Cryo-EM structure of the autoinhibited state of myosin-2

Abstract

We solved the near-atomic resolution structure of smooth muscle myosin-2 in the autoinhibited state (10S) using single-particle cryo–electron microscopy. The 3.4-Å structure reveals the precise molecular architecture of 10S and the structural basis for myosin-2 regulation. We reveal the position of the phosphorylation sites that control myosin autoinhibition and activation by phosphorylation of the regulatory light chain. Further, we present a previously unidentified conformational state in myosin-2 that traps ADP and P_i produced by the hydrolysis of ATP in the active site. This noncanonical state represents a branch of the myosin enzyme cycle and explains the autoinhibition of the enzyme function of 10S along with its reduced affinity for actin. Together, our structure defines the molecular mechanisms that drive 10S formation, stabilization, and relief by phosphorylation of the regulatory light chain.

Fig. 1.. Cryo-EM structure of 10S.

(A) Cryo-EM reconstruction of 10S in the front, side, and back view. Individual subunits are segmented and color-coded. The two heavy chains (blue and yellow) and bound ELC (green) and RLC (pink) are shown. (B) Atomic model of 10S fitted in the final electron density map. Individual subunits are segmented and color-coded according to (A). (C) Representative cryo-EM densities of different regions of 10S. Densities of the nucleotide (3.4 Å) and P_i displayed at 12.5 σ contour level. Densities of segment 1 (4.5 Å), segment 2 (5 Å), and segment 3 (5.5 Å) displayed at 8.0 σ contour level.

Fig. 2.. Interactions of the myosin heads.

(A) Interactions of loop-4_BH at the BH-FH interface. Potential interactions (distance > 4 Å) between R371_BH and Q771_FH and Q385_BH and Q728_FH are shown. (B) Interactions of CM-loop_BH at the BH-FH interface. Potential interactions (distance > 4 Å) between D412_BH and R253_FH and R406_BH and E169_FH are shown. (C) Interaction between the FH and segment 1. (D) Interaction between the converter_BH and segment 2. (E) Interaction between the NTD_BH and segment 2. (F) Interaction between the HO linker_BH and segment 3. The views in (C) to (F) are rotated 180° relative to the overview figure to highlight key interactions at the respective interfaces.

Fig. 3.. Active site and nucleotide interactions.

(A) Orientation of the lever arm in BH and FH in comparison to the pre-powerstroke state (PDB ID: 1QVI). (B) Active site and nucleotide interactions in the FH. Key residues are shown in stick representation. The distance between R247 and E470 is >6.1 Å. (C) Active site and nucleotide interactions in the BH. Key residues are shown in stick representation. (D) Comparison between the active sites in BH and FH. Key residues are shown in stick representation. (E) Comparison between the active sites in BH, FH, and the pre-powerstroke state (PDB ID: 2XEL). The salt bridge between switch-1 R247/R238 and switch-2 E470/E459 is only formed in the pre-powerstroke state and absent in BH and FH. Key residues are shown in stick representation. (F) Comparison of the phosphate release tube in the BH and FH.

Fig. 4.. Light chain interactions.

(A) Interactions between the RLC_BH (salmon) and the RLC_FH (dark purple). The PD of the RLC_FH forms extensive intra and intersubunit interactions. (B) RLC_BH interacts with segment 3. (C) Position of the phosphorylatable residue S19 is shown for RLC_BH (wheat spheres) and RLC_FH (orange spheres). The resolved PDs of the RLCs are shown in red. S19_FH is sandwiched between both RLCs; S19_BH is in close proximity to segment 3 (gray). (D) Different binding modes of the RLC_FH and the RLC_FH to the respective lever arms. (E) Interactions between the ELC_BH with the RLC_BH and the lever arm_BH. (F) Interactions between the ELC_BH and segment 2. (G) Interactions between the ELC_FH and the RLC_FH. (H) Interactions between the ELC_BH, the BH, and the lever arm_BH.

Fig. 5.. Model for 10S relief by RLC phosphorylation.

In the off state, phosphorylation of the RLC_BH on S19 (pS19) causes structural transitions to the pre-powerstroke state (RLC_BH,PPS, PDB ID: 1QVI; pS19 modeled) that make the RLC_FH available for phosphorylation. Collectively, this is expected to result in 10S relief and the subsequent activation of myosin motor activity.