title: Structure-function relationships in human testis-determining factor SRY: an aromatic buttress underlies the specific DNA-bending surface of a high mobility group (HMG) box.
abstract: Human testis determination is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in SRY cause 46 XY gonadal dysgenesis with female somatic phenotype (Swyer syndrome) and confer a high risk of malignancy (gonadoblastoma). Such mutations cluster in the SRY high mobility group (HMG) box, a conserved motif of specific DNA binding and bending. To explore structure-function relationships, we constructed all possible substitutions at a site of clinical mutation (W70L). Our studies thus focused on a core aromatic residue (position 15 of the consensus HMG box) that is invariant among SRY-related HMG box transcription factors (the SOX family) and conserved as aromatic (Phe or Tyr) among other sequence-specific boxes. In a yeast one-hybrid system sensitive to specific SRY-DNA binding, the variant domains exhibited reduced (Phe and Tyr) or absent activity (the remaining 17 substitutions). Representative nonpolar variants with partial or absent activity (Tyr, Phe, Leu, and Ala in order of decreasing side-chain volume) were chosen for study in vitro and in mammalian cell culture. The clinical mutation (Leu) was found to markedly impair multiple biochemical and cellular activities as respectively probed through the following: (i) in vitro assays of specific DNA binding and protein stability, and (ii) cell culture-based assays of proteosomal degradation, nuclear import, enhancer DNA occupancy, and SRY-dependent transcriptional activation. Surprisingly, however, DNA bending is robust to this or the related Ala substitution that profoundly impairs box stability. Together, our findings demonstrate that the folding, trafficking, and gene-regulatory function of SRY requires an invariant aromatic "buttress" beneath its specific DNA-bending surface.
authors.affiliation: From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Racca
authors.affiliation: From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Yen-Shan
authors.initials: YS
authors.lastname: Chen
authors.affiliation: From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: James D
authors.initials: JD
authors.lastname: Maloy
authors.affiliation: From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Nalinda
authors.initials: N
authors.lastname: Wickramasinghe
authors.affiliation: From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Nelson B
authors.initials: NB
authors.lastname: Phillips
authors.affiliation: From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106 maw21@case.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D051876:Sex-Determining Region Y Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D015533:Transcriptional Activation
chemical_list: D024322:Amino Acids, Aromatic; D004268:DNA-Binding Proteins; D051876:Sex-Determining Region Y Protein; D004247:DNA
keywords: DNA-binding Protein; Development; Disorders of Sexual Development; Intersex; Protein Conformation; Swyer Syndrome; Testis; Transcription