New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment

Abstract

CK2 is a Ser/Thr protein kinase that regulates the activity of the Drosophila basic-helix-loop-helix (bHLH) repressor M8 encoded by the Enhancer of split Complex (E(spl)C) during neurogenesis. Specifically, phosphorylation appears to elicit a conformational change in an autoinhibited state of M8 to one that is permissive for repression. We describe biochemical and molecular modeling studies that provide new insights into repression by M8. Our studies implicate the phosphorylation domain in autoinhibition, and indicate that binding of the co-repressor Groucho (Gro) is context-dependent. Molecular modeling indicates that the Orange domain, proposed to be a specificity-determinant, may instead play a structural role, and that a conformational rearrangement of this domain may be necessary for repression. This model also provides a structural mechanism for the behavior of mutant alleles of the m8 gene. The insights gained from these studies should be applicable to the conserved metazoan bHLH repressors of the Hairy and Enhancer of Split (HES) family that are related to Drosophila M8.

Essential Roles of Da Transactivation Domains in Neurogenesis and in E(spl)-Mediated Repression

“…Our demonstration that Da AD1 interacts with the Orange domain of E(spl) proteins adds a new function to that elusive domain, which defines the bHLH-O subfamily and to date has been implicated in dimerization (21,65) and Sens binding (31). The recently proposed structure of the Orange domain as a dimeric hairpin of two ␣ helices (Protein Data Bank accession number 2DB7) is consistent with both its role in dimerization and its role as a protein interaction surface.…”

Multilayered gene control drives timely exit from the stem cell state in uncommitted progenitors during Drosophila asymmetric neural stem cell division

“…5E). The crystal structure of the Hey1 Orange motif suggested that Orange motif monomers can dimerize (Protein Data Bank [PDB] file 2DB7) (Eastwood et al 2011 revealed significant homology among the residues located at the interaction interface of Orange monomers (Fig. 5F, highlighted in green).…”

E(spl)

“…Like the HLH domain, the Orange domain consists of two amphipathic alpha-helices and mediates protein-protein interactions, including dimerization of the basic helix-loop-helix-orange (bHLH-O) proteins, where it acts as an "extension" of the HLH domain (Dawson, Turner, Weintraub, & Parkhurst, 1995;Eastwood, Yin, Bandyopadhyay, & Bidwai, 2011;Knust et al, 1992;Taelman et al, 2004;Zarifi et al, 2012). Like the HLH domain, the Orange domain consists of two amphipathic alpha-helices and mediates protein-protein interactions, including dimerization of the basic helix-loop-helix-orange (bHLH-O) proteins, where it acts as an "extension" of the HLH domain (Dawson, Turner, Weintraub, & Parkhurst, 1995;Eastwood, Yin, Bandyopadhyay, & Bidwai, 2011;Knust et al, 1992;Taelman et al, 2004;Zarifi et al, 2012).…”