Myosins Active Site Closure via Switch-1 Allosterically Regulates the Motor-Actin Interaction during the ATPase Cycle for Productive Force Generation

Myosins are essential for many cellular processes including cell motility and muscle contraction. These motors have active site motifs (P-loop, switch-1, switch-2) that bind ATP and a divalent metal cofactor to coordinate ATP hydrolysis. ATP hydrolysis is activated through allosteric communication between the ATP and polymer binding sites to couple the free energy of ATP hydrolysis to force generation. Our lab seeks to understand how changes in enzymatic properties leads to different mechanical properties in molecular motors. We hypothesize that myosins depend on active site closure via switch-1 to regulate the motor-actin interaction during the ATPase cycle for productive force generation. We developed a strategy to control enzymatic activity and motility of myosin by substituting a switch-1 serine with cysteine, diminishing its interaction with the Mg+2 metal thus inhibiting ATPase activity. Substituting Mg+2 with Mn+2, which strongly interacts with cysteine, restores metal interaction and ATPase activity. Thus swapping of the divalent metals provides a direct and experimentally reversible link between switch-1 and the actin binding cleft. We have shown that the S237C mutant of non-muscle myosin II has rapid basal steady-state ATP turnover, but upon binding actin, its MgATPase is inhibited. Mn+2 relieves this inhibition and restores ATP turnover near WT values. The acto•S237C complex has slow, rate limiting MgATP binding which is rescued to near WT activity in the presence of Mn+2. Mant-ADP release experiments show the need for switch-1 interaction with the metal for tight ADP binding. Finally, to test whether this allosteric communication is necessary for force generation, we will test S237C motility in the presence of Mg2+ and Mn2+. These results support a strong reciprocal coupling of nucleotide and F-actin binding in myosin.