Dimerization of Doublesex Is Mediated by a Cryptic Ubiquitin-associated Domain Fold: IMPLICATIONS FOR SEX-SPECIFIC GENE REGULATION

James R Bayrer,Wei Zhang,Michael A Weiss

doi:10.1074/jbc.m507990200

James R Bayrer, Wei Zhang + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m507990200

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Abstract

Male- and female-specific isoforms of the Doublesex (DSX) transcription factor regulate somatic sexual differentiation in Drosophila. The isoforms (DSX(M) and DSX(F)) share an N-terminal DNA binding domain (the DM motif), broadly conserved among metazoan sex-determining pathways. DM-DNA recognition is enhanced by a C-terminal dimerization domain. The crystal structure of this domain, determined at a resolution of 1.6 A, reveals a novel dimeric arrangement of ubiquitin-associated (UBA) folds. Although this alpha-helical motif is well characterized in pathways of DNA repair and subcellular trafficking, to our knowledge this is its first report in a transcription factor. Dimerization is mediated by a non-canonical hydrophobic interface extrinsic to the putative ubiquitin binding surface. Key side chains at this interface, identified by alanine scanning mutagenesis, are conserved among DSX homologs. The mechanism of dimerization is thus unrelated to the low affinity domain swapping observed among ubiquitin-associated CUE domains. The unexpected observation of a ubiquitin-associated fold in DSX extends the repertoire of alpha-helical dimerization elements in transcription factors. The possibility that the ubiquitination machinery participates in the regulation of sexual dimorphism is discussed.

Highlights

Sexual differentiation in Drosophila is regulated by the X:autosome ratio and a sex-specific RNA-splicing pathway (Fig. 1A; Ref. 1)
The DSX isoforms are encoded by mRNAs sharing the first three exons; the C-terminal segment of DSXF is encoded by exon 4, whereas that of DSXM is encoded by exons 5 and 6
In this article we describe the crystal structure of a dimeric fragment of CTDF at 1.6 Å resolution and its scanning mutagenesis in a yeast two-hybrid (Y2H) system

Summary

EXPERIMENTAL PROCEDURES

Protein Crystallization—CTDF-p (65 residues; GS followed by DSXF residues 350 – 412) was designed based on Y2H studies [7, 10] and expressed in Escherichia coli (strain B834(DE3)pLysS) as a thrombincleavable fusion protein and purified as described [11]. The structure of CTDF-p forms an ellipsoidal dimer with protomers oriented head to tail (Fig. 2B). Despite limited sequence homology (Fig. 4A and supplemental Fig. S4), structural alignment of a CTDF-p protomer with 18 UBA domains yields a mean pairwise root mean square deviation of 1.7 Å (range 1.2–2.3 Å) among main-chain atoms (Fig. 4B); the mean base-line pairwise root mean square deviation among these 18 domains is 1.3 Å. Each protomer contains a small hydrophobic core centered about the side chains of Met-377 and Cys-360 (Fig. 5A). This “mini-core” contains residues from each of the three helixes, the predominant contribution is from ␣2. Whereas ␣1 and ␣3 define the surface of the dimer, ␣2 and ␣2Ј pack at the interface; they are parallel and nearly perpendicular to

TABLE ONE

Most favored

DISCUSSION

Findings

Wild type ϩ