title: Nucleotide-dependent shape changes in the reverse direction motor, myosin VI.
abstract: We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca(2+)). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO(4), which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix.
authors.affiliation: Institute of Molecular and Cellular Biology, Leeds University, Leeds, UK.
authors.forename: Chun Feng
authors.initials: CF
authors.lastname: Song
authors.forename: Kasim
authors.initials: K
authors.lastname: Sader
authors.forename: Howard
authors.initials: H
authors.lastname: White
authors.forename: John
authors.initials: J
authors.lastname: Kendrick-Jones
authors.forename: John
authors.initials: J
authors.lastname: Trinick
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000648:ultrastructure; D009711:Nucleotides / Q000494:pharmacology*; D055503:Protein Multimerization / Q000187:drug effects; D017434:Protein Structure, Tertiary
chemical_list: D001059:Apoproteins; D009711:Nucleotides; C089785:myosin VI; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D018995:Myosin Heavy Chains; D002118:Calcium