Recognition of Anesthetic Barbiturates by a Protein Binding Site: A High Resolution Structural Analysis

Abstract

Barbiturates potentiate GABA actions at the GABAA receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.

Crystallographic Studies with Xenon and Nitrous Oxide Provide Evidence for Protein-dependent Processes in the Mechanisms of General Anesthesia

“…For instance, apoferritin has been used as a model for the type A γ-aminobutyric acid receptor. 57,58 Also, there is evidence that membrane proteins such as the Gloeobacter violaceus and Erwinia chrysanthemi pentameric ligand-gated ion channels possess anesthetic binding sites similar to those found in globular proteins that are hydrophobic pockets or cavities of various sizes. In the E. chrysanthemi channel, xenon bound to a hydrophobic cavity within the pore, 59 and bromoform has multiple hydrophobic binding sites.…”

Ketamine Metabolite (2R,6R)-Hydroxynorketamine Interacts with μ and κ Opioid Receptors

“…As a validation of our methodology in a well-characterized protein, we first studied the interaction of S -ketamine with horse spleen apoferritin (HSAF). This 24-mer binds a variety of psychoactive molecules, such as propofol, isoflurane, sevoflurane, barbiturates, and the fluorescent general anesthetic 1-aminoanthracene (1-AMA), at an interfacial site between monomers similar to transmembrane binding sites in the GABA A receptor. − …”

Molecular Mechanics Parameterization of Anesthetic Molecules

“…Dissociation constants of the ligand with a known protein can be determined by experiment, and in some cases these can be compared in simulation. For example, many diazirine photoaffinity ligands bind to horse spleen apoferritin (HSAF) in a presumed binding site with known affinity (Oakley et al, 2012; Vedula et al, 2009). A standard free energy of binding can be computed using alchemical FEP.…”