Structural insight into the inhibition of tubulin by vinca domain peptide ligands

Abstract

The tubulin vinca domain is the target of widely different microtubule inhibitors that interfere with the binding of vinblastine. Although all these ligands inhibit the hydrolysis of GTP, they affect nucleotide exchange to variable extents. The structures of two vinca domain antimitotic peptides--phomopsin A and soblidotin (a dolastatin 10 analogue)--bound to tubulin in a complex with a stathmin-like domain show that their sites partly overlap with that of vinblastine and extend the definition of the vinca domain. The structural data, together with the biochemical results from the ligands we studied, highlight two main contributors in nucleotide exchange: the flexibility of the tubulin subunits' arrangement at their interfaces and the residues in the carboxy-terminal part of the beta-tubulin H6-H7 loop. The structures also highlight common features of the mechanisms by which vinca domain ligands favour curved tubulin assemblies and destabilize microtubules.

Figure 1

The tubulin vinca domain. (A) Chemical formulas of vinblastine, phomopsin A and soblidotin. The amino-terminal tripeptide of soblidotin is highlighted in darker blue. (B) View of the vinca domain in (Tc)₂R, with bound phomopsin A and its σ_a-weighted F_obs−F_calc omit map; the map is contoured at 3.5σ and overlapped with phomopsin A. (C) View of the vinca domain in (Tc)₂R, with bound soblidotin and its σ_a-weighted F_obs−F_calc omit map contoured at 4σ (magenta). The σ_a-weighted F_obs−F_calc omit map of crystals soaked with the soblidotin N-terminal peptide, contoured at 3.5σ (green), is overlapped with it. A stereoscopic version of this panel is provided as Supplementary Fig 6 online. (D) The structure of (Tc)₂R with bound phomopsin A (cyan), represented as a space-filling model. Two sites are identified, one at the β1–α2 interface and the other one at the β2 end of the complex. The RB3-SLD (dark blue) and colchicine (yellow) domains are also represented. Colch, colchicine; RB3-SLD, RB3 stathmin-like domain.

Figure 2

The overlap of tubulin-bound soblidotin (A, cyan) and phomopsin A (B, cyan) with vinblastine (magenta); all are shown as sticks together with the β-tubulin structural elements with which they are in contact. A semitransparent vinblastine surface is drawn to aid visualization of the overlap.

Figure 3

Effects of vinca domain ligand binding on (Tc)₂R nucleotides. (A) Inhibition of nucleotide exchange. The variations of the normalized percentage of (Tc)₂R nucleotide exchanged as a function of phomopsin A (filled circles), soblidotin (open circles) and vinblastine (open squares) concentrations are presented. Each data point is the average of three measurements, performed as described in Methods. (B) Inhibition of GTPase activity. The variation of the specific GTPase activity of (Tc)₂R is presented as a function of phomopsin A concentration (filled circles, dashed), soblidotin (open circles, dotted) and its amino-terminal tripeptide (open triangles, alternating dash–dot). The lines represent fits of the data, calculated as described by Wang et al (2007), from which the vinblastine data (open squares, continuous line) are taken. Insert: equilibrium binding constants K_a of these ligands, deduced from the fit of the inhibition curves, as described (Wang et al, 2007). For phomopsin A and soblidotin, at the concentration of (Tc)₂R at which data were measured (5 μM), the variation of the GTPase activity is linear over most of the range explored, which allows only a lower limit of K_a to be determined.