Allosteric communication in Dictyostelium myosin II
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Myosin’s affinities for nucleotides and actin are reciprocal. Actin-binding substantially reduces the affinity of ATP for myosin, but the effect of actin on myosin’s ADP affinity is quite variable among myosin isoforms, serving as the principal mechanism for tuning the actomyosin system to specific physiological purposes. To understand the structural basis of this variable relationship between actin and ADP binding, we studied several constructs of the catalytic domain of Dictyostelium myosin II, varying their length (from the N-terminal origin) and cysteine content. The constructs varied considerably in their actin-activated ATPase activity and in the effect of actin on ADP affinity. Actin had no significant effect on ADP affinity for a single-cysteine catalytic domain construct, a double-cysteine construct partially restored the actin-dependence of ADP binding, and restoration of all native Cys restored it further, but full restoration of function (similar to that of skeletal muscle myosin II) was obtained only by adding all native Cys and an artificial lever arm extension. Pyrene-actin fluorescence confirmed these effects on ADP binding to actomyosin. We conclude that myosin’s Cys content and lever arm both allosterically modulate the reciprocal affinities of myosin for ADP and actin, a key determinant of the biological functions of myosin isoforms.
KeywordsActin Cysteine Lever arm ADP Fluorescence
Fluorescence measurements were performed in the Biophysical Spectroscopy Facility, University of Minnesota. We thank Sarah Blakeley, Christina Yi, and Evan Smith for technical assistance and to Octavian Cornea for assistance with manuscript preparation. This work was supported by NIH grants to DDT (AR32961 and AG26160), and PG was supported by the Minnesota Muscle Training Grant, NIH T32 AR007612.
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