out from thick filaments and binds to thin filaments in the intact filament lattice30
, suggesting that any steric interference in relaxed muscle in vivo
is minimal. Such interactions may serve in vivo
to organize and stabilize the filament lattice in the relaxed state. The strong binding of S1 to actin that occurs in the absence of ATP has been shown to greatly reduce MyBP-C binding23, 32
, consistent with our observation of overlapping binding sites. Indeed, S1's high affinity for actin in the ATP-free state is sufficient to displace MyBP-C23, 32
. The extension of MyBP-C axially over two actin subunits deduced from our fitting readily explains why S1 bound to actin at only half saturation is able to compete off essentially all MyBP-C attached to actin filaments23
. In the presence of ATP, when myosin heads are actively cycling and bind more weakly to actin, both whole MyBPC and N-terminal fragments inhibit actomyosin ATPase activity and S1 binding23, 27, 32
, again consistent with the location of cMyBP-C's N-terminal binding site on actin SD1.
motility assays of the effect of C0C2 on the movement of regulated thin filaments (containing tropomyosin and troponin) show that thin filaments at high Ca2+
(and also F-actin alone) are slowed by the presence of C0C2, whereas at low Ca2+
their velocity is increased
in the presence of C0C227
. Our reconstruction suggests a possible explanation in terms of the binding sites of cMyBP-C and tropomyosin-troponin. At high Ca2+
(tropomyosin in the closed position45
) cMyBP-C and tropomyosin binding sites do not overlap, which would allow uninhibited binding of cMyBP-C to its site on actin SD1. Its binding could thus exert a significant drag on thin filament sliding. In contrast, the reconstruction suggests that cMyBP-C and tropomyosin compete for part of the same SD1 surface of actin when tropomyosin is in the low Ca2+
(blocking) position. If so, cMyBP-C could destabilize tropomyosin in its blocking position, tending to activate the thin filament, thus enhancing its motility.
If comparable effects occur in vivo
, this would suggest that cMyBP-C may contribute (along with troponin) to a delicate balance between the blocked and closed positions of tropomyosin, poising the thin filament between its switched-off and switched-on regulatory states. In the case of cardiac MyBP-C, phosphorylation weakens MyBP-C actin binding, which could contribute to cMyBP-C's role in modulating cardiac activity. Other factors also come into play in intact muscle. The MyBP-C:actin molar ratio in the myofibril is approximately 1:30, and thus thin filaments could never be highly decorated in muscle as they are in our in vitro
experiments. In addition, MyBP-C is confined to the middle third of each half sarcomere and here occurs only every 43 nm (similar to the actin helical repeat). Thus only a limited region of the thin filament can interact with MyBP-C at any particular sarcomere length, and here the interactions must be relatively sparse. Nevertheless, in light of the substantial stiffness of tropomyosin47, 48
and the consequent ability of structural changes to be transmitted considerable distances along the thin filament46
, even these limited interactions could have significant effects in vivo
. Such interactions, of course, must necessarily be relatively weak and in rapid equilibrium to enable the sliding of filaments that is essential for muscle shortening.
The arrangement of the C0C3 fragment on F-actin is such that the C0, C1 and M domains bind on or near SD1 while C2 and C3 extend to higher radius, lying above the next actin subunit in the direction of the pointed end (, Supplementary Movies 2
). This domain arrangement is quite similar to that proposed by Whitten et al.37
based on modeling of neutron scattering data from C0C2-decorated F-actin, except that the M-domain in their model is well above, and does not bind to, the actin surface. Our fitting is consistent with the conclusion from comparative binding studies of different N-terminal fragments that the M-domain is critical for actin binding, and may interact with actin through electrostatic attraction between its high concentration of positively charged residues and negative charge on actin SD125, 27, 36
. The stronger and more obvious binding that we see with C0C1f compared with C0C1 (which lacks the first part of the M-domain), supports a role of the M-domain in direct binding to actin. As suggested by Shaffer et al.25
, the higher pH (8.0) of the neutron scattering experiments may cause the M-domain to dissociate from actin, accounting for its different positioning compared with our reconstruction (pH 7.4). The potential clash between the C0 and C1 domains and tropomyosin at low but not high Ca2+
that we observe is also supported by the model of Whitten et al.37
based on neutron scattering.
We conclude that cMyBP-C binds by its N-terminal domains to SD1 of actin at a site that can potentially modulate tropomyosin and myosin head binding in different physiological states. These findings, together with recent information showing that MyBP-C can bind to thin filaments in intact muscle30
and its known ability to bind to myosin subfragment 2 in a phosphorylation-dependent way21, 22
, add support for and insight into the mounting evidence that MyBP-C plays a key role in muscle contraction.