EPR Spectra and Molecular Dynamics Agree That the Nucleotide Pocket of Myosin V Is Closed and That It Opens on Binding Actin

JOURNAL OF MOLECULAR BIOLOGY

卷:411

Article

Purcell, Thomas J.²  Naber, Nariman²  Sutton, Shirley³  Cooke, Roger^2,4  Pate, Edward¹ 

[1] Washington State Univ, Voiland Sch Chem Engn & Bioengn, Pullman, WA 99164 USA

[2] Univ Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94158 USA

[3] Stanford Univ, Sch Med, Dept Biochem, Stanford, CA 94305 USA

[4] Univ Calif San Francisco, Cardiovasc Res Inst, San Francisco, CA 94158 USA

关键词: myosin V;    electron paramagnetic resonance spectroscopy;    spin probes;    Switch 1;    molecular dynamics simulation;   

DOI  :  10.1016/j.jmb.2011.05.019

来源: Elsevier

【 摘 要 】

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 center dot SLADP complex (MV = myosin V) binds to actin, implying an opening of the active site in the A center dot MV center dot 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 center dot ADP center dot BeFx shows a closed nucleotide site and has been proposed to be the detached state. The MV center dot ADP structure shows an open nucleotide site and has been proposed to be the A center dot MV center dot 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 center dot SLADP complex. The simulation of the open conformation gave a probe mobility that was 35-40 degrees greater than that observed experimentally for the A center dot MV center dot 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 center dot 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. (C) 2011 Elsevier Ltd. All rights reserved.

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