C. Denise Okafor

RNA–Magnesium–Protein Interactions in Large Ribosomal Subunit

Some of the magnesium ions in the ribosome are coordinated by multiple rRNA phosphate groups. These magnesium ions link distal sequences of rRNA, primarily by incorporating phosphate groups into the first coordination shell. Less frequently, magnesium interacts with ribosomal proteins. Ribosomal protein L2 appears to be unique by forming specific magnesium-mediated interactions with rRNA. Using optimized models derived from X-ray structures, we subjected rRNA/magnesium/water/rProtein L2 assemblies to quantum mechanical analysis using the density functional theory and natural energy decomposition analysis. The combined results provide estimates of energies of formation of these assemblies, and allow us to decompose the energies of interaction. The results indicated that RNA immobilizes magnesium by multidentate chelation with phosphate, and that the magnesium ions in turn localize and polarize water molecules, increasing energies and specificities of interaction of these water molecules with L2 protein. Thus, magnesium plays subtle, yet important, roles in ribosomal assembly beyond neutralization of electrostatic repulsion and direct coordination of RNA functional groups.

Ribosomal small subunit domains radiate from a central core

The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

Nonenzymatic Ligation of DNA with a Reversible Step and a Final Linkage that Can Be Used in PCR

Nonenzymatic DNA ligation chemistries containing a reversible step allow thermodynamic control of product formation, but they are not necessarily compatible with polymerase enzymes. We report a ligation system that uses commercially available reagents, includes a reversible step, and results in a linkage that can function as a template for PCR amplification with accurate sequence transfer.

Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event

Abstract Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions.

Crystal structures of the nuclear receptor, liver receptor homolog 1, bound to synthetic agonists

Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Although preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth nor do they expand the pocket like phospholipids. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable face-to-face π-π-stacking with the LRH-1 residue His-390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.

Structure and Dynamics of the Liver Receptor Homolog 1-PGC1 alpha Complex.

Peroxisome proliferator-activated gamma coactivator 1-α (PGC1α) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1α binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1α is known to bind and activate LRH-1, mechanisms through which PGC1α changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1–PGC1α complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1α with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), in coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1–PGC1α complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1α induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1α but promoted allosteric signaling from the helix 6/β-sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1–PGC1α interaction and may illuminate strategies for selective therapeutic targeting of PGC1α-dependent LRH-1 signaling pathways.

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. Author summary Proteins 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.

The first crystal structure of a DNA-free nuclear receptor DNA binding domain sheds light on DNA-driven allostery in the glucocorticoid receptor

The glucocorticoid receptor (GR) is a steroid hormone receptor of the nuclear receptor family that regulates gene expression in response to glucocorticoid hormone signaling. Interaction with specific GR DNA binding sequences causes conformational changes in the GR DNA binding domain (DBD) that result in recruitment of specific sets of co-regulators that determine transcriptional outcomes. We have solved the crystal structure of GR DBD in its DNA-free state, the first such crystal structure from any nuclear receptor. In contrast to previous NMR structures, this crystal structure reveals that free GR DBD adopts a conformation very similar to DNA-bound states. The lever arm region is the most variable element in the free GR DBD. Molecular dynamics of the free GR DBD as well as GR DBD bound to activating and repressive DNA elements confirm lever arm flexibility in all functional states. Cluster analysis of lever arm conformations during simulations shows that DNA binding and dimerization cause a reduction in the number of conformations sampled by the lever arm. These results reveal that DNA binding and dimerization drive conformational selection in the GR DBD lever arm region and show how DNA allosterically controls GR structure and dynamics.

Folding and Catalysis Near Life’s Origin: Support for Fe 2+ as a Dominant Divalent Cation

There is broad consensus that during and immediately following the origin of life, RNA was the single biopolymer or was among a small group of cooperating biopolymers. During the origin of life, the Archean Earth was anoxic; Fe2+ was abundant and relatively benign. We hypothesize that RNA used Fe2+ as a cofactor instead of, or along with, Mg2+ during the inception and early phases of biology, until the Great Oxidation Event (GOE). In this model, RNA participated in a metal substitution during the GOE, whereby Mg2+ replaced Fe2+ as the dominant RNA cofactor. A GOE-induced Fe2+ to Mg2+ substitution predicts that under ‘early Earth’ (anoxic) conditions, Fe2+ can participate in a variety of functions, including mediation of RNA folding and catalysis by ribozymes and proteins. Understanding the influence of Fe2+ on nucleic acid structure and function could provide an important link between the geological record and the ancestral biological world. This review focuses on experimental work investigating the interactions and functions of RNA and nucleic acid processing proteins with Fe2+ under anoxic, early Earth conditions.

Targeting the nuclear receptor LRH-1 with synthetic agonists

Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including lipid and glucose metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Preclinical studies have demonstrated a great therapeutic potential of targeting LRH-1 for treatment of metabolic diseases, such as diabetes; however, development of LRH-1 modulators has been challenging. In a recent study, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography, molecular dynamics simulations, and cellular activation assays to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid (PL) LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth, nor do they expand the pocket like PLs. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable, face-to-face π-π-stacking with LRH1 residue H390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.