Vestigialization of an Allosteric Switch: Genetic and Structural Mechanisms for the Evolution of Constitutive Activity in a Steroid Hormone Receptor

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.</p

Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Phylogenetic reconstruction of the structure and function of the ancestor of the nuclear receptor protein family reveals how functional diversity evolves by subtle tinkering with an ancestral template

Evolution of a New Function by Degenerative Mutation in Cephalochordate Steroid Receptors

Gene duplication is the predominant mechanism for the evolution of new genes. Major existing models of this process assume that duplicate genes are redundant; degenerative mutations in one copy can therefore accumulate close to neutrally, usually leading to loss from the genome. When gene products dimerize or interact with other molecules for their functions, however, degenerative mutations in one copy may produce repressor alleles that inhibit the function of the other and are therefore exposed to selection. Here, we describe the evolution of a duplicate repressor by simple degenerative mutations in the steroid hormone receptors (SRs), a biologically crucial vertebrate gene family. We isolated and characterized the SRs of the cephalochordate Branchiostoma floridae, which diverged from other chordates just after duplication of the ancestral SR. The B. floridae genome contains two SRs: BfER, an ortholog of the vertebrate estrogen receptors, and BfSR, an ortholog of the vertebrate receptors for androgens, progestins, and corticosteroids. BfSR is specifically activated by estrogens and recognizes estrogen response elements (EREs) in DNA; BfER does not activate transcription in response to steroid hormones but binds EREs, where it competitively represses BfSR. The two genes are partially coexpressed, particularly in ovary and testis, suggesting an ancient role in germ cell development. These results corroborate previous findings that the ancestral steroid receptor was estrogen-sensitive and indicate that, after duplication, BfSR retained the ancestral function, while BfER evolved the capacity to negatively regulate BfSR. Either of two historical mutations that occurred during BfER evolution is sufficient to generate a competitive repressor. Our findings suggest that after duplication of genes whose functions depend on specific molecular interactions, high-probability degenerative mutations can yield novel functions, which are then exposed to positive or negative selection; in either case, the probability of neofunctionalization relative to gene loss is increased compared to existing models

Data_Sheet_2.FASTA

<p>Toll-like receptor 4 (TLR4) induces inflammation in response to both pathogen- and host-derived molecules. Lipopolysaccharide (LPS) recognition by TLR4 has been shown to occur across the amniotes, but endogenous signaling through TLR4 has not been validated outside of placental mammals. To determine whether endogenous danger signaling is also shared across amniotes, we studied the evolution of TLR4-activation by the calgranulin proteins (S100A8, S100A9, and S100A12), a clade of host molecules that potently activate TLR4 in placental mammals. We performed phylogenetic and syntenic analysis and found MRP-126—a gene in birds and reptiles—is likely orthologous to the mammalian calgranulins. We then used an ex vivo TLR4 activation assay to establish that calgranulin pro-inflammatory activity is not specific to placental mammals, but is also exhibited by representative marsupial and sauropsid species. This activity is strongly dependent on the cofactors CD14 and MD-2 for all species studied, suggesting a conserved mode of activation across the amniotes. Ortholog complementation experiments between the calgranulins, TLR4, CD14, and MD-2 revealed extensive lineage specific-coevolution and multi-way interactions between components that are necessary for the activation of NF-κB signaling by calgranulins and LPS. Our work demonstrates that calgranulin activation of TLR4 evolved at least ~320 million years ago and has been conserved in the amniote innate immune system.</p

Data_Sheet_1.FASTA

<p>Toll-like receptor 4 (TLR4) induces inflammation in response to both pathogen- and host-derived molecules. Lipopolysaccharide (LPS) recognition by TLR4 has been shown to occur across the amniotes, but endogenous signaling through TLR4 has not been validated outside of placental mammals. To determine whether endogenous danger signaling is also shared across amniotes, we studied the evolution of TLR4-activation by the calgranulin proteins (S100A8, S100A9, and S100A12), a clade of host molecules that potently activate TLR4 in placental mammals. We performed phylogenetic and syntenic analysis and found MRP-126—a gene in birds and reptiles—is likely orthologous to the mammalian calgranulins. We then used an ex vivo TLR4 activation assay to establish that calgranulin pro-inflammatory activity is not specific to placental mammals, but is also exhibited by representative marsupial and sauropsid species. This activity is strongly dependent on the cofactors CD14 and MD-2 for all species studied, suggesting a conserved mode of activation across the amniotes. Ortholog complementation experiments between the calgranulins, TLR4, CD14, and MD-2 revealed extensive lineage specific-coevolution and multi-way interactions between components that are necessary for the activation of NF-κB signaling by calgranulins and LPS. Our work demonstrates that calgranulin activation of TLR4 evolved at least ~320 million years ago and has been conserved in the amniote innate immune system.</p