Mechanism of nucleotide binding to actomyosin VI - Evidence for allosteric head-head communication

被引:43
作者
Robblee, JP [1 ]
Olivares, AO [1 ]
De la Cruz, EM [1 ]
机构
[1] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA
关键词
D O I
10.1074/jbc.M403504200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 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.
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页码:38608 / 38617
页数:10
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