Biology subjects

Okafor, C. D.

Publications and source records attributed to Okafor, C. D..

16 recordsLinked to original sources

bioRxiv2026

A novel SXXLF motif in the FXR N-terminal domain mediates coregulator and interdomain interactions

The nuclear receptor superfamily is comprised of ligand-regulated transcription factors that contain an intrinsically disordered domain at the amino-terminal end, known as the N-terminal domain (NTD). While this poorly conserved domain is known to possess ligand-independent activation function (AF-1), few NTD functions are conserved between nuclear receptors (NRs). Identified roles in other receptors include androgen receptor (AR), estrogen receptor (ER) and mineralocorticoid receptor (MR). Here, we aim to define the function of the NTD of the farnesoid X receptor (FXR), a crucial regulator of lipid and bile acid metabolism. We show that the NTD engages in interdomain contact with other FXR domains. We also observe that the NTD interacts directly with coregulator proteins. Using mutagenesis, mammalian two-hybrid assays and molecular dynamics simulations, we identify and validate a novel SXXLF motif in the NTD which mediates interactions with both coregulators and the ligand binding domain. Mutation of the motif induces large changes in conformational and allosteric coupling in FXR. Our study identifies a new nuclear receptor-interacting motif that modulates the transcriptional activity of FXR. Graphical AbstractFXR-NTD regulates transcriptional activity through interdomain communication with the LBD and is also involved in co-activator recruitment. The SENLF motif is the first defined functional element within the FXR-NTD and mediates both NTD-LBD interaction and selective co-activator engagements to drive NTD-mediated transcriptional activity. O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/724725v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5a37aorg.highwire.dtl.DTLVardef@2fa9e1org.highwire.dtl.DTLVardef@13a19daorg.highwire.dtl.DTLVardef@1775ed2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Molecular Dynamics Simulations Reveal the Importance of Non-Native Interactions in Modulating the Inactive to Active Conformational Transition in Progesterone Receptor

Nuclear receptors are a family of transcription factors that activate respective genes when bound to specific ligands. Certain ligands induce conformational changes in the receptor which helps them recruit co-activators. The conformational changes induced by these ligands conformationally transition inactive conformation into an active conformation by changing the orientation of the C-terminal helix (H12). Despite their immense physiological importance, very few questions have been solved about the kinetics and the molecular mechanism of this transition from the inactive to active conformation. In this study, we have used extensive unbiased atomistic molecular dynamics simulations of Progesterone receptor bound to a partial agonist asoprisnil to investigate these two questions. Two different crystal structures for this complex provide us with a unique opportunity to study the conformational transition at the molecular level. Apart from elucidating several important dynamical information from these simulations, we used Markov state modeling to calculate the rate of the transition between the inactive and active-like states. More importantly, we have also shown the importance of non-native interactions in this conformational transition, which were seen to be formed during the transition from inactive to active-like conformation but not present in the active conformation itself. Apart from contributing to our fundamental understanding about the structure and dynamics of nuclear receptor at the molecular level, this study might be able to contribute to the larger problem of protein-folding itself.

biophysics↗

bioRxiv2024

Simulations reveal the flexible domain architecture of full-length nuclear receptor complexes

Nuclear receptors are multidomain transcription factors whose full-length quaternary architecture is poorly described and understood. Most nuclear receptors bind DNA as heterodimers or homodimers, which could encompass a variety of arrangements of the individual domains. Only a handful of experimental structures currently exist describing these architectures. Given that domain interactions and protein-DNA interactions within transcriptional complexes are tightly linked to function, understanding the arrangement of nuclear receptor domains on DNA is of utmost importance. Here, we employ modeling and molecular dynamics (MD) simulations to describe the structure of the full-length farnesoid X receptor (FXR) and retinoid X receptor alpha (RXR) heterodimer bound to DNA. Combining over 100 microseconds of atomistic MD simulations with enhanced sampling simulations, we characterize the dynamic behavior of eight FXR-RXR-DNA complexes, showing that these complexes support a range of quaternary architectures. Our simulations reveal critical roles for the hinge in the DNA-bound dimer in facilitating interdomain allostery, mediating DNA binding and driving the dynamic flexibility of the complex. These roles have been hard to appreciate previously due to experimental limitations in studying the flexible hinge. These studies provide a much-needed framework that will enable the field to obtain a complete understanding of nuclear receptor quaternary architectures.

