Direct Observation of the Myosin Va Recovery Stroke
That Contributes to Unidirectional Stepping along Actin
Katsuyuki Shiroguchi1, 4,
Harvey F. Chin2,
Diane E. Hannemann2,
Eiro Muneyuki3,
Enrique M. De La Cruz2,
Kazuhiko Kinosita Jr.1
Journal name: Plos Biology
Volume: 9
Article number: e1001031
DOI:doi: doi:10.1371/journal.pbio.1001031
Received
Accepted
Published
Abstract
Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These
functions are driven by large-scale nucleotide-dependent conformational changes, termed ‘‘strokes’’; the ‘‘power stroke’’ is
the force-generating swinging of the myosin light chain–binding ‘‘neck’’ domain relative to the motor domain ‘‘head’’ while
bound to actin; the ‘‘recovery stroke’’ is the necessary initial motion that primes, or ‘‘cocks,’’ myosin while detached from
actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a ‘‘hand over hand’’ mechanism in
which the trailing head detaches and steps forward ,72 nm. Despite large rotational Brownian motion of the detached
head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the
attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward
site binding since the orientation and angle stability of the detached head, which is determined by the properties of the
recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and
fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through
stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and
observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to
the post-recovery stroke conformation, where it remains for ,40 s, until ATP hydrolysis products are released. Angle
distributions indicate that the post-recovery stroke conformation is stabilized by >5 kBT of energy. The high kinetic and
energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward
site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.
Affiliations
Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Japan.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America
Department of Physics, Faculty of Science and Technology, Chuo University, Tokyo, Japan
Current address: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America