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 actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower 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 similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for 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 communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.
Department of Biophysics, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany.
M L
ML
Knetsch
T Q
TQ
Uyeda
D J
DJ
Manstein
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004023:Dictyostelium / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D017931:DNA Primers; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins