Resolution of Conformational States of Dictyostelium Myosin II Motor Domain Using Tryptophan (W501) Mutants: Implications for the Open-Closed Transition Identified by Crystallography † Andra ´s Ma ´lna ´si-Csizmadia, Robert J. Woolley, and Clive R. Bagshaw* Department of Biochemistry, UniVersity of Leicester, Leicester LE1 7RH. U.K. ReceiVed May 17, 2000; ReVised Manuscript ReceiVed October 18, 2000 ABSTRACT: When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide- free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20° C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions. Tryptophan fluorescence has long been used to character- ize myosin-nucleotide complexes in steady-state or transient kinetic studies (1, 2). ATP binding and hydrolysis by rabbit skeletal muscle myosin subfragments is associated with a 10-20% enhancement in tryptophan emission intensity while, at the end of the reaction, the product ADP remains bound to give about a 5% enhancement. These studies, in conjunction with quenched-flow methods, showed that the predominant nucleotide state during the steady-state hydroly- sis of ATP was a M**‚ADP‚Pi complex in a conformation distinct from the binary M*‚ADP state formed after complete turnover of the ATP or by direct addition of ADP to myosin. Nonhydrolyzable (e.g., AMP‚PNP) 1 or slowly hydrolyzable (e.g., ATPγS) nucleotides induced a fluorescence enhance- ment of amplitude between that observed for the M**‚ADP‚ Pi and M*‚ADP states (1, 2). Stopped-flow studies established that the nucleotide bind- ing process was at least two-step with a fluorescence change occurring during a first order isomerization step, most likely of a myosin-nucleotide binary complex (3). The higher amplitude observed on ATP binding and hydrolysis to give M**‚ADP‚Pi indicated part of the fluorescence change coincided with the hydrolysis step itself (or an additional conformation change required for rapid hydrolysis to occur). These studies led to a commonly accepted mechanism for the ATPase as where the asterisk (*) distinguishes conformational states and relates approximately to the level of tryptophan fluorescence enhancement (3, 4). An exact assignment of fluorescence level of M*‚ATP remained ambiguous because the enhance- ment by nonhydrolyzable ATP analogues is slightly greater than that of the corresponding M*‚ADP state (1). With ATP itself, M*ATP remains a minor species, at least at 20° C, and the observed fluorescence is dominated by M**‚ADP‚ Pi. These studies were performed before the high-resolution structure of the motor domain was solved by X-ray crystal- lography. Interest in this field has been rekindled by the need to relate nucleotide states observed in solution with those in crystals (5-8). In the latter case, nucleotide complexes (ADP‚AlF 4 , ADP‚Vi) which are thought to mimic the M**‚ ADP‚Pi state are associated with a movement of the switch II region toward the P i analogue group. This triggers a movement in residues at the C terminus of the motor domain and a large swing in the angle of the regulatory domain. This so-called open-closed transition at the active site is † This work was supported by BBSRC and the Wellcome Trust. * To whom correspondence should be addressed. Phone: +44 (0)- 116 252 3454. Fax: +44 (0)116 252 3369. E-mail: crb5@le.ac.uk. 1 Abbreviations: AMP‚PNP, adenosine 5′-(γ-imidotriphosphate); ATPγS, adenosine 5′-O-(3-thiotriphosphate); Dd, Dictyostelium dis- coideum; mant-, 2′(3′)-O-(N-methylanthraniloyl)-; Vi, vanadate; wt, wild-type. 16135 Biochemistry 2000, 39, 16135-16146 10.1021/bi001125j CCC: $19.00 © 2000 American Chemical Society Published on Web 12/02/2000