EPR spectra and molecular dynamics agree that the nucleotide pocket of myosin V is closed and that it opens on binding actin.

We have used EPR spectroscopy and computational modeling of nucleotide-analog spin probes to investigate conformational changes at the nucleotide site of myosin V. We find that, in the absence of actin, the mobility of a spin-labeled diphosphate analog [spin-labeled ADP (SLADP)] bound at the active site is strongly hindered, suggesting a closed nucleotide pocket. The mobility of the analog increases when the MV·SLADP complex (MV=myosin V) binds to actin, implying an opening of the active site in the A·MV·SLADP complex (A=actin). The probe mobilities are similar to those seen with myosin II, despite the fact that myosin V has dramatically altered kinetics. Molecular dynamics (MD) simulation was used to understand the EPR spectra in terms of the X-ray database. The X-ray structure of MV·ADP·BeFx shows a closed nucleotide site and has been proposed to be the detached state. The MV·ADP structure shows an open nucleotide site and has been proposed to be the A·MV·ADP state at the end of the working powerstroke. MD simulation of SLADP docked in the closed conformation gave a probe mobility comparable to that seen in the EPR spectrum of the MV·SLADP complex. The simulation of the open conformation gave a probe mobility that was 35-40° greater than that observed experimentally for the A·MV·SLADP state. Thus, EPR, X-ray diffraction, and computational analysis support the closed conformation as a myosin V state that is detached from actin. The MD results indicate that the MV·ADP crystal structure, which may correspond to the strained actin-bound post-powerstroke conformation resulting from head-head interaction in the dimeric processive motor, is superopened. Copyright © 2011 Elsevier Ltd. All rights reserved.

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