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Padrón Lab Research Summary

How the thick filaments of striated muscle become activated during muscle contraction?

 

Padrón Lab research focuses on how thick filaments of striated muscle are activated during muscle contraction,
studying the molecular mechanism of myosin-linked regulation.


Muscle is formed by the overlapping of two sets of filaments, the thin actin-containing and the thick myosin-containing filaments. The thick filaments of striated muscle are polymers of myosin II. The tails of myosin molecules are packed together forming a backbone, whereas the two heads from each myosin molecule are arranged on the surface, forming helices in the relaxed state. The contraction of muscle occurs when these sets slide actively one against the other, shortening the sarcomere. Muscle contraction needs to be controlled in an effective way such that this force production could be useful for programmed movement. Nervous stimulation causes release of calcium from the sarcoplasmic reticulum. Calcium can control the initiation of muscular contraction either by acting on actin filaments (thin filament regulation) and/or by acting on myosin filaments (thick filament regulation).

 

The molecular basis of actin-linked regulation is well understood, following the advances on the X-ray diffraction and electron microscopic (EM) studies of the thin filaments. Thin filament regulation is mediated by calcium binding to troponin-C, and involves -via the troponin complex- the movement of tropomyosin towards the thin filament groove removing the steric block that hinder the attachment of myosin heads to actin molecules. There are two structural states for thin filaments: the switched-OFF state, in which tropomyosin blocks the myosin binding site of actin molecules; and the switched-ON state in which the tropomyosin move away into the groove releasing the myosin binding site.  On the other hand, the molecular basis of the myosin-linked regulation is not well understood. Myosin-linked regulation occurs either by direct calcium binding to the myosin light chains (as in scallops); or by phosphorylation of the myosin regulatory light chains (RLC), (as in arthropod chelicerates, vertebrate striated and smooth muscle).  The limiting step to advance towards the understanding of the molecular mechanism of  myosin-linked regulation is the determination of the molecular structure of thick filaments. The species used has been an important factor: the thick filaments from the tarantula leg muscles have proved to be the most easily preserved specimens.

 

In spite of advances on the elucidation of the molecular mechanism of actin-linked regulation, the myosin-linked regulation mechanism has been less amenable to structural studies, due to the low resolution of 3D-maps. A combination of adequate specimens, a negative staining method that preserve the helices and the use of improved reconstruction techniques, allowed the calculation of a 5 nm resolution tarantula 3D-map. This 3D-map was interpreted with the available myosin head structural information, but fitting of atomic structures remained ambiguous due to limited resolution. Cryo-electron microscopy of frozen-hydrated tarantula thick filaments extended resolution up to 2.5 nm, and use of single particle averaging techniques enabled calculation of a 3D-map. This 3D-map was interpreted unambiguously by fitting the heavy meromyosin (HMM) atomic structure to it, leading to the atomic model of the relaxed thick filament, which revealed intra- and intermolecular interactions that keep myosin heads forming helices closer to the backbone surface (Nature 436:1195-1199, 2005).

 

The new map showed a detailed repeating motif on the filament surface (fig.1a) representing myosin heads pairs.  This map was interpreted when realizing the close similarity with the reported interacting-heads atomic model of HMM, obtained by EM from 2D crystals of smooth muscle. This interacting-heads atomic model is in the switched-OFF state, with dephosphorylated RLC, and the myosin shows an asymmetric interaction between its two heads, with the actin-binding region of one (blocked) head interacting with the converter and ELC regions of the (free) head (fig 1b). This heads-down, pointing to bare zone interpretation was a completely unexpected surprise. The interacting-heads atomic structure fits precisely into the map as confirmed by fitting the motor and light chain domains of both heads, indicating that this structure derived from smooth muscle is also present in striated muscle. The strong similarity between structures of myosin molecules isolated from smooth chicken muscle and from striated tarantula muscle suggest that this interacting-heads structure may underlie the relaxed state of thick filaments, over a wide range of species. Figure 2 shows our current thinking on the structure of thick filaments of tarantula striated muscle in the relaxed state. Myosin molecules, with heads in the asymmetric interacting-heads configuration are packed together forming four helices of myosin head pairs which form the relaxed thick filament.

Figure 1
Atomic model of the tarantula thick filament

(a) The atomic model of the tarantula thick filament reveal  three intramolecular interactions, two between the blocked-head actin binding domain (green) and the ELC (purple), and the converter domain (blue) of the free-head of the same molecule (yellow ellipsis); and, another between the actin binding domain (green) and the S2 (red) of the same molecule (yellow arrow); and two intermolecular interactions, one interaction between the actin binding domain (blue) of the free-head of a myosin head and the ELC (orange) of the neighboring blocked-head (red ellipsis), with another interaction between the myosin tail (red)  and the SH3 domain (green) of the neighboring blocked-head (red bracket).

