Evolution of CPEB4 Dynamics Across its Liquid-Liquid Phase Separation Transition

Abstract

Knowledge about the structural and dynamic properties of proteins that form membrane-less organelles in cells via liquid-liquid phase separation (LLPS) is required for understanding the process at a molecular level. We used spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the dynamic properties (rotational diffusion) of the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein (CPEB4_NTD) across its LLPS transition, which takes place with increasing temperature. We report the coexistence of three spin labeled CPEB4_NTD (CPEB4*) populations with distinct dynamic properties representing different conformational spaces, both before and within the LLPS state. Monomeric CPEB4* exhibiting fast motion defines population I and shows low abundance prior to and following LLPS. Populations II and III are part of CPEB4* assemblies where II corresponds to loose conformations with intermediate range motions and population III represents compact conformations with strongly attenuated motions. As the temperature increased the population of component II increased reversibly at the expense of component III, indicating the existence of an III ⇌ II equilibrium. We correlated the macroscopic LLPS properties with the III ⇌ II exchange process upon varying temperature and CPEB4* and salt concentrations. We hypothesized that weak transient intermolecular interactions facilitated by component II lead to LLPS, with the small assemblies integrated within the droplets. The LLPS transition, however, was not associated with a clear discontinuity in the correlation times and populations of the three components. Importantly, CPEB4_NTD exhibits LLPS properties where droplet formation occurs from a preformed microscopic assembly rather than the monomeric protein molecules.

Figure 1

(A) RT microscope image of 10 μM CPEB4* in 100 mM NaCl. (B) Absorbance of 10 μM CPEB4* in 100 mM NaCl as a function of temperature and the corresponding fit to a sigmoidal curve.

Figure 2

(A) Comparison of RT EPR spectra of denatured (28 μM, in 3 M GdmCl, black) and phosphorylated CPEB4* (20 μM, red.). The * marks a cavity background signal. For the phosphorylation experiments 3-malemide proxyl (MSL) was used as spin label. (B) EPR spectrum of CPEB4* (112 μM, 100 mM NaCl, pH 8) in a non-LLPS state (2 °C) and the corresponding simulations (black) with three components: a fast motion (I) in blue, an intermediate motion (II) in green, and a slow motion (III) in purple. Black and blue arrows indicate the characteristic features of slow and fast motion species, respectively. The simulation parameters are presented in Table S2. (C) W-band DEER data in logarithmic scale, measured at 25 K, of 80 μM CPEB4* in 3 M GdmCl (blue), 60 μM CPEB4* frozen after incubating at RT (LLPS, black), and frozen after incubating over ice (LT, non-LLPS, red). The corresponding straight lines represent a linear fit.

Figure 3

(A) Temperature dependent EPR spectra of CPEB4* (112 μM, 100 mM NaCl, pH 8) and the corresponding simulations (black). Arrows indicate signatures of the slow motion spectrum of component III. (B) Relative populations of components I, II and III as a function of temperature. The arrow indicates LLPS transition temperature (17.5 ± 2 °C). (C) Same EPR spectra as in part A of CPEB4* after subtracting the simulated component I spectrum. Black and red arrows mark the decay and rise of component III and II, respectively, with temperature.

Figure 4

Dependence of K_II/III on [CPEB4*] at 2 °C (red) and 30 °C (black) for 100 mM (sphere) and 500 mM NaCl (square) at 2 °C (green) and 30 °C (blue).

Figure 5

RT EPR spectra of 20 μM CPEB4* (100 mM NaCl, pH 8) in the presence of different percentage of 1,6-hexanediol (HD). The black spectrum is CPEB4* in the absence of any HD. The red and blue arrows point to spectral signatures of component III and II, respectively. * marks a cavity background signal.

Scheme 1. Suggested Model for LLPS Formation for CPEB4_NTD

Key: (A) Equilibria between components I, II and III. (B) On the left, the non-LLPS state showing the presence of small assemblies containing compact and loose conformations. On the right hand side, droplets formed via interaction of loose conformations. Small red arrows in B, in both LLPS and non-LLPS states, indicate exchange between II and III, and the black arrows point to the corresponding forms. The schematic drawings are not to scale; the diameter of LLPS droplets ≫ the diameter of non-LLPS assemblies.