biophysics↗

bioRxiv2024

Describing interdomain communication and allostery in full-length FXR

Nuclear receptors are ligand-induced transcription factors that bind directly to target genes and regulate their expression. Ligand binding initiates conformational changes that propagate to other domains, allosterically regulating their activity. The nature of this interdomain communication in nuclear receptors is poorly understood, largely owing to the difficulty of experimentally characterizing full-length structures. We have applied computational modeling approaches to describe and study the structure of the full length farnesoid X receptor (FXR), approximated by the DNA binding domain (DBD) and ligand binding domain (LBD) connected by the flexible hinge region. Using extended molecular dynamics simulations (> 10 microseconds) and enhanced sampling simulations, we provide evidence that ligands selectively induce domain rearrangement, leading to interdomain contact. We use protein-protein interaction assays to provide experimental evidence of these interactions, identifying a critical role of the hinge in mediating interdomain contact. Our results illuminate previously unknown aspects of interdomain communication in FXR and provide a framework to enable characterization of other full length nuclear receptors.

biochemistry↗

bioRxiv2023

The nuclear receptor LRH-1 discriminates between ligands using distinct allosteric signaling circuits

Nuclear receptors (NRs) are transcription factors that regulate essential biological processes in response to cognate ligands. An important part of NR function involves ligand-induced conformational changes that recruit coregulator proteins to the activation function surface (AFS), ~15 [A] away from the ligand binding pocket. Ligands must communicate with the AFS to recruit appropriate coregulators and elicit different transcriptional outcomes, but this communication is poorly understood. These studies illuminate allosteric communication networks underlying activation of liver receptor homolog-1 (LRH-1), a NR that regulates development, metabolism, cancer progression and intestinal inflammation. Using >100 microseconds of all-atom molecular dynamics simulations involving 69 LRH-1 complexes, we identify distinct signaling circuits used by active and inactive ligands for AFS communication. Inactive ligands communicate via strong, coordinated motions along paths through the receptor to the AFS. Activating ligands disrupt the "inactive" circuit by inducing connectivity elsewhere. Ligand-contacting residues in helix 7 help mediate the switch between circuits, suggesting new avenues for developing LRH-1-targeted therapeutics. We also elucidate aspects of coregulator signaling, showing that localized, destabilizing fluctuations are induced by inappropriate ligand-coregulator pairings. These studies have uncovered novel features of LRH-1 allostery, and the quantitative approach used to analyze many simulations provides a framework to study allosteric signaling in other receptors.

biochemistry↗

bioRxiv2022

Ligand-induced shifts in conformational ensembles that predict transcriptional activation

Nuclear receptors function as ligand-regulated transcription factors whose ability to regulate diverse physiological processes is closely linked with conformational changes induced upon ligand binding. Understanding how conformational populations of nuclear receptors are shifted by various ligands could illuminate strategies for the design of synthetic modulators to regulate specific transcriptional programs. Here, we investigate ligand-induced conformational changes using a reconstructed, ancestral nuclear receptor. By making substitutions at a key position, we engineer receptor variants with altered ligand specificities. We use atomistic molecular dynamics (MD) simulations with enhanced sampling to generate ensembles of wildtype and engineered receptors in combination with multiple ligands, followed by conformational analysis and prediction of ligand activity. We combine cellular and biophysical experiments to allow correlation of MD-based predictions with functional ligand profiles, as well as elucidation of mechanisms underlying altered transcription in receptor variants. We determine that conformational ensembles accurately predict ligand responses based on observed population shifts, even within engineered receptors that were constitutively active or transcriptionally unresponsive in experiments. These studies provide a platform which will allow structural characterization of physiologically-relevant conformational ensembles, as well as provide the ability to design and predict transcriptional responses in novel ligands.

