Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle

Mingxuan Sun, Michael B. Rose, Shobana K. Ananthanarayanan, Donald J. Jacobs, Christopher M. Yengo

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP-Pi) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer (FRET) to monitor conformational changes in the upper-50-kDa domain of the actin binding cleft in the weak and strong actin binding states. Steady-state and lifetime data monitoring the FRET signal suggest that the cleft is in a more open conformation in the weak actin binding states. Transient kinetic experiments suggest that a rapid conformational change occurs, which is consistent with cleft closure before actin-activated phosphate release. Our results have identified a pre-force-generation actomyosin ADP-Pi state, and suggest force generation may occur from a state not yet seen by crystallography in which the actin binding cleft and the nucleotide binding pocket are closed. Computational modeling uncovers dramatic changes in the rigidity of the upper-50-kDa domain in different nucleotide states, which suggests that the intrinsic flexibility of this domain allows myosin motors to accomplish simultaneous tight nucleotide binding (closed nucleotide binding pocket) and high-affinity actin binding (closed actin binding cleft).

Original languageEnglish (US)
Pages (from-to)8631-8636
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number25
DOIs
StatePublished - Jun 24 2008

All Science Journal Classification (ASJC) codes

  • General

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