The Allosteric Mechanisms Driving the Evolution of Androgen Specificity

C. Denise Okafor, Jennifer Colucci, Kirsten Cottrill, Alexandra Towner, Eric Ortlund

 

Emory University

 

Understanding the genetic and biophysical mechanisms by which proteins evolve ligand specificity is crucial for continued progress in evolutionary biology and biochemistry. Androgen receptor is an essential hormone-controlled transcription factor, a member of the steroid receptor subfamily. AR modulates expression of essential genes by relaying allosteric signals to coregulator proteins, specifically in response to binding of androgenic hormones (e.g. dihydrotestosterone, DHT) in its ligand binding pocket. This preference was fine-tuned over evolution, as AR’s immediate phylogenetic ancestor was able to bind and respond to both progestogens and androgens. Here, we seek to understand the evolution of ligand specificity in this receptor using ancAR1, the ancestor of extant androgen receptors. Three historical substitutions have been identified that restore progesterone activation to AR1 in luciferase reporter assays. Our lab has used x-ray crystallography and biophysical methods to characterize the effects of the substitutions on AR1 interactions with both progesterone and DHT. Furthermore, microsecond-long molecular dynamics simulations and network analyses were employed to probe the effects of the replacements on the allosteric networks governing ligand activation within the receptor. This work reveals that allosteric communication between the AR1 ligand binding pocket and activation function helix, a key regulatory region of the receptor, is severely weakened in the non-activating AR1-progesterone complex. The historical substitutions reverse this effect, potentially illuminating the allosteric mechanism that drove the evolution of ligand-specificity in AR1. This work has application for understanding the allosteric network that governs ligand activation of modern-day steroid receptors.