biochemistry↗

bioRxiv2022

Two enhancer binding proteins activate σ54 -dependent transcription of a quorum regulatory RNA in a bacterial symbiont

To colonize a host, bacteria depend on an ensemble of signaling systems to convert information about the various environments encountered within the host into specific cellular activities. How these signaling systems coordinate transitions between cellular states in vivo remains poorly understood. To address this knowledge gap, we investigated how the bacterial symbiont Vibrio fischeri initially colonizes the light organ of the Hawaiian bobtail squid Euprymna scolopes. Previous work has shown that the small RNA Qrr1, which is a regulatory component of the quorum-sensing system in V. fischeri, promotes host colonization. Here, we report that transcriptional activation of Qrr1 is repressed by the regulator BinK, which inhibits cellular aggregation by V. fischeri prior to light organ entry. We show that Qrr1 expression depends on the alternative sigma factor {sigma}54 and the transcription factors LuxO and SypG, which function together as an OR logic gate, thereby ensuring Qrr1 is expressed during colonization. Finally, we provide evidence that this regulatory mechanism is widespread throughout the Vibrionaceae family. Together, our work reveals how coordination between the signaling pathways underlying aggregation and quorum sensing promotes host colonization, which provides insight into how integration among signaling systems facilitates complex processes in bacteria.

microbiology↗

bioRxiv2022

Identification of Antimalarial Compounds that Inhibit Apicomplexan AP2 Proteins

Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified compounds that interact with this domain. Four compounds were found to compete for DNA binding with AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that are targeted against the ApiAP2 family of proteins. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics. Author SummaryPlasmodium parasites are the causative agent of malaria, which resulted in over 600,000 deaths in 2021. Due to resistance arising for every antimalarial therapeutic deployed to date, new drug targets and druggable pathways must be explored. To address this concern, we used a molecular docking screen to predict competitors of DNA binding by the parasite specific family of Apicomplexan AP2 (ApiAP2) transcription factor proteins for testing in vitro and in vivo. We find that ApiAP2 competing compounds have antimalarial activity consistent with the disruption of gene regulation. This work will further our understanding of both the biological role and targetability of parasite transcriptional regulation.

molecular biology↗

bioRxiv2021

Evolutionary tuning of a key helix drove androgen selectivity

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 receptor 2 (AncSR2), the common ancestor of all 3-ketosteroids and the ancestral androgen receptor 1 (AncAR1), the seminal androgen receptor, to explore the biophysical mechanisms that drove the evolution of androgen specificity. We determine that ligand specificity in androgen receptors is driven by changes in the conformational dynamics of the receptor as well as altered binding pocket interactions, with helix 10 (H10) playing a critical role in tuning ligand specificity.

evolutionary biology↗

bioRxiv2021

Molecular mechanism regulating transcriptional control of the hig toxin-antitoxin locus of antibiotic-resistance plasmid Rts1 from Proteus vulgaris

