Vancraenenbroeck R et al. (2019), Polymer effects modulate binding affinities in ...

XB-ART-56917

Proc Natl Acad Sci U S A 2019 Sep 24;11639:19506-19512. doi: 10.1073/pnas.1904997116.

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Polymer effects modulate binding affinities in disordered proteins.

Vancraenenbroeck R , Harel YS , Zheng W , Hofmann H .

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Structural disorder is widespread in regulatory protein networks. Weak and transient interactions render disordered proteins particularly sensitive to fluctuations in solution conditions such as ion and crowder concentrations. How this sensitivity alters folding coupled binding reactions, however, has not been fully understood. Here, we demonstrate that salt jointly modulates polymer properties and binding affinities of 5 disordered proteins from a transcription factor network. A combination of single-molecule Förster resonance energy transfer experiments, polymer theory, and molecular simulations shows that all 5 proteins expand with increasing ionic strengths due to Debye-Hückel charge screening. Simultaneously, pairwise affinities between the proteins increase by an order of magnitude within physiological salt limits. A quantitative analysis shows that 50% of the affinity increase can be explained by changes in the disordered state. Disordered state properties therefore have a functional relevance even if these states are not directly involved in biological functions. Numerical solutions of coupled binding equilibria with our results show that networks of homologous disordered proteins can function surprisingly robustly in fluctuating cellular environments, despite the sensitivity of its individual proteins.

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Genes referenced: myc

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	Fig. 1. smFRET of a disordered protein network. (A) Illustration of interactions among 5 disordered bHLH-LZ proteins. Pairwise interactions allow the formation of 5 dimers. (B) Heat map of pairwise sequence identities (above diagonal) and sequence similarities (below diagonal) of the 5 disordered bHLH-LZ proteins (SI Appendix, Tables S1 and S2). (C) Solvent-driven changes in the free energy of disordered states (red) with respect to that of folded dimers (blue) will alter the affinity. The affinity, expressed by the dissociation constant K, is directly related to the binding free energy via K∝exp(−ΔGbinding). (D) FRET histograms of the disordered bHLH-LZ proteins at a physiological salt concentration (100 mM KCl).
	Fig. 2. Salt sensitivity of the folding-coupled binding reactions. (A) Disordered proteins labeled with donor (D) and acceptor (A) dyes are mixed with unlabeled proteins (Left) to form the folded dimer (Right). The positions of the terminal labeling sites are indicated in the NMR structure of the MAX homodimer (Right; PDB file 1r05). (B) FRET histograms of MYC at 0.5 M KCl in the presence of unlabeled MAX (concentration is indicated). The gray area indicates molecules without an active acceptor dye. (C) Salt dependence of the binding free energies ΔGbinding (Left) and dissociation constants K (Right) of all dimers. Colored lines are fits with the counterion binding model (colored line) and a hyperbolic salt dependence of Δn. Examples of Δn are shown for clarity (Inset). The change in K within the physiological salt regime (dashed lines) is indicated for MYC–MAX (blue) and MAX–MAX complexes (green). (D) FRET histograms of MYC with 1 nM unlabeled MAX at different KCl concentrations. The expansion and collapse of disordered MYC occur concomitantly with a stabilization of the folded dimer.
	Fig. 3. Polymer behavior of the disordered proteins. (A) Contributions to the polyampholyte model. Interactions are treated as perturbation of an ideal chain without volume. (B) Donor-acceptor distances (RDA) of the disordered proteins as a function of KCl with and without 2.5 M urea. Bands are the uncertainty (±2 SD) in the FRET–RDA conversion (SI Appendix). Black lines are global fits with the polyampholyte theory. Dashed lines are predictions for chains without dye-charges (−4). (C) RDA for MYC (blue circles) and the modified ΔMYC sequence with less hydrophobicity (green circles). The prediction for a salt-independent hydrophobic effect is shown as a solid gray line. (Inset) RDA of MYC as function of 3 salts: LiCl, NaCl, and CsCl. The relative ion radius is shown for comparison. (D) CD spectra of MYC at low salt (0 M KCl, blue) and high salt (2 M KCl) with (red) and without (black) urea. (E) Changes in Δω2 = ω2(c) – ω2(0), with c being the KCl concentration for MYC (blue circles) and ΔMYC (green circles). Solid lines are linear fits. (Inset) Ratio of hydrophobic surface area of MYC vs. ΔMYC (dashed line) in comparison to the ratio of Δω2 (solid line). (F) Molecular simulations of unlabeled MYC at 4 concentrations of NaCl. The root-mean-squared end-to-end distances (Ree) from the simulation (circles) are compared with the average donor–acceptor distance from the experiment (shaded band).
	Fig. 4. Linking disordered state properties to binding affinities. (A) Thermodynamic cycle that dissects the binding process into 3 contributions: 1) stretching of the disordered proteins to the distance in a folded dimer (ΔG1), 2) folding of the 2 stretched proteins (ΔG2), and 3) association of the folded proteins (ΔG3). The contribution of stretching (ΔG1) is calculated from the distance distributions obtained from polyampholyte theory (Top). ΔG1 is the free energy of stretching the disordered chains to at least the distance in a folded dimer (dashed line). (B) Results of the calculations schematically depicted in A. The 3 contributions: ΔG1 obtained from smFRET and polyampholyte theory (blue), ΔGbinding obtained from the determined affinities (Fig. 2C) (green), and the joint contribution of folding and association (ΔG2 + ΔG3) calculated from ΔGbinding − ΔG1 (black dashed line) are shown as function of the KCl concentration for MAX. The arrows indicate the salt-induced changes within the physiological limits of 75 and 300 mM. (C) Comparison of the free energy changes between 75 and 300 mM KCl for the stretching of the disordered state (Bottom, ΔΔG1) and dimer formation (Top, ΔΔGbinding).
	Fig. 5. Global network behavior. (A) Fluctuation amplitude of an i–k complex (computed for MYC–MAX) in the presence of different concentrations of competitor j (MAD) in the hypothetical Anti (Left) and the experimentally found Syn (Right) case. The signs indicate the direction of affinity changes in the schemes (Top). The salt variation was 0.2 ± 0.1 M. (B) Illustration of networks in Syn (Left) and Anti (Right) configuration. The signs indicate positive (red) or negative (blue) values for the affinity change with salt, i.e., for the gradient. (C) Relative variation of dimer concentrations in linear and centralized networks for the Syn (blue) and Anti (red) scenarios when sampling 1,100 different salt concentrations Δs from a Gaussian with a width of 0.05 M. (D) Comparison of charge fractions of IDPs in the Disprot database (blue) with those of 119 bHLH sequences (red). (Inset) Distribution of electrostatic interaction energies per amino acid for proteins from the Disprot database (blue) and for bHLH sequences (red), computed with polyampholyte theory assuming the donor–acceptor distance of an ideal chain.

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