Evolution of Minimal Specificity and Promiscuity in Steroid Hormone Receptors (original) (raw)
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Biophysical mechanisms for large-effect mutations in the evolution of steroid hormone receptors
Proceedings of the National Academy of Sciences, 2013
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.
Evolution of steroid receptors from an estrogen-sensitive ancestral receptor
Molecular and Cellular Endocrinology, 2011
Members of the steroid hormone receptor (SR) family activate transcription from different DNA response elements and are regulated by distinct hormonal ligands. Understanding the evolutionary process by which this diversity arose can provide insight into how and why SRs function as they do. Here we review the characteristics of the ancient receptor protein from which the SR family descends by a process of gene duplication and divergence. Several orthogonal lines of evidence -bioinformatic, phylogenetic, and experimental -indicate that this ancient SR had the capacity to activate transcription from DNA estrogen response elements in response to estrogens. Duplication and divergence of the ancestral SR gene subsequently generated new receptors that were activated by other steroid hormones, including progestagens, androgens, and corticosteroids. The androgen and progesterone receptors recruited as their ligands steroids that were previously present as biochemical intermediates in the synthesis of estrogens. This process is an example of molecular exploitation-the evolution of new molecular interactions when an older molecule, which previously had a different function, is co-opted as a binding partner by a newly evolved molecule. The primordial interaction between the ancestral steroid receptor and estrogens may itself have evolved due to an early molecular exploitation event.
Origin of an ancient hormone/receptor couple revealed by resurrection of an ancestral estrogen
Science advances, 2017
The origin of ancient ligand/receptor couples is often analyzed via reconstruction of ancient receptors and, when ligands are products of metabolic pathways, they are not supposed to evolve. However, because metabolic pathways are inherited by descent with modification, their structure can be compared using cladistic analysis. Using this approach, we studied the evolution of steroid hormones. We show that side-chain cleavage is common to most vertebrate steroids, whereas aromatization was co-opted for estrogen synthesis from a more ancient pathway. The ancestral products of aromatic activity were aromatized steroids with a side chain, which we named "paraestrols." We synthesized paraestrol A and show that it effectively binds and activates the ancestral steroid receptor. Our study opens the way to comparative studies of biologically active small molecules.
Molecular and Cellular Endocrinology, 2008
Comparative endocrinology considers the evolution of bioregulatory systems and the anatomical structures and molecules that constitute the neuroendocrine and endocrine systems. One aim of comparative endocrinology is to trace the origins of the main endocrine systems. The understanding of the evolution of the ligand/receptor couple is central to this objective. One classical approach to tackle this question is the characterization of receptors and ligands in various types of non-model organisms using as a starting point the knowledge accumulated on classical models such as mammals (mainly human and mouse) and arthropods (with Drosophila among other insects). In this review we discuss the potential caveats associated to this two-by-two comparison between a classical model and non-model organisms. We suggest that the use of an evolutionary approach involving comparisons of several organisms in a coherent framework permits reconstruction of the most probable scenarios. The use of the vast amount of genomic data now available, coupled to functional experiments, offers unprecedented possibilities to trace back the origins of the main ligand/receptor couples.
Evolutionary tuning of a key helix drove androgen selectivity
2021
The genetic and biophysical mechanisms by which new protein functions evolve are central concerns in evolutionary biology and molecular evolution. Despite much speculation, we know little about how protein function evolves. Here, we use ancestral proteins to trace the evolutionary history of ligand recognition in a sub-class of steroid receptors (SRs), an ancient family of ligand-regulated transcription factors that enable long-range cellular communication central to multicellular life. The most ancestral members of this family display promiscuous ligand binding due to their large ligand binding pockets, while more recently evolved SRs tend to have smaller cavities. Less obvious, however, are the forces driving the selectivity of highly similar ligands. A key example is the divergence between the progesterone and androgen receptors (PR, AR), which display a high degree of sequence similarity and yet display differential ligand preferences. This work uses the ancestral steroid recept...
Ligand modulation of allosteric networks in an ancestral steroid receptor
2018
Understanding the evolution of binding specificity, a heavily studied area of research, is key for determining how protein sequence changes alter function. Ligand-activation in the steroid receptor subfamily of transcription factors operates via a common allosteric mechanism which permits extant receptors to respond specifically to their cognate hormones. Here, we combine atomistic simulations with graph theory-based modeling of the inter-residue interactions within protein complexes to gain insight into how allostery drove selectivity in an ancestral receptor. An inactive ligand complex displays weakened allosteric communication, as quantified by suboptimal paths linking two functional surfaces. When function-switching mutations are incorporated, responses in allosteric networks are consistent with ligand activation status. Further analysis reveals residues that modulate features distinguishing active and inactive complexes, identifying a key, conserved residue that is crucial for ...
PLoS Genetics, 2014
An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become ''stuck'' in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations to trigger a profound evolutionary change in the protein's function and shaped the genetics of evolutionary reversibility.
In the last decade, there has been important progress in understanding the origins and evolution of receptors for adrenal steroids (aldosterone, cortisol) and sex steroids (estradiol, progesterone, testosterone) due to the sequencing of genomes from animals that are at key sites in vertebrate evolution. Although the estrogen receptor [ER] appears to be the ancestral vertebrate steroid receptor and estradiol [E2] is the physiological ligand for vertebrate ERs, the identity of the ancestral ligand(s) for the ER remains unknown. Here, using an analysis of crystal structures of human ERa with E2 and other chemicals and 3D models of human ERa with 27-hydroxycholesterol and 5-androsten-3b,17b-diol, I propose that one or more D5 steroids were the ancestral ligands for the ER, with E2 evolving later as the canonical estrogen. The evidence that chemicals with a b-hydroxy at C3 in a saturated A ring can act as estrogens and the conformational flexibility of the vertebrate ER can explain the diversity of synthetic chemicals that disrupt estrogen responses by binding to vertebrate ERs.
Directed evolution of estrogen receptor proteins with altered ligand-binding specificities
Protein Engineering Design and Selection, 2008
Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor a (hERa) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast twohybrid system that reports the ligand-dependent interaction between hERa and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 mM. The optimized receptor mutant (L384F/L387M/ Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 mM) and a 10-fold decreased activity to 17ß-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.
The Journal of Steroid Biochemistry and Molecular Biology
Present-day nuclear receptors (NRs) responding to adrenal and sex steroids are key regulators of reproduction and growth in mammals, and are thought to have evolved from an ancestral NR most closely related to extant estrogen-related receptors (ERRs). The molecular events (and ligands) that distinguish steroid-activated NRs (SRs) from their inferred ancestor, that gave rise to both the ERRs and SRs, remain unknown. We report that target sequences for fatty-acylation (palmitoylation) at a key cysteine residue (corresponding to Cys447 in human estrogen receptor ERα) in helix 8 of the ligand-binding domain accurately demarcate SRs from ERRs. Docking studies are consistent with the hypothesis that palmitate embeds into a key groove in the receptor surface. The implications of lipidation, and of potential alternative ligands for the key cysteine residue, for receptor function and the evolution of SRs are discussed.