Binding of the general anesthetic sevoflurane to ion channels.

The direct-site hypothesis assumes general anesthetics bind ion channels to impact protein equilibrium and function, inducing anesthesia. Despite advancements in the field, a first principle all-atom demonstration of this structure-function premise is still missing. We focus on the clinically used sevoflurane interaction to anesthetic-sensitive Kv1.2 mammalian channel to resolve if sevoflurane binds protein's well-characterized open and closed structures in a conformation-dependent manner to shift channel equilibrium. We employ an innovative approach relying on extensive docking calculations and free-energy perturbation of all potential binding sites revealed by the latter, and find sevoflurane binds open and closed structures at multiple sites under complex saturation and concentration effects. Results point to a non-trivial interplay of site and conformation-dependent modes of action involving distinct binding sites that increase channel open-probability at diluted ligand concentrations. Given the challenge in exploring more complex processes potentially impacting channel-anesthetic interaction, the result is revealing as it demonstrates the process of multiple anesthetic binding events alone may account for open-probability shifts recorded in measurements.

The binding and mechanism of a positive allosteric modulator of Kv3 channels.

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel's tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation.

Concentration-dependent thermodynamic analysis of the partition process of small ligands into proteins.

In the category of functional low-affinity interactions, small ligands may interact with multiple protein sites in a highly degenerate manner. Better conceived as a partition phenomenon at the molecular interface of proteins, such low-affinity interactions appear to be hidden to our current experimental resolution making their structural and functional characterization difficult in the low concentration regime of physiological processes. Characterization of the partition phenomenon under higher chemical forces could be a relevant strategy to tackle the problem provided the results can be scaled back to the low concentration range. Far from being trivial, such scaling demands a concentration-dependent understanding of self-interactions of the ligands, structural perturbations of the protein, among other molecular effects. Accordingly, we elaborate a novel and detailed concentration-dependent thermodynamic analysis of the partition process of small ligands aiming at characterizing the stability and structure of the dilute phenomenon from high concentrations. In analogy to an "aggregate" binding constant of a small molecule over multiple sites of a protein receptor, the model defines the stability of the process as a macroscopic equilibrium constant for the partition number of ligands that can be used to analyze biochemical and functional data of two-component systems driven by low-affinity interactions. Acquisition of such modeling-based structural information is expected to be highly welcome by revealing more traceable protein-binding spots for non-specific ligands.

Atomistic Model for Simulations of the Sedative Hypnotic Drug 2,2,2-Trichloroethanol.

2,2,2-Trichloroethanol (TCE) is the active form of the sedative hypnotic drug chloral hydrate, one of the oldest sleep medications in the market. Understanding of TCE's action mechanisms to its many targets, particularly within the ion channel family, could benefit from the state-of-the-art computational molecular studies. In this direction, we employed de novo modeling aided by the force field toolkit to develop CHARMM36-compatible TCE parameters. The classical potential energy function was calibrated targeting molecular conformations, local interactions with water molecules, and liquid bulk properties. Reference data comes from both tabulated thermodynamic properties and ab initio calculations at the MP2 level. TCE solvation free energy calculations in water and oil reproduce a lipophilic, yet nonhydrophobic, behavior. Indeed, the potential mean force profile for TCE partition through the phospholipid bilayer reveals the sedative's preference for the interfacial region. The calculated partition coefficient also matches experimental measures. Further validation of the proposed parameters is supported by the model's ability to recapitulate quenching experiments demonstrating TCE binding to bovine serum albumin.