X-ray Structures of the MgADP, MgATPγS, and MgAMPPNP Complexes of the Dictyostelium discoideum Myosin Motor Domain,

Abstract

The three-dimensional structures of the truncated myosin head from Dictyosteliumdiscoideum myosin II (S1dC) complexed with MgAMPPNP, MgATPγS, and MgADP are reported at 2.1, 1.9, and 2.1 Å resolution, respectively. Crystals were obtained by cocrystallization and were isomorphous with respect to those of S1dC·MgADP·BeF_x [Fisher, A. J., et al. (1995) Biochemistry34, 8960−8972]. In all three structures, the electron density for the entire nucleotide was clearly discernible. The overall structures of all three complexes are very similar to that of the beryllium fluoride complex which suggests that the differences in the physiological effects of ATPγS and AMPPNP are due to the changes in the equilibrium between the actin-bound and actin-free states of myosin caused by the lower affinity of AMPPNP for myosin. In S1dC·MgAMPPNP, the presence of the bridging nitrogen prompts the side chain of Asn²³³ to rotate which disrupts the hydrogen bonding pattern in the nucleotide binding pocket and alters the water structure surrounding the ribose hydroxyl groups. It appears that this change is responsible for the reduced affinity of AMPPNP for myosin relative to ATPγS. In contrast to the G-proteins, there is no major change in the conformation of the ligands that coordinate the nucleotide in S1dC·MgADP. This is due to three water molecules that adopt the approximate positions of the three oxygens on the γ-phosphate and maintain the interactions with the Mg²⁺ ion and protein molecule. Interestingly, the thiophosphate group is evident in S1dC·MgATPγS even though it is slowly hydrolyzed by myosin. This suggests that the conformation observed here and in chicken skeletal myosin subfragment-1 [Rayment, I., et al. (1993) Science261, 50−58] is unable to hydrolyze ATP and represents the structure of the prehydrolysis weak binding state of myosin.