111 research outputs found
Protein evolution
On the origin of proteins A series of mistakes over the past 3.7 billion years or so has left us with a spectacular array of protein structures and functions, which are responsible for nothing less than life itself, writes Bea Perk
Theophany and Chaoskampf : the interpretation of Theophanic imagery in the Baal Epic, Isaiah, and the Twelve
This dissertation concerns the interpretation of theophanic imagery in the Prophets. In
essence, my argument is that the imagery which attends and describes theophany in the poetic
books of the Hebrew Bible is mythic and not metaphorical-that it should be interpreted in
relation to the Chaoskampf myth, as Yahweh defeats chaos and restores order, rather than being
understood as a metaphorical comparison with natural phenomena. In arguing this way,
however, I am taking a new approach to theophanic imagery, for such imagery has almost
uniformly been understood in Old Testament scholarship as metaphor. Before examining in
detail this traditional and more widespread approach to theophanic imagery and the alternative to
it which I wish to suggest, however, it will be helpful to state at the outset two foundations upon
which my argument rests and the specific texts which will be enlisted to support it
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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
Crystallization and preliminary X-ray diffraction analysis of Sfh3, a member of the Sec14 protein superfamily
Sec14 is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the yeast Saccharomyces cerevisiae and is the founding member of the Sec14 protein superfamily. Recent functional data suggest that Sec14 functions as a nanoreactor for PtdCho-regulated presentation of PtdIns to PtdIns kinase to affect membrane trafficking. Extrapolation of this concept to other members of the Sec14 superfamily suggests a mechanism by which a comprehensive cohort of Sec14-like nanoreactors sense correspondingly diverse pools of lipid metabolites. In turn, metabolic information is translated to signaling circuits driven by phosphoinositide metabolism. Sfh3, one of five Sec14 homologs in yeast, exhibits several interesting functional features, including its unique localization to lipid particles and microsomes. This localization forecasts novel regulatory interfaces between neutral lipid metabolism and phosphoinositide signaling. To launch a detailed structural and functional characterization of Sfh3, the recombinant protein was purified to homogeneity, diffraction-quality crystals were produced and a native X-ray data set was collected to 2.2 Å resolution. To aid in phasing, SAD X-ray diffraction data were collected to 1.93 Å resolution from an SeMet-labeled crystal at the Southeast Regional Collaborative Access Team at the Advanced Photon Source. Here, the cloning and purification of Sfh3 and the preliminary diffraction of Sfh3 crystals are reported, enabling structural analyses that are expected to reveal novel principles governing ligand binding and functional specificity for Sec14-superfamily proteins
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
Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor
Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution
A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis
The Sec14-like phosphatidylinositol transfer protein Sfh3 associates with bulk LDs in vegetative cells but targets to a neutral lipid hydrolase-rich LD pool during sporulation. Sfh3 inhibits LD utilization by a PtdIns-4-phosphate–dependent mechanism, and this inhibition prevents prospore membrane biogenesis in sporulating cells.Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches
Distal substitutions alter conformational space to create new functions among paralogous transcription factors
Non UBCUnreviewedAuthor affiliation: Emory UniversityResearche
X-ray crystal structure of the ancestral 3-ketosteroid receptor-progesterone-mifepristone complex shows mifepristone bound at the coactivator binding interface.
Steroid receptors are a subfamily of nuclear receptors found throughout all metazoans. They are highly important in the regulation of development, inflammation, and reproduction and their misregulation has been implicated in hormone insensitivity syndromes and cancer. Steroid binding to SRs drives a conformational change in the ligand binding domain that promotes nuclear localization and subsequent interaction with coregulator proteins to affect gene regulation. SRs are important pharmaceutical targets, yet most SR-targeting drugs have off-target pharmacology leading to unwanted side effects. A better understanding of the structural mechanisms dictating ligand specificity and the evolution of the forces that created the SR-hormone pairs will enable the design of better pharmaceutical ligands. In order to investigate this relationship, we attempted to crystallize the ancestral 3-ketosteroid receptor (ancSR2) with mifepristone, a SR antagonist. Here, we present the x-ray crystal structure of the ancestral 3-keto steroid receptor (ancSR2)-progesterone complex at a resolution of 2.05 Å. This improves upon our previously reported structure of the ancSR2-progesterone complex, permitting unambiguous assignment of the ligand conformation within the binding pocket. Surprisingly, we find mifepristone, fortuitously docked at the protein surface, poised to interfere with coregulator binding. Recent attention has been given to generating pharmaceuticals that block the coregulator binding site in order to obstruct coregulator binding and achieve tissue-specific SR regulation independent of hormone binding. Mifepristone's interaction with the coactivator cleft of this SR suggests that it may be a useful molecular scaffold for further coactivator binding inhibitor development
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