Fluorescence resonance energy transfer and molecular modeling studies on 4',6-diamidino-2-phenylindole (DAPI) complexes with tubulin

Abstract

The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2+/-0.4 microM for the DAPI-tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a kappa2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20+/-2 A and 43+/-2 A, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.

Figure 1.

Structures of 4′-6-diamidino-2-phenylindole (DAPI) and 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene)-GTP (TNP-GTP) at neutral or basic pH.

Figure 2.

Changes in anisotropy (A) and DAPI fraction bound (B) during DAPI titration with tubulin, tubulin S, and the complex DAPI–tubulin S with the C-terminal peptides. Titration of 0.5 μM DAPI with tubulin (•) and tubulin S (○). The buffer used was 10 mM Tris-HCl (pH 7.5). The inset shows the titration of 0.5 μM DAPI plus 1.5 μM tubulin S with increasing concentrations of C-terminal peptides (▴). Excitation wavelength 350 nm; emission wavelength 455 nm; excitation and emission bandwidths were 2.5 and 12 nm, respectively.

Figure 3.

Overlap of the absorption spectra of tubulin–cobalt and tubulin TNP–GTP complexes with the fluorescence emission spectrum of DAPI–tubulin complex. The spectra were obtained in 10 mM triethanolamine, 0.1 mM GTP (pH 7.0), at 25°C. The absorption spectrum of 0.16 mM tubulin–cobalt complex containing 0.8 mol/mol of cobalt (segmented line) was recorded using an identical concentration of tubulin–magnesium as a control. The absorption spectrum of one mole of TNP–GTP, bound at the E-nucleotide binding site, was recorded (solid line). The fluorescence emission spectrum of 2 μM DAPI with 7.9 μM tubulin (dashed line) was recorded using an excitation wavelength of 350 nm and excitation and emission bandwidth of 2.5 nm.

Figure 4.

DAPI surrounding amino acid group selected on the basis of the FRET results shown in the ribbon three-dimensional structure of tubulin heterodimer. The three-dimensional structure of tubulin heterodimer was drawn with the computer program package DS Modelling 1.1. The nucleotides are shown in orange at the E-site in β-tubulin (GDP) and at the N-site in α-tubulin (GTP). The amino acids indicated were selected as described in the text. The selected groups of amino acids are indicated by different colors. Group 1 (green); Group 2 (yellow); Group 3 (fuchsia); Group 4 (blue). The tryptophan residues closest to Groups 1 and 2 are also indicated.

Figure 5.

Computer molecular models of DAPI-binding sites on tubulin heterodimer. The models and location of DAPI-binding sites in the three-dimensional structure of tubulin heterodimer were built by computer molecular docking with the program Autodock v. 3.0.5 as explained in the Material and Methods. (A) The three-dimensional structure of tubulin heterodimer and DAPI were drawn (backbone) with the program Viewer Lite 5.0, Accelrys Inc. The binding sites with DAPI bound are indicated by different colors and with Roman numerals. Site I (red); site II (green); site III (dark blue); site IV (yellow); site V (light blue); site VI (fuchsia); site VII (brown); site VIII (black). (B) Part of the three-dimensional structure of tubulin and the binding site II with DAPI (green) were drawn (ribbons) with the program Mol Mol using surface potentials. This site is shown as van der Waals surface, colored according to the electrostatic potentials. As a reference of the molecule position, the helix H3 of α-tubulin is indicated.