Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication.
We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is approximately five times greater than for ATP binding because of a higher affinity in the collision complex (1/K(5b)' = 2.2 mm) and faster isomerization rate constant (k(+5a)' = 366 s(-1)). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.
Robblee
James P
JP
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Olivares
Adrian O
AO
de la Cruz
Enrique M
EM
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D015233:Models, Statistical; D018482:Muscle, Skeletal / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011721:Pyrenes / Q000737:chemistry; D011817:Rabbits; D013997:Time Factors
D000199:Actins; D009711:Nucleotides; D011721:Pyrenes; C089785:myosin VI; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; C030984:pyrene; D000251:Adenosine Triphosphatases; D018995:Myosin Heavy Chains