Researchers discover water-based ‘Humpty-Dumpty’ mechanism to reverse humankind at brink of developmental mystery

Researchers from Indiana University School of Medicine have discovered a molecular “stabilization” mechanism within a male-specific protein-DNA complex whose mutation causes a sex-reversal: babies with XY chromosomes but female bodies, a condition called Swyer syndrome. Synapse exploits a water molecule that binds the male factor (designated SRY) and DNA control sites in the vulnerable beginnings of male development.

The study focuses on the exact replacement of the conserved aromatic residue in SRY (tyrosine) with a closely related aromatic residue (phenylalanine). The clinical mutation, shared by an XY fertile father and an XY sterile daughter, places the embryonic male switch at the cut-off point of genetic function. The two aromatic rings appear to be interchangeable in protein structure, but they differ in their ability to stabilize a conductive water molecule in the protein-DNA complex.

“Losing a single atom in SRY, an oxygen atom in critical tyrosine, impairs male developmental robustness,” said Michael Weiss, MD, chair of the Department of Biochemistry and Molecular Biology.

Normally, the father has XY chromosomes and the daughter has XX chromosomes but in some families, daughters can have XY chromosomes due to an SRY mutation. The sex chromosomes can deteriorate over evolutionary time scales, leading to new switches being mapped upstream as they grow. Male-determination pathways are backwards. And these initial steps can be biochemically vulnerable.”

In the study, the researchers focused on position 72 in the DNA-binding domain of SRY, which was not previously considered particularly important. However, the researchers discovered that tyrosine at this position enables water-mediated motor synapse operation, which extends the life of the protein-DNA complex.

This mechanism is conserved in all mammalian SRY factors and is widely observed in a related family of switching factors in multicellular (and some unicellular) animals. The last family, named ‘SOX’ (SRY-related HMG box) is central to the modeling and evolution of metazoans.

The researchers published two recent papers about their work in Frontiers in endocrinology. The first describes their findings regarding square 72 focus, and the second describes how the water-mediated grating mechanism works. Weiss said they call it the “Humpty-Dumpty” model because of the accelerated disassembly of the male-specific protein-DNA complex in the absence of a water-mediated synapse.

Said Joseph D. “It took several years for the water-mediated mechanism to be revealed. Crucial insight was provided by molecular dynamics (MD) simulations of the boundary water molecules in this system.”

“In the MD simulation, a distinct water molecule is stabilized by a tyrosine as a bridge to the DNA: this particular hydration site is occupied for thousands of picoseconds, then it leaves,” Weiss said. “But then, another water molecule in the bulk solvent will almost immediately jump into place, restoring the bridge.”

A subtle change from tyrosine to phenylalanine alters this hydration, a perturbation propagating from position 72 that is expected to destabilize the cascade of protein-DNA contacts in the tail of the domain. Tail detachment may accelerate dissociation of the protein-DNA complex and likely male-specific gene regulatory assemblies of target genes.

XY females with differences in sexual differentiation due to Swyer syndrome lack functional ovaries and are at risk of developing rare forms of early gonadal cancer. Recognizing this syndrome is important to allow surgical removal of the gonads before cancer begins. Otherwise, the affected woman has a normal uterus and birth canal, and thus may have children after in vitro fertilization of a donor egg.

Similar mutations can occur in the SOX genes, Weiss said, causing a variety of birth defects or diseases.

“Swyer mutations provide clues to help us understand a wide range of SOX diseases and may lead to much improved protocols for different areas of medicine, such as regenerative medicine or cancer,” said Weiss. “This finding therefore goes beyond sex determination because SRY is a prototype switch.”