Dimensions and Dynamics of Highly Cooperative Sic1-WD40 Binding: smFRET through a Polymer Physics Lens

Abstract

Sic1 is a cyclin-dependent kinase inhibitor which must be phosphorylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physicochemical/mechanistic basis of its highly cooperative binding.We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex. Using time-resolved fluorescence anisotropy, we find local segment specific rotational correlation times which are complexly modulated by chain compactness and electrostatics (charge screening and phosphorylation).Previous single molecule Fӧrster Resonance Energy Transfer (smFRET) measurements [1] observed end-to-end reconfiguration on timescales larger than ∼1ms; resulting in FRET histograms with multiple conformational sub-ensembles. These sub-ensembles and their dynamics are further explored by trapping single Sic1, pSic1 and WD40 in nanometer sized surface-tethered lipid vesicles and modulating their electrostatics and compactness. In a refinement to the conventional approaches for inferring dimensions from smFRET experiments, we use distance distributions from Monte Carlo simulations which extensively sample coarse-grained protein conformations. The application of polymer physics theory/simulation towards smFRET data interpretation, and towards IDP binding, contributes to the growing toolkit for understanding the diverse behaviours of IDPs.[1] Liu B. et al., J. Phys. Chem. B. 2014 118(15):4088-97.