(b) The  atomic model of tarantula heavy meromysoin.

(c) Diagram summarizing the behaviour of the mysoin heads. Left: in the relaxed (switched OFF) state, in the absence of calcium (or with the RLC dephosphorylated), the heads are rigid, pointing back toward the tail and interacting bewten them. Right: following activation (switched ON), after RLC phosphorylation (or in thepresence of calcium), the heads become flexible, and get disordered.

Reprinted from Padrón, R. & Alamo, L. Review: The use of negative staining and cryo-electron microscopy to understand the molecular mechanism of myosin-linked regulation of striated muscle contraction. Acta Microsc. 13: 1 – 29, 2006, with permission.
Credit: Raúl Padrón & Lorenzo Alamo.

Why do myosin heads form helices in the relaxed state? The myosin heads form helices because they are in the switched-OFF conformation in which specific interactions occurs between both heads by forming three intramolecular interactions, stabilizing the head pairs; and because the head pairs interact between then by forming two intermolecular interactions. Therefore due to these interactions the thick filament has the myosin heads helically arranged, packed down on the surface, away from the thin filaments.
Figure 2
How thick filaments are switched OFF or ON
Our current interpretation of the structure of the thick filament of tarantula muscle formed by myosin molecules (top left) with their heads rigid pointing back to the myosin tail when in the relaxed OFF state (RLC dephosphorylated). This switched OFF structure of the thick filament warrants that the myosin heads are held down close to the filament backbone (top right), such that they can not interact with the thin filaments, and shown in the relaxed sarcomere. When calcium, via calmodulin and the myosin light chain kinase phosphorylates the myosin RLC, they switched ON  the myosin heads become flexible, and disordered, and move away from the filament surface, protruding towards the thin filaments, and activating the thick filament. The activated filaments can interact with the thin filaments, if they are also switched ON, producing force, and sliding of both sets of filaments one against the other, shortening the sarcomere, as shown in the contracting sarcomere.

Reprinted from Investigación y Ciencia (Spanish version of Scientific American). Padrón, R. El modelo atómico del filamento de miosina. 2007, with permission from Prensa Científica, S. A.
Credit: Lorenzo Alamo


How this increased understanding on the atomic structure of the tarantula thick filaments can help understanding the molecular mechanism of myosin-linked regulation of muscle contraction?  How are thick filaments activated? We think that the determination of the molecular structure of these thick filaments can help understanding the phosphorylation-type of myosin-linked regulation. For a myosin head to interact with the thin filaments, to produce force, the thick filaments must first become activated. EM and X-ray evidences suggests that RLC phosphorylation  is involved in breaking the interactions, activating thick filaments by disordering and releasing the heads, enabling their interaction with thin filaments. We envisage the activation of  thick filaments as the physiological release of heads from the surface before force development; leading to the actual interaction between activated heads and thin filaments, and producing contraction and shortening of sarcomere. In tarantula muscle, it is still not clear if RLC phosphorylation directly regulate (as in smooth muscle and Limulus) or just modulate the number of heads that become activated. This last mechanism could be a way to modulate the number of heads involved actively in producing force after a series of twitches in tetanus.

 

Why on activation the myosin heads get disordered and protrude away from the surface following RLC phosphorylation? Isolated myosin molecules of  scallop striated muscle in the switched OFF-state have their two heads held down towards it tails forming a rigid structure with low ATPase activity, and when they are switched-ON by  calcium binding the heads become flexed randomly around the tail junctionl. In tarantula thick filament, a similar situation can take place, suggesting that intra and intermolecular interactions, as well as intermolecular interaction are diminished after RLCs are phosphorylated. Thus head–head interactions previously observed in single molecules, and suggested to be the structural mechanism by which actomyosin ATPase is switched-OFF, is also a key feature of relaxed native filament. Further experiments in which the resolution of the tarantula 3D map is increased enough using field emission gun (FEG) cryo-electron microscopy or tomography should help to define more precisely the specific nature of the intra- and intermolecular interactions involved in the activation mechanism.

Figure 3
Animated crossbridge cycle, showing the activation step

Adapted from from Investigación y Ciencia (Spanish version of Scientific American). Padrón, R. El modelo atómico del filamento de miosina. 2007, with permission from Prensa Científica, S. A.
Credit of animation: Lorenzo Alamo

In summary, the structure of thick filaments of striated muscle in the relaxed state has been finally understood at the molecular level. The structure reveals intra- and intermolecular interactions that keep heads together forming helices close to the surface of the thick filament. The RLC phosphorylation induces the weakening of these interactions allowing the activation of thick filaments, producing disordering and releasing out of heads, and enabling their interaction with thin filaments. These results have opened the way to understand the molecular mechanism of myosin-linked regulation of muscle contraction, which is important as mutations associated with mid-ventricular cardiomiopathy occurs in the RLC near the phosphorylation site.