A Metal Switch for Controlling the Activity of Molecular Motor Proteins

Abstract

Molecular motors, such as kinesins and myosins, as well as molecular switches such as G-proteins, use the energy from nucleotide hydrolysis to carry out cellular tasks. In addition to the P-loop, these proteins use similar structural motifs, called switch 1 and switch 2, to sense and respond to the presence or absence of the gamma-phosphate of the nucleotides and coordinate nucleotide hydrolysis. We have developed a strategy to probe metal interactions within kinesin and myosin molecular motors, by taking advantage of the differential affinities of Mg(II) and Mn(II) for serine (-OH) and cysteine (−SH). We present the crystal structure of a kinesin motor domain bound to MnADP and report on a serine-to-cysteine substitution in the switch 1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn(II) to Mg(II). This mutant kinesin binds ATP similarly in the presence of either metal ion, but its ATP hydrolysis activity is greatly diminished in the presence of Mg(II). In kinesin-1, kinesin-5, kinesin-10 and kinesin-14, this defect is rescued by Mn(II), providing a way to control both the enzymatic activity and force-generating ability of these nanomachines. We also present ATPase data for an analogous substitution in the switch 1 of non-muscle myosin-2 from Dictyostelium discoideum. This mutant myosin shows aberrant actin interaction whereby actomyosin dissociation becomes slow and rate-limiting in the presence of Mg(II), yet is rescued to wild-type activity by Mn(II). These data are consistent with switch 1 being unable to efficiently close upon MgATP binding, thus affecting the opening of the upper 50K domain. There are several relevant and important applications to this metal switch technology that will allow further biophysical characterization of molecular motors and molecular switch proteins.