title: The cryptophycin-tubulin ring structure indicates two points of curvature in the tubulin dimer.
abstract: Cryptophycin-1 is the parent compound of a group of cyclic peptides with potent antineoplastic activity. Cryptophycins are thought to function by modulating the dynamic instability of spindle microtubules, and in vitro are known to bind in an equimolar ratio to the beta-tubulin subunit and to induce the formation of ring-like complexes. However, the detailed mechanisms whereby the cryptophycins interact with tubulin are not known. We have investigated the origin of the conformational changes in tubulin both biochemically and by electron microscopy and image analysis. Cryptophycin was found to protect both alpha- and beta-tubulin against proteolysis by trypsin, indicating conformational changes in specific regions of both subunits. The ring mass was determined to be approximately 0.81 MDa by sedimentation velocity combined with dynamic light scattering and by STEM, indicating a complex of eight alphabeta dimers. Statistical analysis of rings imaged by cryoelectron microscopy revealed 16-fold symmetry, corresponding to eight dimers. Computational averaging based on this symmetry yielded an image of a 24 nm diameter ring, at 2.6 nm resolution, that clearly distinguishes intradimer contacts from interdimer contacts, and allows discrimination of alpha-subunits from beta-subunits. Fitting of the tubulin dimer crystal structure into this projected density map indicates two points of curvature: a 13 degrees intradimer bend and a 32 degrees interdimer bend. We conclude that drug binding to one subunit (beta) results in two bends per dimer, affecting both subunits.
authors.affiliation: Protein Expression Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Norman R
authors.initials: NR
authors.lastname: Watts
authors.forename: Naiqian
authors.initials: N
authors.lastname: Cheng
authors.forename: Wendy
authors.initials: W
authors.lastname: West
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
authors.forename: Dan L
authors.initials: DL
authors.lastname: Sackett
mesh_terms: D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D019295:Computational Biology; D047630:Depsipeptides; D019281:Dimerization; D006868:Hydrolysis; D007089:Image Enhancement; D008027:Light; D008855:Microscopy, Electron, Scanning; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits; D051381:Rats; D012542:Scattering, Radiation; D014357:Trypsin / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D014461:Ultracentrifugation
chemical_list: D001704:Biopolymers; D047630:Depsipeptides; D010456:Peptides, Cyclic; D021122:Protein Subunits; D014404:Tubulin; C088419:cryptophycin; D014357:Trypsin