Myosin regulatory light chain and nucleotide modulation of actin binding site electric charge.

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, K(A), 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, K(A) values at 0 M ionic strength for S1(elc) and S1(elc,rlc) are 12 and 4.9 x 10(6) M(-1), respectively, and /ZMZA/ values are 3.9 +/- 0.3 and 3.6 +/- 0.2 esu2. In the presence of ATP, K(A) values at 0 M ionic strength for S1(elc) and S1(elc,rlc) are 81 and 7.3 x 10(4) 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, K(M), for the actin activation of S1 steady-state MgATPase activity was significantly smaller for S1(elc), consistent with its greater K(A) 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. K(A) and /ZMZA/ were also measured at 25 degrees C for S1(elc,rlc) binding to actin in the presence of the ATP analog ATPgammaS. At 0 M ionic strength, K(A) is 8.0 x 10(4) M(-1), and /ZMZA/ is 0, within experimental uncertainty, suggesting that for S1 x 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 x MgATP dissociating from one actin and S1 x MgADP x Pi being guided electrostatically to bind to another.