Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: Implications for the open-closed transition identified by crystallography

When myosin interacts with ATP there is a characteristic enhancement in try ptophan fluorescence which has been widely exploited in kinetic studies. Us ing Dictyostelium motor domain mutants, we show that W501, located at the e nd of the relay helix close to the converter region, responds to two indepe ndent conformational events on nucleotide binding. First, a rapid isomeriza tion gives a small fluorescence quench and then a slower reversible step wh ich controls the hydrolysis rate (and corresponds to the open-closed transi tion identified by crystallography) gives a large enhancement. A mutant lac king W501 shows no ATP-induced enhancement in the fluorescence, yet quenche d-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a pr oducts complex as in the wild-type. The nucleotidefree, open and closed sta tes of a single tryptophan-containing construct, W501+, show distinct fluor escence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transi tion does not require hydrolysis per se and can be induced by a nonhydrolyz able analogue. At 20 degrees C, the equilibrium may favor the open state, b ut with ATP as substrate, the subsequent hydrolysis step pulls the equilibr ium toward the closed state such that a tryptophan mutant containing only W 501 yields an overall 80% enhancement. These studies allow solution-based a ssays to be rationalized with the crystal structures of the myosin motor do main and show that three different states can be distinguished at the inter face of the relay and converter regions.