Myosin Regulatory Light Chain and Nucleotide Modulation of Actin Binding Site Electric Charge

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Abstract
The ionic strength dependence of skeletal muscle myosin subfragment 1 (S1) binding to actin in the presence of ADP and ATP was measured for S1 with either only an essential light chain [S1(elc)] or with both an essential and the regulatory light chains [S1(elc,rlc)] bound. The data were analyzed to determine the apparent association constant, KA, for actin binding and the absolute value of the product of the net effective electric charges at the actin−myosin interface, |zMzA|. When MgADP is bound at the myosin active site, KA values at 0 M ionic strength for S1(elc) and S1(elc,rlc) are 12 and 4.9 × 106 M-1, respectively, and |zMzA| values are 3.9 ± 0.3 and 3.6 ± 0.2 esu2. In the presence of ATP, KA values at 0 M ionic strength for S1(elc) and S1(elc,rlc) are 81 and 7.3 × 104 M-1, respectively, and |zMzA| values are 14.7 esu2 for S1(elc) but only 6.4 esu2 for S1(elc,rlc). The Michaelis constant, KM, for the actin activation of S1 steady-state MgATPase activity was significantly smaller for S1(elc), consistent with its greater KA and |zMzA|. These data indicate that the regulatory light chain can allosterically regulate the interactions of myosin and actin by modulating the electric charge at the actin binding site. KA and |zMzA| were also measured at 25 °C for S1(elc,rlc) binding to actin in the presence of the ATP analog ATPγS. At 0 M ionic strength, KA is 8.0 × 104 M-1, and |zMzA| is 0, within experimental uncertainty, suggesting that for S1·MgATP the electric charge at the actin binding site is abolished. The results are interpreted in terms of possible roles of electrostatic interactions in mechanisms for S1·MgATP dissociating from one actin and S1·MgADP·Pi being guided electrostatically to bind to another.