Regulation of ubiquitous bacterial type II toxin-antitoxin (TA) gene pairs occurs via a negative feedback loop whereby their expression is typically responsive to changing levels of toxins at the transcriptional level similar to a molecular rheostat. While this mechanism can explain how certain TA complexes are regulated, accumulating evidence suggests diversity in this regulation. One system for which the negative feedback loop is not well defined is the plasmid-encoded HigBHigA TA pair originally identified in a post-operative infection with antibiotic resistant Proteus vulgaris. In contrast to other type II TA modules, each hig operator functions independently and excess toxin does not contribute to increased transcription in vivo. Structures of two different oligomeric complexes of HigBHigA bound to its operator DNA reveal similar interactions are maintained suggesting plasticity in how hig is repressed. Consistent with this result, molecular dynamic simulations reveal both oligomeric states exhibit similar dynamics. Further, engineering a dedicated trimeric HigBHigA complex does not regulate transcriptional repression. We propose that HigBHigA functions via a simple on/off transcriptional switch regulated by antitoxin proteolysis rather than a molecular rheostat. The present studies thus expand the known diversity of how these abundant bacterial protein pairs are regulated. IMPORTANCEBacteria respond to various stimuli by rapidly regulating gene expression to control growth. The diversity in how bacteria inhibit growth is exemplified by the abundance and diversity of toxin-antitoxin (TA) gene pairs. To tightly regulate their own expression, antitoxin proteins function as transcriptional autorepressors with additional regulation imparted by responsiveness of the system to toxin concentrations, similar to a molecular rheostat. However, some TAs do not appear to be responsive to changing levels of toxin. To expand our understanding of diverse TAs, we studied the regulation of a structurally distinct TA called host inhibition of growth (HigBA) originally discovered on the antibiotic resistance Rts1 plasmid associated with Proteus vulgaris. We find that the hig operon is regulated via a simple on/off transcriptional switch that is incalcitrant to changing toxin levels. These results expand the known mechanistic diversity of how TA pairs regulate their expression.

microbiology↗

bioRxiv2020

Tapping into a phospholipid-LRH-1 axis yields a powerful anti-inflammatory agent with in vivo activity against colitis

Phospholipids are ligands for nuclear hormone receptors (NRs) and regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions greatly improves agonists of liver receptor homolog-1 (LRH-1), a promising therapeutic target for diabetes and colitis. Conventional LRH-1 modulators partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of hybrid molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. The phospholipid-mimicking group improves binding affinity, increases LRH-1 transcriptional activity, promotes coregulator recruitment, and interacts with the targeted LRH-1 residues in crystal structures. The best new agonist markedly improves colonic histopathology and disease-related weight loss in a humanized LRH-1 murine T-cell transfer model of colitis. This is the first evidence of in vivo efficacy for an LRH-1 modulator in colitis, a leap forward in agonist development.

biochemistry↗

bioRxiv2020

Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF

In biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain nonheme-iron oxygenase, SznF, hydroxylates N{delta} and N{omega} of N{omega}-methyl-L-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the mono-iron cofactor in the enzymes C-terminal cupin domain, which effects the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. Here we leverage our recent report of an intensely absorbing {micro}-peroxodiiron(III/III) intermediate formed from the Fe2(II/II) complex and O2 to understand assembly of the diiron cofactor in the HO-like domain and to obtain structures with both SznF iron cofactors bound. Tight binding at one diiron subsite is associated with a conformational change, which is followed by weak binding at the second subsite and rapid capture of O2 by the Fe2(II/II) complex. Differences between iron-deficient and iron-replete structures reveal both the conformational change required to form the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability, showing that a ligand-harboring core helix dynamically refolds during metal acquisition and release. The cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulation. The additional ligand is conserved in another experimentally validated HO-like N-oxygenase but not in two known HO-like diiron desaturases. Among [~]9600 sequences identified bioinformatically as belonging to the emerging HO-like diiron protein (HDO) superfamily, [~]25% have this carboxylate residue and are thus tentatively assigned as N-oxygenases. Significance statementThe enzyme SznF assembles the N-nitrosourea pharmacophore of the drug streptozotocin. Its central N-oxygenase domain resembles heme-oxygenase (HO) and belongs to an emerging superfamily of HO-like diiron enzymes (HDOs) with unstable metallocofactors that have resisted structural characterization. We investigated assembly of the O2-reactive diiron complex from metal-free SznF and Fe(II) and leveraged this insight to obtain the first structure of a functionally assigned HDO with intact cofactor. Conformational changes accompanying cofactor acquisition explain its instability, and the observation of an unanticipated glutamate ligand that is conserved in only a subset of the HDO sequences provides a potential basis for top-level assignment of enzymatic function. Our results thus provide a roadmap for structural and functional characterization of novel HDOs.

