Anesthetic interaction with ketosteroid isomerase: insights from molecular dynamics simulations

Abstract

The nature and the sites of interactions between anesthetic halothane and homodimeric Delta5-3-ketosteroid isomerase (KSI) are characterized by flexible ligand docking and confirmed by 1H-15N NMR. The dynamics consequence of halothane interaction and the implication of the dynamic changes to KSI function are studied by multiple 5-ns molecular dynamics simulations in the presence and absence of halothane. Both docking and MD simulations show that halothane prefer the amphiphilic dimeric interface to the hydrophobic active site of KSI. Halothane occupancy at the dimer interface disrupted the intersubunit hydrogen bonding formed either directly through side chains of polar residues or indirectly through the mediation of the interfacial water molecules. Moreover, in the presence of halothane, the exchange rate of the bound waters with bulk water was increased. Halothane perturbation to the dimer interface affected the overall flexibility of the active site. This action is likely to contribute to the halothane-induced reduction of the KSI activity. The allosteric halothane modulation of the dynamics-function relationship of KSI without direct competition at the enzymatic active sites may be generalized to offer a unifying explanation of anesthetic action on a diverse range of multidomain neuronal proteins that are potentially relevant to clinical general anesthesia.

FIGURE 1

(A) Halothane motion trajectories over the 5-ns all-atom simulations in a fully hydrated KSI (system C). For clarity, water molecules are not shown. The initial positions of halothane are marked by their CPK representations. The residues whose chemical shifts are affected by halothane in NMR experiments are highlighted either in purple (strongly) or yellow (weakly). Notice reasonably good overlap between the halothane trajectories and halothane-perturbed chemical shift regions. System B contains only a single halothane molecule at location No. 1. (B) System D, where both subunits have a bound substrate analog 19-NTHS in the active site. Notice that halothane No. 4 has a much more restricted motion trajectory compared to that in system C. (C) Chemical structure of a halothane molecule.

FIGURE 2

Electrostatic interactions between residues E77 and R113 at the dimer interface of KSI. The distances from the carboxylate oxygen of E77 of subunit A to the side-chain amine of R113 of subunit B, or vice versa, in the presence (shaded) or absence (solid) of halothane, are plotted as a function of simulation time.

FIGURE 3

Halothane effects on water behavior at the dimer interface of KSI. The number of water molecules at the interface at an arbitrarily defined time point was counted and the identity of these water molecules was registered. The number of these registered water molecules was plotted as a function of subsequent simulation time and the data were fit using a single exponential decay function. The decay of the number of registered water along the simulation time reflects the exchange rate of the bound water with the bulk water. Halothane accelerates the apparent water exchange rates.

FIGURE 4

Comparison of structural drift revealed by the root mean-square deviation of the C_α atoms in the control system (solid) and the system having 4 halothane molecules (shaded) over the duration of 5-ns MD simulations: (A) subunit A, (B) subunit B, and (C) average for both subunits.

FIGURE 5

Structural flexibility shown as the root mean-square fluctuations of the C_α atoms in system A (solid) and system C (shaded) from the last 3 ns of simulations.