Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction

The kinetic and functional consequences of deleting nine residues from an a ctin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosi n were not altered by the deletion. However, the deletion affected actin bi nding and the communication between the actin- and nucleotide-binding sites . The affinity of M765NL for actin (644 nM) was approximately 100-fold lowe r than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a si milar extent for both mutant and wild-type constructs. The addition of 0.5 mu M actin decreased ADP affinity from 0.6 to 34 mu M for M765NL and from 1 .6 to 39 mu M for M765. In contrast, communication between the actin- and n ucleotide-binding sites appears disturbed in regard to phosphate release: t hus, basal ATPase activity for M765NL (0.19 s(-1)) was 3-fold larger than f or M765 (0.06 s(-1)), and the stimulation of ATPase activity by actin was 5 -fold lower for M765NL. These results indicate different paths of communica tion between the actin- and nucleotide-binding sites, in regard to ADP and P-i release, and they confirm that loop 2 is involved in high affinity acti n binding.