Cooperative unfolding of compact conformations of the intrinsically disordered protein osteopontin

Abstract

Intrinsically disordered proteins (IDPs) constitute a class of biologically active proteins that lack defined tertiary and often secondary structure. The IDP Osteopontin (OPN), a cytokine involved in metastasis of several types of cancer, is shown to simultaneously sample extended, random coil-like conformations and stable, cooperatively folded conformations. By a combination of two magnetic resonance methods, electron paramagnetic resonance and nuclear magnetic resonance spectroscopy, we demonstrate that the OPN ensemble exhibits not only characteristics of an extended and flexible polypeptide, as expected for an IDP, but also simultaneously those of globular proteins, in particular sigmoidal structural denaturation profiles. Both types of states, extended and cooperatively folded, are populated simultaneously by OPN in its apo state. The heterogeneity of the structural properties of IDPs is thus shown to even involve cooperative folding and unfolding events.

Figure 1

Scheme of spin labeling sites along the protein backbone and sketch of residues spanned by each of the six OPN double mutants.

Figure 2

DEER time traces of C108–C188 (1/3 L) and C54–C247 (3/3 S) at different urea concentrations. Δ_eff is defined as the signal decay at 3 μs, as indicated by the double-headed arrow [note the different V(t)/V(0) scales].

Figure 3

(a) Δ_eff for selected double mutants as a function of urea concentration. All the data for all double mutants under investigation can be found in Figures S2–S4 of the Supporting Information. (b) Detailed representation of Δ_eff for C54–C247 (3/3 S) as a function of urea concentration. Error bars stem from signal noise. The gray curve is based on a sigmoidal data fit to confirm the visual observation of sigmoidality. The fit is based on the relationship Δ_eff = a + b/{1 + exp[−c(urea) – m]/s}.

Figure 4

Sketch of OPN double mutants assessed by DEER. Labels E (exponential), L (linear), and S (sigmoidal) denote the profile shapes of the respective Δ_eff functions (see Figure 3 and Figures S2 and S4 of the Supporting Information).

Figure 5

(a) Δ_eff values for the different double mutants under different denaturing conditions (4 M NaCl, 8 M urea, or 4 M NaCl with 8 M urea). (b) Exemplary (for C108–C188 (1/3 L)) DEER time traces for different denaturation conditions (4 M NaCl, 8 M urea, or 4 M NaCl with 8 M urea).

Figure 6

(a) ¹⁵N–¹H NMR chemical shift changes {calculated as cs[c(urea) = 0 M] – cs[c(urea) = x M]} of selected backbone positions as a function of urea concentration. (b) ¹³C–¹H HSQC of ¹³CH₃-Lys-labeled side chains: green for 0 M urea, pink for 2 M urea, blue for 4 M urea, and black for 6 M urea. Note that the shift in the ¹H dimension is merely a consequence of readjusting the transmitter offset in the dependence of the urea concentration to achieve suppression of water signals.

Figure 7

(a) PRE data for the four single mutants C54, C108, C188, and C247. Superimposed in blue are PREs calculated for random coils with a Flory characteristic ratio of 2 by the Solomon–Bloembergen relation. The red dots mark the different labeling sites. The asterisks mark stretches comprising larger numbers of unassigned resonances. (b) Charge map of OPN (top; blue corresponds to patches of primarily basic residues, red to patches of acidic residues, and gray to primarily hydrophobic patches) and PRE changes (ΔPRE) for high-salt (center) and high-urea (bottom) conditions obtained for the C188 mutant [ΔPRE = ¹⁵N–¹H HSQC intensity (high urea and salt) – ¹⁵N–¹H HSQC intensity (no urea or salt)].

Figure 8

Sketch of the assumed “average” structure of OPN based on the PRE data. The arrows indicate significant PRE effects. As such, OPN can be pictured as having a more compact core and back-folded termini. The colors refer to the charge map in Figure 7b (blue corresponds to patches of primarily basic residues, red to patches of acidic residues, and gray to primarily hydrophobic patches).