Most proteins are regulated by physical interactions with other molecules; some are highly specific, but others interact with many partners. Despite much speculation, we know little about how and why specificity/promiscuity evolves in natural proteins. It is widely assumed that specific proteins evolved from more promiscuous ancient forms and that most proteins' specificity has been tuned to an optimal state by selection. Here we use ancestral protein reconstruction to trace the evolutionary history of ligand recognition in the steroid hormone receptors (SRs), a family of hormone-regulated animal transcription factors. We resurrected the deepest ancestral proteins in the SR family and characterized the structure-activity relationships by which they distinguished among ligands. We found that that the most ancient split in SR evolution involved a discrete switch from an ancient receptor for aromatized estrogens—including xenobiotics—to a derived receptor that recognized non-aromatized progestagens and corticosteroids. The family's history, viewed in relation to the evolution of their ligands, suggests that SRs evolved according to a principle of minimal specificity: at each point in time, receptors evolved ligand recognition criteria that were just specific enough to parse the set of endogenous substances to which they were exposed. By studying the atomic structures of resurrected SR proteins, we found that their promiscuity evolved because the ancestral binding cavity was larger than the primary ligand and contained excess hydrogen bonding capacity, allowing adventitious recognition of larger molecules with additional functional groups. Our findings provide an historical explanation for the sensitivity of modern SRs to natural and synthetic ligands—including endocrine-disrupting drugs and pollutants—and show that knowledge of history can contribute to ligand prediction. They suggest that SR promiscuity may reflect the limited power of selection within real biological systems to discriminate between perfect and “good enough.”

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca 2؉ flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca 2؉ flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated leukocytes following an inflammatory episode. Several factors, including members of the galectin and tumor necrosis factor families (1, 2), regulate leukocyte turnover by inducing apoptotic cell death. In contrast, several galectin family members, in particular galectin-1 (Gal-1), 2 uniquely regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure, which normally sensitizes apoptotic cells to phagocytic removal (3, 4), independent of apoptosis, a process recently termed preaparesis (5).Previous studies suggested that dimerization may be required for Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays impaired Gal-1 dimerization and fails to induce PS exposure (6). However, the manner in which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear. Previous studies suggest that dimerization may be required for efficient cross-linking of functional receptors or the formation of signaling lattices (7-9). Consistent with this, monomeric mutants of several other galectins fail to induce PS exposure or signal leukocytes (4, 8). Gal-1 signaling of PS exposure requires initial signaling events, such as mobilization of intracellular Ca 2ϩ followed by sustained receptor engagement (10). Although mGal-1 fails to induce PS exposure (6), whether mGal-1 can induce these initial signaling events remains unknown (10).In addition to directly regulating signaling, monomer-dimer equilibrium may ...

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...

Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

The synthetic glucocorticoids (GCs) dexamethasone, mometasone furoate, and triamcinolone acetonide are pharmaceutical mainstays to treat chronic inflammatory diseases. These drugs bind to the glucocorticoid receptor (GR), a ligand-activated transcription factor and member of the nuclear receptor superfamily. The GR is widely recognized as a therapeutic target for its ability to counter proinflammatory signaling. Despite the popularity of GCs in the clinic, long-term use leads to numerous side effects, driving the need for new and improved drugs with less off-target pharmacology. X-ray crystal structures have played an important role in the drug-design process, permitting the characterization of robust structure-function relationships. However, steroid receptor ligand-binding domains (LBDs) are inherently unstable, and their crystallization requires extensive mutagenesis to enhance expression and crystallization. Here, we use an ancestral variant of GR as a tool to generate a high-resolution crystal structure of GR in complex with the potent glucocorticoid triamcinolone acetonide (TA) and a fragment of the small heterodimer partner (SHP). Using structural analysis, molecular dynamics, and biochemistry, we show that TA increases intramolecular contacts within the LBD to drive affinity and enhance stability of the receptor-ligand complex. These data support the emerging theme that ligand-induced receptor conformational dynamics at the mouth of the pocket play a major role in steroid receptor activation. This work also represents the first GR structure in complex with SHP, which has been suggested to play a role in modulating hepatic GR function.

