Most proteins are regulated by physical interactions with other molecules; some are highly specific, but others interact with many partners. Despite much speculation, we know little about how and why specificity/promiscuity evolves in natural proteins. It is widely assumed that specific proteins evolved from more promiscuous ancient forms and that most proteins' specificity has been tuned to an optimal state by selection. Here we use ancestral protein reconstruction to trace the evolutionary history of ligand recognition in the steroid hormone receptors (SRs), a family of hormone-regulated animal transcription factors. We resurrected the deepest ancestral proteins in the SR family and characterized the structure-activity relationships by which they distinguished among ligands. We found that that the most ancient split in SR evolution involved a discrete switch from an ancient receptor for aromatized estrogens—including xenobiotics—to a derived receptor that recognized non-aromatized progestagens and corticosteroids. The family's history, viewed in relation to the evolution of their ligands, suggests that SRs evolved according to a principle of minimal specificity: at each point in time, receptors evolved ligand recognition criteria that were just specific enough to parse the set of endogenous substances to which they were exposed. By studying the atomic structures of resurrected SR proteins, we found that their promiscuity evolved because the ancestral binding cavity was larger than the primary ligand and contained excess hydrogen bonding capacity, allowing adventitious recognition of larger molecules with additional functional groups. Our findings provide an historical explanation for the sensitivity of modern SRs to natural and synthetic ligands—including endocrine-disrupting drugs and pollutants—and show that knowledge of history can contribute to ligand prediction. They suggest that SR promiscuity may reflect the limited power of selection within real biological systems to discriminate between perfect and “good enough.”
The functions of most proteins are defined by their interactions with other biological substances, such as DNA, nutrients, hormones, or other proteins. Some proteins are highly specific, but others are more promiscuous and can interact with a variety of natural substances, as well as drugs and pollutants. Understanding molecular interactions is a key goal in pharmacology and toxicology, but there are few general principles to help explain or predict protein specificity. Because every biological entity is the result of evolution, understanding a protein's history might help explain why it interacts with the substances to which it is sensitive. In this paper, we used ancestral protein reconstruction to experimentally trace how specificity evolved in an ancient group of proteins, the steroid hormone receptors (SRs), a family of proteins that regulate reproduction and other biological processes in animals. We show that SRs evolved according to a principle of minimal specificity: at each point in time, these proteins evolved to be specific enough to distinguish among the substances to which they were naturally exposed, but not more so. Our findings provide an historical explanation for modern SRs' diverse sensitivities to natural and man-made substances; they show that knowledge of history can contribute to predicting the ligands to which a modern protein will respond and indicate that promiscuity reflects the limited power of natural selection to discriminate between perfect and “good enough.”