Analysis of Nucleotide Binding to DictyosteliumMyosin II Motor Domains Containing a Single Tryptophan Near the Active Site

Mihály Kovács,András Málnási-Csizmadia,Robert J Woolley,Clive R Bagshaw

doi:10.1074/jbc.m202180200

Mihály Kovács, András Málnási-Csizmadia + Show 2 more

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https://doi.org/10.1074/jbc.m202180200

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Abstract

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop.

Highlights

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding
We found that tryptophan at position 113 in a D. discoideum construct does respond with a small enhancement (3–5%) upon nucleotide binding; the residue at 131 gives a large (30%) quench, so overall the enhancement observed with vertebrate skeletal myosin cannot be accounted by simple analogy with the D. discoideum model constructs
We focus on the response of tryptophan residues located near the myosin nucleotide binding site

Summary

Introduction

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. We demonstrated that tryptophan at this position shows a small quench on nucleotide binding (10 –20%) followed by a large enhancement (50 – 80%) associated with a major conformational change (4), presumably equivalent to the open-closed transition identified by x-ray crystallography (6). The latter step is freely reversible and slightly favors the open state (low fluorescence M†1⁄7ATP) in the presence of ATP under ambient conditions, but coupling to the hydrolysis step pulls the equilibrium over to the more stable M*ADP1⁄7Pi state with enhanced fluorescence (5). The large quench associated with Trp[131] and of another construct, Trp[129], provided valuable signals to characterize the nucleotide binding steps that were otherwise unob-

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