Visualization of head-head interactions in the inhibited state of smooth muscle myosin

Abstract

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.

Figure 1

Docking of the S1 coordinates into the 2-D EM map of unphosphorylated HMM. The motor domain (red for one head, pink for the other), the converter domain (green), the long alpha helix (yellow, and magenta for the hook), the ELC (orange), and the RLC (cream) are indicated by a ribbon diagram in a–c. a, One contour from the 2-D map with two S1 molecules docked before modification of the light chain binding domain orientation. b, An overview of the same orientation shown in a, but after rebuilding the light chain binding domain of the upper S1 molecule. c, The arrangement of the unphosphorylated myosin heads is shown in the half unit cell of the map. d, The projection map (left) was obtained by averaging the structure factors obtained from ten electron micrographs after correction for the CTF. Middle, The atomic coordinates are projected into a 2-D image for comparison with the original electron density map. Right, This 2-D projection is shown filtered to 2-nm resolution. One HMM motif is outlined in each map in d.

Figure 2

Docking of the S1 coordinates into the 2-D EM map of thiophosphorylated HMM. The myosin head structure in a–c is color coded as in Fig. 1. a, Two S1 molecules docked into one contour from the 2-D map. The same orientation is shown in b after rebuilding the light chain binding domain of the left S1 molecule. The lack of intramolecular interactions between two heads is shown in c. d, The projection map (left) was obtained from averaging the structure factors of 21 micrographs. The fitted coordinates were then projected into a 2-D image (middle) for comparison with the original electron density map and filtered to 2-nm resolution (right). One HMM molecule is outlined in each map in d.