Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back

Abstract

Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation.

Fig. 1

The NTEs of the smooth and tarantula myosin RLCs. (A) Sequence alignment of the NTEs of chicken smooth (a) and tarantula striated (b) muscles. Phosphorylatable serines are in yellow (Ser35) and red (Ser45). Helix L (yellow), coil Pro/Ala linker (orange), helix P formed by PCK (blue) and MLCK (green) target consensus sequences, adjacent to the EF helix A (purple, Gln53-Ile66). (c) Vectors and angles as defined for the flexural rigidity analysis (see Methods). (B) Straightened tarantula RLC fragment used as the starting structure for MD simulations and flexural rigidity analysis. The 52-aa NTE (formed by helix L, linker and helix P) is adjacent to the RLC helix A (purple). The helix L has 9 positively charged lysines, and is connected by a flexible linker to the helix P, with includes the PKC (P_PKC, blue) and MLCK (P_MLCK, green) consensus sequences with their phosphorylatable Ser35 and Ser45.

Fig. 2

Snapshots showing key structural features of trajectories. The starting structure was assumed to be a straight α-helix. This peptide was equilibrated for the un-P (shown on the left part of A), pSer45, pSer35 and di-P MD simulations. Final simulation states of the stabilized structures are shown on A–D. Helix P bends bent over helix A in A–C but stays straightened on D. There is a linker extension at Pro31-Lys32 becomes coil/turn in A and C but not in B and D. Helix P is always bend at Ser45 except in D, where helices P and A remain straight. No salt bridges are established in un-P state A. Salt bridges (yellow arrows) are established for the mono-P states: pSer35/Arg38, Arg39 in C or pSer45/Arg39 in B; and salt bridges pSer35/Arg38, Arg39, and pSer45/Arg42 in D. Helix L (shadowed) moves freely around the flexible linker in all conditions, interacting eventually with helix-A in A–C, but in di-P (D) inter-helix pSer45/Lys15 salt bridge between helix L and A is established (blue arrow).

Fig. 3

op panels Evolution of secondary structure of NTE helix P along their trajectories: un-P (A), Ser45 (B) or Ser35 (C) mono-P, and di-P (D). Snapshots illustrating the final key structural features are shown in Fig. 2. Helix to coil/turns: at Pro31-Lys32 (A,C) (red arrowheads). HCH: at Ser43-Gly44 (A, B) and Gly44 (C) (black arrows). Vertical axis: one-letter code aa number. Secondary structure key colours: α-helix (pink), turn (cyan), coil (white). Centre panel: root mean square deviation (RMSD) of the NTE peptides trajectories of each phosphorylated state shown in Fig. 2. Arrows show that peptides stabilized after 100 (A), 530 (B), 300 (C) and 80 (D) ns. Bottom panel: Persistence length analysis (|θ| vs. time) of the NTEs. The angle |θ| captures the bending of the α-helix of the three vectors regions (Fig. 1Ac). The angle |θ| changed to ~90° for the un-P (A), to ~80° for Ser45 (B) or Ser35 (C) mono-P cases, and does not changed much (~10°) for the Di-P state (D). The result in (D) shows an important increase of straightening and rigidification (Table 1) only induced by diphosphorylation.

Fig. 4

The cooperative phosphorylation-controlled mechanism for recruiting active heads in tarantula thick filament activation.^,, The three myosin interacting-heads motifs on the left are shown along one thick filament helix with their entire RLC NTEs unphosphorylated (bare zone at the top). This mechanism shows how the Ser45 phosphorylation of the constitutive Ser35 monophosphorylated swaying free head NTE (b, centre IHM) hinders its docking back making it it permanently mobile (b, arrow). Two actuators (red and yellow boxes) control the sequential release of myosin heads on tarantula thick filament activation. Activating actuator (red boxes): according to our MD simulations the activating actuator is based on a disorder-to-order transition of the RLC NTE, which induces its elongation in accordance with the increase of the static and dynamic persistence length ξ_s and ξ_d. This implies a substantial straightening and rigidification of the diphosphorylated NTE, modifying the free head regulatory domain, and producing the release of these heads (b). The >16-fold increase in the free head NTE straightness and rigidity upon diphosphorylation would hinder docking back of this head after swaying away as it could not recover its original interacting stereospecific disposition, hindering as well the S2 intramolecular interaction so becoming release and mobile (b, arrow). The constitutively Ser35 monophosphorylated free heads diphosphorylation induces a disorder-to-order transition which fully elongates the helix P along the helix A by establishing three salt bridges pSer35/Arg38, Arg39, and pSer45/Arg42. Potentiating actuator (yellow boxes): In contrast to the activating actuator on the free head, the potentiation actuator on the blocked head is not based on a disorder-to-order transition of the RLC NTE. The blocked head Ser45 monophosphorylation does not produce any conformational change on the NTE, except a salt bridge between pSer45 and Lys39 or pSer45 and Lys37. a net negative charge reduction of -2 and an ~1.8-fold increase in dynamic persistence length (ξ_d) (Table 1), suggesting that blocked head NTE monophosphorylation at Ser45 makes it more rigid, hindering the docking back of its partner free head, making it also release and mobile (d, top arrow). This in turn could weaken the blocked head RLC NTE electrostatic interaction with a loop on the motor domain of the neighbour free head, making the blocked head to sway away (c). This is functionally important since the blocked heads monophosphorylation at Ser45 by MLCK is an effective way to recruit potentiating heads (Fig. 4c). FH and BH: free and blocked heads.