Tubulin electron crystallography

Figure 7-33 Stereoscopic ribbon diagram of the tubulin dimer with a-tubulin with bound GTP at the top and P-tubulin with bound GDP at the bottom. The p-tubulin subunit also contains a bound molecule of taxotere (see Box 7-D) which is labeled TAX. This model is based upon electron crystallography of zinc-induced tubulin sheets at 0.37-nm resolution and is thought to approximate closely the packing of the tubulin monomers in microtubules.315 The arrow at the left points toward the plus end of the microtubule. Courtesy of Kenneth H. Downing.

The atomic structure of tubulin protofilaments (Figs. 3 and 4) is known from 3.5 A resolution maps, the first of which came from electron crystallography of zinc-induced 2D sheets (Nogales et al., 1998b), subsequently refined (Lowe et al., 2001). Recent data, including important information... 

Nogales, E., Wolf, S., and Downing, K. H. (1998b). Structure of the a//-tubulin dimer by electron crystallography. Nature 391, 199-203. 

The first structural location of the taxane binding site [42] placed it in the interprotofilament space, thus supporting the biochemical results. However, this changed when the first high resolution 3D structure of the paclitaxel-tubulin complex was solved by electron-crystallography of a two-dimensional zinc-induced tubulin polymer [5]. The fitting of this structure into a three-dimensional reconstruction of microtubules from cryoelectron microscopy allowed a pseudo atomic resolution model of microtubules [43] in which the paclitaxel binding site was placed inside the lumen of the microtubules hidden from the outer solvent. 

Structures Derived by Electron Crystallography Straight Tubulin... 

Fig. 11 Three-dimensional structures of epothilones determined in different environments (O red, S yellow, N dark blue). Top structures of free EpoA determined by X-ray crystallography from dichloromethane/petroleum ether (top left [9 8] (a)) and from methanol/water (top right [143](b)). Bottom structures of EpoA bound to tubulin determined by solution NMR in aqueous medium (bottom left [96]) and by electron crystallography from zinc-stabilized tubulin sheets (bottom right [26]).(a) The crystal structure data have been available from the author to interested research groups since October 1995.(b) H.-J. Hecht, G. Hofle, unpublished results CCDC 241333 and CCDC 241334 contain the crystallographic data of this structure. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retiieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK fax (+ 44) 1223-336-033 or deposit cede. cam. ac. uk)...

Due to this lack of information, we and others invented the INPHARMA method that has been described above [75, 76], The prerequisites for INPHARMA are well fulfilled for EpoA and tubulin since the binding affinity is in the correct range, namely fast exchange. In addition, there is a 3D electron crystallography structure of the MT/taxol complex [11], Thus, INPHARMA could rely on this structure and derive from the interligand NOEs the orientation of EpoA in the binding pocket of tubulin. 

Keywords Diffraction, Dynamics, Electron crystallography, Epothilone, Lauli-malide, Microtubules, Modeling, Paclitaxel, SAR, Taxol, Tubulin... 

Fig. 18 The solution of epothilone bound to tubulin by electron crystallography, a 2Fo-Fc map and model of epothilone A bound to tubulin at la (ltvk) [3], b Energy optimized model derived from ltvk through MAID protocol used as template for analysis of SAR and design of new analogs...

Electron crystallography has proven to be a valuable tool for analysis of tubulin structure and drug interactions. Likewise, tubulin and its ability to form stable 2D crystals have driven development of EC methods that are now being used to study... 

The First Electron Crystallography Structure of a,P-Tubulin/ Paclitaxel Complex... 

Based on analysis of electronic crystallography and NMR data for the bindings of Taxol and epothilone A to tubulin subunits, it was proposed that they did not share a common pharmacophore (similar binding mode and sites) as hypothesized for a long time, because they bind to their receptors uniquely and independently. Also, the T-shape conformation of Taxol binding to tubulin was supported from this study. 

The importance of the tubulin-polymerization activity of taxol has led to numerous studies of the taxol— tubulin interaction. The nature of the binding site of taxol on tubulin could not be determined by X-ray crystallography because the complex cannot be crystallized, but stabilized zinc sheets of microtubules can be prejjtared and the structure of these sheets has been determined by electron crystallography at a resolution of... 

Based on the structure of ap-tubulin dimer, resolved by electron crystallography of zinc-induced tubulin sheets, a previously unrecognised... 

Figure Bl.17.11. Reconstructed density of an a,p-tubulin protein dimer as obtained from electron crystallography (Nogales etal 1997). Note the appearance of the P-sheets ((a), marked B) and the a-helices ((b), marked H) in the density. In particular the right-handed a-helix H6 is very clear. Pictures by courtesy of E Nogales and Academic Press.

Nogales E, Wolf S G and Downing K 1998 Structure of a,(3-tubulin dimer by electron crystallography Nature 391 199-202... 

Nogales E, Wolf 5G, Downing KH. 5tructure of the ap tubulin dimer by electron crystallography. Nature 1998 391 199-203. 

Initially, data were collected on crystal structure and conformation of epothilone B in solution [13], together with information on the binding site of epothilone A on a/p-tubulin, using electron crystallography and molecular modelling (Fig. 16.2)... 

Figure 3.12 A X-ray crystal structure of Epo A (crystals obtained from MeOH). B NMR-derived structure of tubulin-bound Epo A. C Electron crystallography-derived structure of tubulin-bound Epo A. ...

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