Scientists Retrace Evolution With First Atomic Structure Of An Ancient Protein

"Never before have we seen so clearly, so far back in time," said project leader Joe Thornton, an evolutionary biologist at the University of Oregon. "We were able to see the precise mechanisms by which evolution molded a tiny molecular machine at the atomic level, and to reconstruct the order of events by which history unfolded."

A detailed understanding of how proteins -- the workhorses of every cell -- have evolved has long eluded evolutionary biologists, in large part because ancient proteins have not been available for direct study. So Thornton and Jamie Bridgham, a postdoctoral scientist in his lab, used state-of-the-art computational and molecular techniques to re-create the ancient progenitors of an important human protein.

Thornton then collaborated with University of North Carolina biochemists Eric Ortlund and Matthew Redinbo, who used ultra-high energy X-rays from a stadium-sized Advanced Photon Source at Argonne National Laboratory near Chicago to chart the precise position of each of the 2,000 atoms in the ancient proteins. The groups then worked together to trace how changes in the protein's atomic architecture over millions of years caused it to evolve a crucial new function -- uniquely responding to the hormone that regulates stress.

"This is the ultimate level of detail," Thornton said. "We were able to see exactly how evolution tinkered with the ancient structure to produce a new function that is crucial to our own bodies today. Nobody's ever done that before."

The researchers focused on the glucocorticoid receptor (GR), a protein in humans and other vertebrates that allows cells to respond to the hormone cortisol, which regulates the body's stress response. The scientists' goal was to understand the process of evolution behind the GR's ability to specifically interact with cortisol.

They used computational techniques and a large database of modern receptor sequences to determine the ancient GR's gene sequence from a time just before and just after its specific relationship with cortisol evolved. The ancient genes -- which existed more than 400 million years ago -- were then synthesized, expressed, and their structures determined using X-ray crystallography, a state-of-the art technique that allows scientists to see the atomic architecture of a molecule. The project represents the first time the technique has been applied to an ancient protein.

The structures allowed the scientists to identify exactly how the new function evolved. They found that just seven historical mutations, when introduced into the ancestral receptor gene in the lab, recapitulated the evolution of GR's present-day response to cortisol. They were even able to deduce the order in which these changes occurred, because some mutations caused the protein to lose its function entirely if other "permissive" changes, which otherwise had a negligible effect on the protein, were not in place first.

"These permissive mutations are chance events. If they hadn't happened first, then the path to the new function could have become an evolutionary road not taken," Thornton said. "Imagine if evolution could be rewound and set in motion again: a very different set of genes, functions and processes might be the outcome."The atomic structure revealed exactly how these mutations allowed the new function to evolve. The most radical one remodeled a whole section of the protein, bringing a group of atoms close to the hormone. A second mutation in this repositioned region then created a tight new interaction with cortisol. Other earlier mutations buttressed particular parts of the protein so they could tolerate this eventual remodeling.

"We were able to walk through the evolutionary process from the distant past to the present day," said Ortlund, who is now at Emory University in Atlanta. "Until now, we've always had to look at modern proteins and just guess how they evolved."

The work involving the protein is detailed in a paper appearing online Aug. 16 in Science Express, where the journal Science promotes selected research in advance of regular publication. 

The work was funded by multiple grants from the National Institutes of Health and the National Science Foundation, the UNC Lineberger Comprehensive Cancer Center and an Alfred P. Sloan Research Fellowship to Thornton.