Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation

Abstract

AbstractCandida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Egf1’s prion-like domain (PrLD) and the protein’s ability to undergo phase separation for the white-to-opaque transition ofC. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered though have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding ofC. albicansTF interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.Statement of SignificancePhase separated condensates have been found across the domains of life and many types of cells. To understand their varied functions, seeing the residue-by-residue details of the structure and interactions of component protein constituents is essential. A set of transcription factors that phase-separate controls cell fate of the pathogenic yeast Candida albicans. Here, we examine the structural and interaction details of a main regulator of this process, Efg1, using NMR spectroscopy and biochemical assays. We find Efg1’s phase-separating domains are not entirely disordered as often assumed but in fact contain helical regions that persist upon phase separation. We also reveal the balance of contacts formed in the condensed phase and the importance of specific residues and regions in phase separation.