Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3

Abstract

Liquid–liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana , LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

Molecular mechanisms underlying coordinated responses of plants to shade and environmental stresses

“…The evening complex, a protein complex composed of EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRYTHMO (LUX), links the circadian clock to the diurnal control of hypocotyl elongation and flowering (Nusinow et al, 2011;Zhao et al, 2021). Recently, ELF3 was shown to serve as a thermosensor through liquid-liquid phase separation in response to warm temperatures, which is driven by its prion-like domain (PrLD) (Hutin et al, 2023;Jung et al, 2020) (Figure 2). Under normal temperatures, the evening complex directly binds to the promoters of PIF4 and PIF5 and represses their expression (Nusinow et al, 2011).…”

Structure–function relationships in protein homorepeats

“…This gradual effect is less apparent in AR, probably due to the lack of structural information for pathogenic versions of this protein. Importantly, the increase in helicity has been shown to be a key element in the aggregation propensity of HttExon-1 and other polyQ-hosting proteins, probably by enhancing the formation of productive dimers, tetramers and other oligomers through coiled-coil interactions [60][61][62] that eventually can phase separate [63,64] and/or produce inclusion bodies in neurons [65,66*].…”