Asymmetric allostery in estrogen receptor-α homodimers drives responses to the ensemble of estrogens in the hormonal milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations-receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

Asymmetric Allostery in Estrogen Receptor-α Homodimers Drives Responses to the Ensemble of Estrogens in the Hormonal Milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations -receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. Significance: The estrogen receptor-α (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

Asymmetric Syntheses of (+)- and (-)-Collybolide Enable Reevaluation of kappa-Opioid Receptor Agonism.

The fungal metabolite collybolide has attracted attention as a non-nitrogenous, potent, and biased agonist of the kappa-opioid receptor (KOR). Here, we report a 10-step asymmetric synthesis of this complex sesquiterpene that enables facile access to either enantiomer. The synthesis relies on a diastereoselective α-benzoyloxylation to install the buried C6 benzoate and avoid irreversible translactonization of the congested, functionally dense core. Neither enantiomer, however, exhibited KOR agonism, indicating that collybolide has been mischaracterized as a KOR agonist. Given the pharmaceutical, medical, and societal interest in collybolide as a next-generation antipruritic and analgesic, this refutation of KOR activity has important ramifications for ongoing studies. Classification of collybolide as a new non-nitrogenous, KOR-selective, potent agonist with the same clinical potential as salvinorin A seems to have been premature.

Traumatic Brain Injury Preserves Firing Rates But Disrupts Laminar Oscillatory Coupling and Neuronal Entrainment in Hippocampal CA1.

While hippocampal-dependent learning and memory are particularly vulnerable to traumatic brain injury (TBI), the functional status of individual hippocampal neurons and their interactions with oscillations are unknown following injury. Using the most common rodent TBI model and laminar recordings in CA1, we found a significant reduction in oscillatory input into the radiatum layer of CA1 after TBI. Surprisingly, CA1 neurons maintained normal firing rates despite attenuated input, but did not maintain appropriate synchronization with this oscillatory input or with local high-frequency oscillations. Normal synchronization between these coordinating oscillations was also impaired. Simultaneous recordings of medial septal neurons known to participate in theta oscillations revealed increased GABAergic/glutamatergic firing rates postinjury under anesthesia, potentially because of a loss of modulating feedback from the hippocampus. These results suggest that TBI leads to a profound disruption of connectivity and oscillatory interactions, potentially disrupting the timing of CA1 neuronal ensembles that underlie aspects of learning and memory.

Electrophysiological Signature Reveals Laminar Structure of the Porcine Hippocampus.

The hippocampus is integral to working and episodic memory and is a central region of interest in diseases affecting these processes. Pig models are widely used in translational research and may provide an excellent bridge between rodents and nonhuman primates for CNS disease models because of their gyrencephalic neuroanatomy and significant white matter composition. However, the laminar structure of the pig hippocampus has not been well characterized. Therefore, we histologically characterized the dorsal hippocampus of Yucatan miniature pigs and quantified the cytoarchitecture of the hippocampal layers. We then utilized stereotaxis combined with single-unit electrophysiological mapping to precisely place multichannel laminar silicon probes into the dorsal hippocampus without the need for image guidance. We used in vivo electrophysiological recordings of simultaneous laminar field potentials and single-unit activity in multiple layers of the dorsal hippocampus to physiologically identify and quantify these layers under anesthesia. Consistent with previous reports, we found the porcine hippocampus to have the expected archicortical laminar structure, with some anatomical and histological features comparable to the rodent and others to the primate hippocampus. Importantly, we found these distinct features to be reflected in the laminar electrophysiology. This characterization, as well as our electrophysiology-based methodology targeting the porcine hippocampal lamina combined with high-channel-count silicon probes, will allow for analysis of spike-field interactions during normal and disease states in both anesthetized and future awake behaving neurophysiology in this large animal.