title: Mammalian testis-determining factor SRY and the enigma of inherited human sex reversal: frustrated induced fit in a bent protein-DNA complex.
abstract: Mammalian testis-determining factor SRY contains a high mobility group box, a conserved eukaryotic motif of DNA bending. Mutations in SRY cause XY gonadal dysgenesis and somatic sex reversal. Although such mutations usually arise de novo in spermatogenesis, some are inherited and so specify male development in one genetic background (the father) but not another (the daughter). Here, we describe the biophysical properties of a representative inherited mutation, V60L, within the minor wing of the L-shaped domain (box position 5). Although the stability and DNA binding properties of the mutant domain are similar to those of wild type, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance energy transfer (FRET) revealed enhanced conformational fluctuations leading to long range variation in bend angle. (1)H NMR studies of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the minor wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional regulation in an embryonic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have demonstrated a nonlocal coupling between DNA-directed protein folding and protein-directed DNA bending. Perturbation of this coupling is associated with a genetic switch poised at the threshold of activity.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Nelson B
authors.initials: NB
authors.lastname: Phillips
authors.forename: Joseph
authors.initials: J
authors.lastname: Racca
authors.forename: Yen-Shan
authors.initials: YS
authors.lastname: Chen
authors.forename: Rupinder
authors.initials: R
authors.lastname: Singh
authors.forename: Agnes
authors.initials: A
authors.lastname: Jancso-Radek
authors.forename: James T
authors.initials: JT
authors.lastname: Radek
authors.forename: Nalinda P
authors.initials: NP
authors.lastname: Wickramasinghe
authors.forename: Elisha
authors.initials: E
authors.lastname: Haas
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006061:Gonadal Dysgenesis, 46,XY; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012377:Rodentia; D055758:SOX9 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051876:Sex-Determining Region Y Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C497745:SRY protein, human; D051876:Sex-Determining Region Y Protein; D004247:DNA