biochemistry↗

bioRxiv2020

Allosteric regulation of Thioesterase Superfamily Member 1 by free fatty acids and lysophosphatidylcholine

Non-shivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally upregulated in brown adipose tissue upon cold exposure and suppresses thermogenesis to conserve energy reserves. Them1 hydrolyzes long-chain fatty acyl-CoAs, preventing their use as fuel for thermogenesis. Them1 contains a C-terminal StAR-related lipid transfer domain (StarD) with unknown ligand or function. By complementary biophysical approaches, we show that StarD binds to long-chain fatty acids, products of Them1s enzymatic reaction, as well lysophosphatidylcholine (LPC), which activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the StarD and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the StarD functions to localize Them1 near lipid droplets. These findings define the role of the StarD as a lipid sensor that allosterically regulates Them1 activity and localization.

biochemistry↗

bioRxiv2019

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosomes early evolution in a high Fe2+ environment, and its continued use by obligate anaerobes inhabiting high Fe2+ niches. Here we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order rate constant with respect to divalent cations is more than 200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+, and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because it is tightly coordinated by rRNA and buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity. Key PointsO_LIFe2+ cleaves rRNA by a non-oxidative in-line cleavage mechanism that is more than 200 times faster than in-line cleavage with Mg2+; C_LIO_LIribosomes purified from cells grown under low O2 and high Fe2+ retain ~10 Fe2+ ions per ribosome and produce as much protein as low O2, high Mg2+-grown ribosomes; C_LIO_LIa small fraction (~2%) of Fe2+ that is associated with the ribosome is not exchangeable. C_LI

molecular biology↗

bioRxiv2019

Development of the first low nanomolar Liver Receptor Homolog-1 agonist through structure-guided design

As a key regulator of metabolism and inflammation, the orphan nuclear hormone receptor, Liver Receptor Homolog-1 (LRH-1), has potential as a therapeutic target for diabetes, nonalcoholic fatty liver disease, and inflammatory bowel diseases. Discovery of LRH-1 modulators has been difficult, in part due to the tendency for synthetic compounds to bind unpredictably within the lipophilic binding pocket. Using a structure-guided approach, we exploited a newly-discovered polar interaction to lock agonists in a consistent orientation. This enabled the discovery of the first low nanomolar LRH-1 agonist, one hundred times more potent than the best previous modulator. We elucidate a novel mechanism of action that relies upon specific polar interactions deep in the LRH-1 binding pocket. In an organoid model of inflammatory bowel disease, the new agonist increases expression of LRH-1-conrolled steroidogenic genes and promotes anti-inflammatory gene expression changes. These studies constitute major progress in developing LRH-1 modulators with potential clinical utility.

pharmacology and toxicology↗

bioRxiv2018

Ligand modulation of allosteric networks in an ancestral steroid receptor

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 activation in steroid receptors. We have identified a computational method using dynamic network analysis to probe the allosteric mechanisms driving the evolution of ligand specificity in hormone receptors, determining how residue substitutions altered allosteric networks to permit gain or loss of ligand response. These results may have general utility in elucidating how modern steroid receptors are activated by endogenous and xenobiotic molecules.\n\nAuthor summaryProteins interact with a host of biological partners to mediate their function. These binding partners are able to alter structural properties of the protein to send signals dictating downstream biological activity. This mode of regulation is described as allostery. Here, we perform a computational investigation of allostery in steroid receptors, a family of proteins that regulate a host of important biological processes in response to binding and activation by a steroidal ligand. We leverage a defined evolutionary system where known historical amino acid substitutions within the receptor drive a switch in ligand preference and receptor activation. We show that activating ligands induce stronger allosteric signaling between the ligand and the functional surface on the receptor. In addition, we incorporate evolutionary mutations that are known to alter ligand preference and show that this effect may be explained by allostery. This work provides insight into how amino acid substitutions over evolution affect allostery in proteins, permitting the loss and gain of function.

biophysics↗