Kinetic mechanism and regulation of myosin VI.
Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of P(i) release, which lowers the K(ATPase) but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins.
De La Cruz
E M
EM
Department of Physiology, Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, Pennsylvania 19104-6085, USA.
Ostap
E M
EM
Sweeney
H L
HL
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011721:Pyrenes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013552:Swine; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D011721:Pyrenes; C089785:myosin VI; D013912:Threonine; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C030984:pyrene; D000251:Adenosine Triphosphatases; D018995:Myosin Heavy Chains; D000409:Alanine