H E A L T H I N N O V A T I O N , O V E R C O M I N G D I S E A S E S A N D P A N D E M I C S

S C I E N T I F I C H I G H L I G H T S

2 4 H I G H L I G H T S 2 0 2 3 I

X-ray diffraction shows how titin activates molecular motors in muscle

Muscle contraction is controlled by activation of both the thick filament, containing the myosin motors, and thin filament, containing the actin support. X-ray diffraction experiments show that titin takes part in thick filament activation by biasing the myosin motor orientation towards the actin.

Skeletal and cardiac (striated) muscles are made up of sarcomeres (Figure 12a), ~2 μm-long structural units composed of overlapped thick, myosin-containing and thin, actin-containing filaments. Force generation and shortening are due to the cyclical interactions of myosin motors extending from the thick filament and the nearby thin filaments.

Contraction is triggered by the increase in intracellular calcium concentration elicited by the action potential. Calcium binding to troponin (Figures 12b and c) induces a change in the azimuthal position of tropomyosin along the thin filament, making actin sites available for interaction with myosin motors. In the resting muscle, myosin motors lie folded back on their tails in three- stranded helical tracks on the surface of the thick filament (OFF state, upper panel in Figure 12c), unable to reach the actin filament and split ATP. Previous X-ray diffraction

experiments at beamline ID02 [1,2] showed that during contraction, a mechano-sensing mechanism in the thick filament switches myosin motors ON, moving them away from their energetically convenient OFF state (lower panel in Figure 12c) as a function of the load. The molecular basis of the OFF-ON switch has still to be defined, even if it is known that an important role is played by other sarcomeric proteins (Figure 12b) such as the myosin binding protein-C (MyBP-C) and the cytoskeleton protein titin, and that mutations in these proteins are responsible for skeletal and cardiac myopathies.

Titin is a giant protein that spans the whole half- sarcomere. Titin in the sarcomere I-band acts as a molecular spring, in parallel with myosin motors, and is responsible for passive force developed at long sarcomere length (SL). However, at physiological SL (<2.6 μm), tandem immunoglobulins in the poly-Ig segment (Figure 12b) are randomly bent, so that the I-band titin is quite extensible and cannot represent a mechanical link able to sustain mechano-sensing in myosin filaments unless it becomes much stiffer during contraction.

In this work, the role of titin in thick filament regulation at physiological SL was investigated at beamline ID02 by combining half-sarcomere mechanics and X-ray diffraction in frog (Rana esculenta) single muscle cells (Figure 13a) stimulated in the presence of 20 μM para- nitro-blebbistatin (PNB, [3]), which suppresses the masking action of the in-parallel myosin motors.

Fig. 12: The sarcomere and its relevant protein components. a) Overview of the thick filament (blue), myosin motors (orange), and thin filament (yellow) at 2.2 μm sarcomere length (SL). b) Protein disposition on the thin (yellow) and thick (light blue) filaments in the half-sarcomere at rest at 2.2 μm SL, at which the 49 crowns of motors (orange) are overlapped by the thin

filament and only the half bare zone (HBZ) is excluded. Thin filament with tropomyosin (brown) and troponin complex (grey); MyBP-C (green) in the central 1/3 of the half-thick filament. Titin (magenta) with PEVK segment identified by dark magenta.

c) Overlap region on an enlarged scale for better resolution. Upper panel: motors in the off state. Lower panel: one motor dimer is switched on, moving away from the surface of the filament.