Inherited Human Sex Reversal due to Loss of a Water-Mediated Hydrogen Bond at a Conserved Protein-DNA Interface

Abstract

Male sex determination in mammals is initiated by SRY, a Y-encoded architectural transcription factor. The protein contains a high-mobility-group (HMG) box that mediates sequence-specific DNA bending. Mutations in SRY causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box. Although such mutations usually arise de novo in spermatogenesis, some are inherited: male development occurs in one genetic background (the father) but not another (the sterile XY daughter). Here, we compare de novo and inherited mutations at an invariant Tyr adjoining the motif’s basic tail (consensus position 72; Y127C and Y127F in intact SRY). Crystal structures of homologous SOX-DNA complexes suggest that the wild-type side chain’s para-OH group anchors a water-mediated hydrogen bond to the DNA backbone. In an embryonic gonadal cell line, Y127C (de novo) led to accelerated proteasomal proteolysis and blocked transcriptional activity; activity remained low on rescue of expression by chemical proteasome inhibition. Y127F (inherited) preserved substantial transcriptional activity: 91(±11)% on SRY overexpression and 65(±17)% at physiological expression. Control studies indicated no change in protein lifetime or nuclear localization. Only subtle biophysical perturbations were observed in vitro. Although though inherited variant’s specific DNA affinity was only twofold lower than wild type, stopped-flow kinetic analysis revealed a sevenfold decrease in lifetime of the complex. Time-resolved fluorescence energy transfer (using a 15-base pair DNA site) demonstrated native mean DNA bending but with a slightly widened distribution of end-to-end DNA distances. Our findings highlight the contribution of a single water-mediated hydrogen bond to robustness of a genetic switch in human development.