p25alpha is flexible but natively folded and binds tubulin with oligomeric stoichiometry

Abstract

p25alpha is a 219-residue protein which stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25alpha has been proposed to be natively unfolded, so that folding is coupled to interaction with its physiological partners. Here we show that recombinant human p25alpha is folded under physiological conditions, since it has a well structured and solvent-sequestered aromatic environment and considerable chemical shift dispersion of amide and aliphatic protons. With increasing urea concentrations, p25alpha undergoes clear spectral changes suggesting significant loss of structure. p25alpha unfolds cooperatively in urea according to a simple two-state transition with a stability in water of approximately 5 kcal/mol. The protein behaves as a monomer and refolds with a transient on-pathway folding intermediate. However, high sensitivity to proteolytic attack and abnormal gel filtration migration behavior suggests a relatively extended structure, possibly organized in distinct domains. A deletion mutant of p25alpha lacking residues 3-43 also unfolds cooperatively and with similar stability, suggesting that the N-terminal region is largely unstructured. Both proteins undergo significant loss of structure when bound to monomeric tubulin. The stoichiometry of binding is estimated to be 3-4 molecules of tubulin per p25alpha and is not significantly affected by the deletion of residues 3-43. In conclusion, we dismiss the proposal that p25alpha is natively unfolded, although the protein is relatively flexible. This flexibility may be linked to its tubulin-binding properties.

Figure 1.

Purification of p25α (23.7 kDa) and p25αΔ3-43 (19.6 kDa) after ion exchange and gel filtration chromatography.

Figure 2.

Spectral properties of p25α. (A) Far-UV CD spectrum of native (solid line) and denatured (stippled line [5 M urea]) p25α. Native p25α is predicted to have 15% α-helix, 30% β-sheet, and 55% random coil, although the fit to the predicted spectrum is not good (data not shown). (B) Near-UV CD spectrum of native p25α. The peak at 280 nm indicates that the Trp residue experiences a well defined asymmetrical environment, typical of the native state. (C) Fluorescence emission spectra of native (solid line) and denatured (stippled line [5 M urea]) p25α. (D) Stern-Volmer plot of the quenching of native (•) and denatured (○ [5 M urea]) p25α. The slopes of the plots are 1.14 and 4.30 M⁻¹, respectively, indicating that the Trp is much more accessible to acrylamide in the denatured rather than the native state.

Figure 3.

1D NMR spectra of p25α in buffer (A) and buffer and 7 M urea (B). Note the loss of chemical shift dispersion in the region from 6–10 ppm (amide and aromatic protons) and the region from 0–0.7 ppm (the aliphatic region) in urea, suggesting unfolding.

Figure 4.

Equilibrium and kinetic stability of p25α. (A) Equilibrium denaturation of p25α in urea, followed by the ratio of the emission intensities at 327, 335, and 355 nm.•, 327/335 nm; ○, 355/335 nm. (B) Time profiles of unfolding and refolding of p25α followed by stopped-flow. Both time profiles are fitted to a single exponential decay with offset. (C) Log of the observed rate constants of folding and unfolding of p25α vs. [urea]. The data are fitted to a three-state model (D ↔ I ↔ N) (Scheme 1). Results are given in Table 1. (D) Effect of [Na₂SO₄] on the log of the refolding rate in 0.5 M urea, where the intermediate accumulates transiently. The rise in refolding rates with [Na₂SO₄] is consistent with the scenario that the intermediate is on-pathway.

Figure 5.

(Left panel) Cross-linking of p25α, lysozyme, and α-synuclein with BS3 at different urea concentrations. BS3 cross-linking leads to the formation of a second faster-migrating band in the case of p25α and lysozyme (indicated by arrows), which for p25α disappears at higher urea concentrations. (Right panel) Digestion of p25α and lysozyme in the presence of various concentrations of protease. (A) p25α in PBS buffer with trypsin. (B) p25α in 0.5 M Na₂SO₄ with trypsin. (C) p25α in PBS buffer with chymotrypsin. (D) Lysozyme in PBS buffer with trypsin. The numbers on the gel indicate the fraction of protease relative to p25α or lysozyme (w/w). Note that the digestion pattern for p25α is the same when we stabilize the native state (in 0.5 M Na₂SO₄).

Figure 6.

Biophysical properties of p25αΔ3-43. Data summarized in Table 1. (A) Fluorescence emission spectra of native (solid line) and denatured (stippled line [5 M urea]) protein. (B) Equilibrium denaturation in urea, followed by the ratio of the emission intensities at 327 and 340 nm. (C) Stern-Volmer plot of the quenching of native (•) and denatured (○ [5 M urea]) protein.

Figure 7.

Spectroscopic alterations induced in the p25α:tubulin (A,C) and p25αΔ3-43:tubulin (B,D) complexes as measured by far-UV CD (A,B) and Trp fluorescence (C,D). The actual spectrum of the complex is denoted by the stippled line with ○; the mathematical sum of the spectra of the two protein components is denoted by the joined line with •. The difference is indicated by the stippled line with x. For clarity, spectra of free p25α and tubulin are omitted.

Figure 8.

Titration of tubulin with p25α (•) and p25αΔ3-43 (○). The difference F_exp − F_obs (where F_exp is the mathematical sum of the fluorescence of the two protein components and F_obs is the measured fluorescence) is plotted vs. the ratio of the two protein components.