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 <b>I</b> and shows low abundance prior to and following LLPS. Populations <b>II</b> and <b>III</b> are part of CPEB4* assemblies where <b>II</b> corresponds to loose conformations with intermediate range motions and population <b>III</b> represents compact conformations with strongly attenuated motions. As the temperature increased the population of component <b>II</b> increased reversibly at the expense of component <b>III</b>, indicating the existence of an <b>III</b> ⇌ <b>II</b> equilibrium. We correlated the macroscopic LLPS properties with the <b>III</b> ⇌ <b>II</b> exchange process upon varying temperature and CPEB4* and salt concentrations. We hypothesized that weak transient intermolecular interactions facilitated by component <b>II</b> 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.