Liquid-chromatography mass spectrometry is commonly used to identify and quantify metabolites from biological samples to gain insight into human physiology and pathology. Metabolites and their abundance in biological samples are labile and sensitive to variations in collection conditions, handling and processing. Variations in sample handling could influence metabolite levels in ways not related to biology, ultimately leading to the misinterpretation of results. For example, anticoagulants and preservatives modulate enzyme activity and metabolite oxidization. Temperature may alter both enzymatic and non-enzymatic chemistry. The potential for variation induced by collection conditions is particularly important when samples are collected in remote locations without immediate access to specimen processing. Data are needed regarding the variation introduced by clinical sample collection processes to avoid introducing artifact biases. In this study, we used metabolomics and lipidomics approaches paired with univariate and multivariate statistical analyses to assess the effects of anticoagulant, temperature, and time on healthy human plasma samples collected to provide guidelines on sample collection, handling, and processing for vaccinology. Principal component analyses demonstrated clustering by sample collection procedure and that anticoagulant type had the greatest effect on sample metabolite variation. Lipids such as glycerophospholipids, acylcarnitines, sphingolipids, diacylglycerols, triacylglycerols, and cholesteryl esters are significantly affected by anticoagulant type as are amino acids such as aspartate, histidine, and glutamine. Most plasma metabolites and lipids were unaffected by storage time and temperature. Based on this study, we recommend samples be collected using a single anticoagulant (preferably EDTA) with sample processing at <24 h at 4 °C.

Steroid receptors (SRs) are a class of ligand-regulated transcription factors that regulate gene expression in response to the binding of steroid hormones. Ligand binding drives conformational changes within the SR ligand binding domain that alters the receptors' affinity for coregulator proteins that in turn modulate chromatin state and either promote or block the recruitment of transcriptional machinery to a gene. Structural characterizations of SRs have provided insight into how these conformational rearrangements modulate receptor function, including signaling between the ligand binding pocket and the site of coregulator binding. Here, we review some of the proposed structural mechanisms put forward to explain the ability of ligands to modulate SR function. We also provide a discussion on computational methods that have contributed to the elucidation of SR allosteric regulation. Finally, we consider broader discussions of allostery within the SR family, such as receptor-induced reverse allostery and allosteric binding sites located outside of the canonical ligand interaction site.

31Tularemia is a rare but highly contagious and potentially fatal disease caused by bacteria 32 Francisella tularensis where as few as ten inhaled organisms can lead to an infection, making it 33 one of the most infectious microorganisms known and a potential bioweapon. To better 34 understand the response to a live, attenuated tularemia vaccine and the biological pathways 35 altered post-vaccination, healthy adults were vaccinated by scarification and plasma was 36 collected pre-and post-vaccination for longitudinal lipidomics studies. Using tandem mass 37 spectrometry, we identified and quantified individual lipid molecular species within 38 representative lipid classes in plasma to characterize alterations in the plasma lipidome during 39 the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in 40 inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid 41 classes. These included 5-Hydroxyeicosatetraenoic acid (5-HETE), an eicosanoid produced 42 following arachidonic acid liberation and epoxygenation, which is indicative of lipoxygenase 43 activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a 44 dihydroxyeicosatrienoic acid (DHET) by Day 7 post-vaccination, shedding light on the kinetics 45 of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we 46 observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 47 16:1 cholesteryl ester, and several glycerophospholipids, several of these changes in abundance 48 were correlated with serum cytokines and T cell activation. These data provide new insights into 49 alterations in plasma lipidome post tularemia vaccination, potentially identifying key mediators 50 and pathways involved in vaccine response and efficacy. 51 52 53Vaccines initially trigger innate immune responses that result in development of an adaptive 54 immune response with establishment of immunological memory (1). This process is 55 incompletely understood but is probably best detailed for the Yellow Fever vaccine (2). This 56 vaccine is highly effective and elicits a wide range of immune responses, resulting in greater than 57 30 years of protection from yellow fever. Previously published research has shown that the 58 vaccine produces a biologic signature identified by transcriptomics, cytokine analyses and flow 59 cytometry (3). Such biologic signatures may correlate with immunogenicity to predict the 60 efficacy of novel vaccines. Lipids are key stress-response and immune signaling molecules and 61 changes in their circulating concentrations reflect a host of immune processes(4) (5) (6). Thus, 62 lipids play a critical role in inflammation. Accordingly, performing lipidomics analysis is a key 63 element in the systems-wide multi-omic platform approaches leveraging data from 64 transcriptomics, metabolomics, and proteomics to obtain a comprehensive picture of the vaccine 65 response. 67Tularemia, caused by the intracellular ...