Myosin VI dimerization triggers an unfolding of a three-helix bundle in order to extend its reach.

Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of myosin VI.

[1]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[2]  Zygmunt Gryczynski,et al.  A FRET-Based Sensor Reveals Large ATP Hydrolysis–Induced Conformational Changes and Three Distinct States of the Molecular Motor Myosin , 2000, Cell.

[3]  Toshio Yanagida,et al.  Class VI myosin moves processively along actin filaments backward with large steps. , 2002, Biochemical and biophysical research communications.

[4]  P S Kim,et al.  Buried polar residues in coiled-coil interfaces. , 2001, Biochemistry.

[5]  Sebastian Doniach,et al.  Dynamic charge interactions create surprising rigidity in the ER/K α-helical protein motif , 2008, Proceedings of the National Academy of Sciences.

[6]  James A. Spudich,et al.  The Mechanism of Myosin VI Translocation and Its Load-Induced Anchoring , 2004, Cell.

[7]  P S Kim,et al.  Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4. , 1994, Biochemistry.

[8]  P. Graceffa,et al.  A long helix from the central region of smooth muscle caldesmon. , 1991, The Journal of biological chemistry.

[9]  Sebastian Doniach,et al.  Long single α-helical tail domains bridge the gap between structure and function of myosin VI , 2008, Nature Structural &Molecular Biology.

[10]  Colin Echeverría Aitken,et al.  An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. , 2008, Biophysical journal.

[11]  H. Sweeney,et al.  The Structural Basis for the Large Powerstroke of Myosin VI , 2007, Cell.

[12]  R. Vale,et al.  Distinct conformations of the kinesin Unc104 neck regulate a monomer to dimer motor transition , 2003, The Journal of cell biology.

[13]  H. Sweeney,et al.  What can myosin VI do in cells? , 2007, Current opinion in cell biology.

[14]  P. Selvin,et al.  Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering. , 2006, Molecular cell.

[15]  Clara Franzini-Armstrong,et al.  A flexible domain is essential for the large step size and processivity of myosin VI. , 2005, Molecular cell.

[16]  D. Frank,et al.  Coiled-coil-mediated dimerization is not required for myosin VI to stabilize actin during spermatid individualization in Drosophila melanogaster. , 2009, Molecular biology of the cell.

[17]  J. Sellers,et al.  A FERM domain autoregulates Drosophila myosin 7a activity , 2009, Proceedings of the National Academy of Sciences.

[18]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[19]  S. Rosenfeld,et al.  Kinetic Tuning of Myosin via a Flexible Loop Adjacent to the Nucleotide Binding Pocket* , 1998, The Journal of Biological Chemistry.

[20]  K C Holmes,et al.  The structure of the rigor complex and its implications for the power stroke. , 2004, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[21]  A. Houdusse,et al.  A model of Ca(2+)-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. , 1996, Structure.

[22]  Matthias Rief,et al.  Myosin-V is a processive actin-based motor , 1999, Nature.

[23]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[24]  Carl A. Morris,et al.  The structure of the myosin VI motor reveals the mechanism of directionality reversal , 2005, Nature.

[25]  Amber L. Wells,et al.  Myosin VI is a processive motor with a large step size , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Daniel Safer,et al.  Myosin VI is an actin-based motor that moves backwards , 1999, Nature.

[27]  Paul R Selvin,et al.  Myosin VI Steps via a Hand-over-Hand Mechanism with Its Lever Arm Undergoing Fluctuations when Attached to Actin* , 2004, Journal of Biological Chemistry.

[28]  Wolfgang Kabsch,et al.  Automatic indexing of rotation diffraction patterns , 1988 .

[29]  Florian Odronitz,et al.  Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species , 2007, Genome Biology.

[30]  Michelle Peckham,et al.  The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head* , 2005, Journal of Biological Chemistry.

[31]  Amber L. Wells,et al.  Calcium Functionally Uncouples the Heads of Myosin VI* , 2003, Journal of Biological Chemistry.

[32]  P. Selvin,et al.  The unique insert at the end of the myosin VI motor is the sole determinant of directionality , 2007, Proceedings of the National Academy of Sciences.

[33]  John Trinick,et al.  A monomeric myosin VI with a large working stroke , 2004, The EMBO journal.

[34]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[35]  Roberto Dominguez,et al.  Structure of the light chain-binding domain of myosin V. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[36]  H. Sweeney,et al.  Kinetic Mechanism and Regulation of Myosin VI* , 2001, The Journal of Biological Chemistry.

[37]  K. Holmes,et al.  The structural basis of muscle contraction. , 2000, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[38]  D. Frank,et al.  Myosin VI: a structural role in actin organization important for protein and organelle localization and trafficking. , 2004, Current opinion in cell biology.

[39]  J. Corrie,et al.  A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein. , 2006, Biochemistry.

[40]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

[41]  G. Spudich,et al.  Myosin VI: cellular functions and motor properties. , 2004, Annual review of cell and developmental biology.

[42]  P. Evans,et al.  Scaling and assessment of data quality. , 2006, Acta crystallographica. Section D, Biological crystallography.