Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor

Abstract

Intrinsically disordered proteins play critical but often poorly understood roles in mediating protein interactions. The interactions of disordered proteins studied to date typically entail structural stabilization, whether as a global disorder-to-order transition or minimal ordering of short linear motifs. The disordered cyclin-dependent kinase (CDK) inhibitor Sic1 interacts with a single site on its receptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The molecular basis for this multisite-dependent interaction with a single receptor site is not known. By NMR analysis, we show that multiple phosphorylated sites on Sic1 interact with Cdc4 in dynamic equilibrium with only local ordering around each site. Regardless of phosphorylation status, Sic1 exists in an intrinsically disordered state but is surprisingly compact with transient structure. The observation of this unusual binding mode between Sic1 and Cdc4 extends the understanding of protein interactions from predominantly static complexes to include dynamic ensembles of intrinsically disordered states.

Fig. 1.

Sic1 is an intrinsically disordered protein. (A) ¹H^N-¹⁵N correlation spectra of Sic1 (black) and pSic1 (red). In the pSic1 sample, resonances of residues Thr-5, Thr-33, Thr-45, Ser-69, Ser-76, and Ser-80 exhibit proton downfield shifts accompanied by small chemical shift changes in adjacent residues. Residues with multiple resonances, primarily from cis- and trans-prolyl isomers, are denoted with a prime. (B) Experimental SSP values (28) for Sic1 (black bars) and pSic1 (open bars). Red circles indicate phosphorylation sites.

Fig. 2.

Sic1 contains transient structure, yet is highly dynamic in both free and complexed states. (A) Transverse ¹⁵N relaxation rate constants of Sic1 (green bars) and pSic1 (black bars). Clusters of residues exhibiting restricted motion described by Gaussian distributions were used to fit (red line) the transverse relaxation rates of pSic1 (31) (see also SI Materials and Methods). (B) HetNOEs of Sic1 (green bars) and pSic1 (black bars). (C) HetNOEs of pSic1 in complex with Skp1–Cdc4 at a molar ratio (Skp1–Cdc4:pSic1) of 1.2, at which 99% of pSic1 is bound. Negative and small positive values demonstrate disorder of pSic1 even in the complex. Trans- and cis-prolyl isomers are reported as black and red bars, respectively. The hetNOE expected for a rigid large complex at this field strength is depicted as a dashed line at 0.82 (46). Red circles indicate phosphorylation sites.

Fig. 3.

Multiple phosphorylation sites interact with Cdc4 in a dynamic equilibrium. (A) TCS effects on ²H,¹⁵N pSic1 (610 μM) in the presence of unlabeled Skp1–Cdc4 for trans- (circles) and cis-prolyl isomers (crosses) at a molar ratio (pSic1:Skp1–Cdc4) of ≈26:1. Ratios of resonance intensities from backbone amide groups with and without irradiation are normalized by a control experiment without Skp1–Cdc4 present [(I_irr/I₀)_+Cdc4/(I_irr/I₀)_−Cdc4] to correct for effects from residual protonation in pSic1. In cases of >2 isomers, only effects for cis- and trans-isomers with respect to the P+1 proline of the CPD are reported (while data are solely from trans-isomers of other prolines). Where only 1 symbol is plotted, cis- and trans-prolyl isomers were not resolved. Because isomers could not be assigned unambiguously in the region around pThr-5, effects for all isomers in this region are depicted as stars. Lines are plotted through the most broadened isomers as a guide. Phosphorylation sites are indicated by red circles. (B) Intensity ratios of HN resonances of Sic1 phosphorylation sites (trans-isomers) as a function of Cdc4:pSic1 molar ratio. (C) Relative intensities of HN resonances of Sic1 phosphorylation sites upon back-titration with a high-affinity cyclin E-derived phosphopeptide (CycE^9pT380, K_d 1μM).

Fig. 4.

Dynamic complex of pSic1 and Cdc4. Multiple suboptimal CPD motifs in pSic1 engage the core Cdc4-binding site in a dynamic equilibrium. Sites not directly bound in the core binding site at any given instant can contribute to the binding energy via a second binding site or via long-range electrostatic interactions. A more detailed view of one possible interaction is shown, with the pThr-45 CPD binding to Cdc4. The linear binding motif that is ordered upon interaction is shown in stick representation [model based on the structure of the Cdc4–cyclin E peptide complex (24)]; the parts of pSic1 remaining disordered are depicted as black lines.