Biophysical mechanisms for large-effect mutations in the evolution of steroid hormone receptors

Abstract

The genetic and biophysical mechanisms by which new protein functions evolve is a central question in evolutionary biology, biochemistry, and biophysics. Of particular interest is whether major shifts in protein function are caused by a few mutations of large effect and, if they are, the mechanisms that mediate these changes. Here we combine ancestral protein reconstruction with genetic manipulation and explicit studies of protein structure and dynamics to dissect an ancient and discrete shift in ligand specificity in the steroid receptors, a family of biologically essential hormone-controlled transcription factors. We previously found that the ancestor of the entire steroid receptor family was highly specific for estrogens, but its immediate phylogenetic descendant was sensitive only to androgens, progestogens, and corticosteroids. Here we show that this shift in function was driven primarily by two historical amino acid changes, which caused a ∼70,000-fold change in the ancestral protein's specificity. These replacements subtly changed the chemistry of two amino acids, but they dramatically reduced estrogen sensitivity by introducing an excess of interaction partners into the receptor/estrogen complex, inducing a frustrated ensemble of suboptimal hydrogen bond networks unique to estrogens. This work shows how the protein's architecture and dynamics shaped its evolution, amplifying a few biochemically subtle mutations into major shifts in the energetics and function of the protein.ancestral reconstruction | molecular evolution | protein evolution | evolutionary biochemistry A central goal in biochemistry/biophysics is to understand how proteins' sequences determine their functional specificity. In molecular evolution, a key objective is to reveal the historical processes by which the diverse functions of extant proteins came to be. These goals, pursued separately, have rarely been achieved in full because of the deep interplay between a protein's history and its physical properties (1-3). A complete explanation of the functional differences among proteins would explicitly identify the historical mutations that caused their functions to diverge, characterize the physical mechanisms that mediated these mutations' effects, and reveal how the architecture of the protein shaped and was shaped by the evolutionary process. Such studies could help explain key questions in evolution, such as the role and mechanisms of large-effect mutations in phenotypic evolution (4-8), and in biophysics, such as the determinants of ligand specificity (9-11).Ancestral sequence reconstruction allows the properties of ancient proteins and the effects of historical mutations to be characterized directly (3,12). It has been used to identify key mutations that led to changes in protein structure and function (13-16), but it has not been used to understand the evolution of proteins as dynamic molecular systems. Scores or hundreds of amino acids participate in a dense network of interactions to determine protein structure, dynamics, and funct...

Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

“…Functional promiscuity and conformational dynamism have been hypothesized to contribute to protein evolvability (33,42), but there have been very few direct studies of the historical evolution of a protein's occupancy of the ensemble of potential conformers (43,44). Our findings reinforce the importance of these factors.…”

Biophysics of protein evolution and evolutionary protein biophysics

“…We have mentioned thioredoxin as one of the proteins that were studied in this manner in the discussion on kinetic stability ( §2.2). Another interesting case is the reconstruction of the evolution of steroid receptors [376,429], revealing that ancient steroid receptors were already able to bind to the hormone aldosterone. But aldosterone only became available to rsif.royalsocietypublishing.org J. R. Soc.…”

Characterization of evolutionary trend in squamate estrogen receptor sensitivity

“…Divergence of various contemporary steroid hormone receptors, including ERs, from the ancestral steroid hormone receptors was in theory initiated by discrete evolutionary shift in the receptor's ligand specificity through few amino acid mutations in the ligand-binding pocket region (Harms et al, 2013). Here, we present evidence for a potential evolution of ER in nature.…”

“…Evolution of the steroid hormone receptors is a much-discussed theme in the field of comparative endocrinology (Harms et al, 2013, Thornton, 2001and Thornton et al, 2003. Divergence of various contemporary steroid hormone receptors, including ERs, from the ancestral steroid hormone receptors was in theory initiated by discrete evolutionary shift in the receptor's ligand specificity through few amino acid mutations in the ligand-binding pocket region (Harms et al, 2013).…”