Concentration-Dependent Binding of Small Ligands to Multiple Saturable Sites in Membrane Proteins

Abstract

Membrane proteins are primary targets for most therapeutic indications in cancer and neurological diseases, binding over 50% of all known small molecule drugs. Understanding how such ligands impact membrane proteins requires knowledge on the molecular structure of ligand binding, a reasoning that has driven relentless efforts in drug discovery and translational research. Binding of small ligands appears however highly complex involving interaction to multiple transmembrane protein sites featuring single or multiple occupancy states. Within this scenario, looking for new developments in the field, we investigate the concentration-dependent binding of ligands to multiple saturable sites in membrane proteins. The study relying on docking and free-energy perturbation provides us with an extensive description of the probability density of protein-ligand states that allows for computation of thermodynamic properties of interest. It also provides one- and three-dimensional spatial descriptions for the ligand density across the protein-membrane system which can be of interest for structural purposes. Illustration and discussion of the results are shown for binding of the general anesthetic sevoflurane against Kv1.2, a mammalian ion channel for which experimental data are available.

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

“…Hence, the equilibrium constant for the process of bringing the ligand from the bulk into the bound state (s1, s2) can be solved by means of an MD/FEP approach. 38 The method requires a harmonic potential coupled to the ligand to guarantee that it is restrained to occupy the effective volume centered at the receptor binding site R *; the restraint k exerts no force when the ligand center of mass is within a distance R from the equilibrium position and exerts a harmonic restoring force when the ligand center of mass is out of this range. In our case, force constants were estimated from the root-mean-square fluctuation of docking solutions.…”

Binding of General Anesthetics to Ion Channels

“…PositionDependent Probability Densities. As demonstrated in reference (Stock et al, 2017), statedependent probabilities ρ X (n 1 ,... ,n s ) for channel structures X ≡{C , O} can be mapped into the probability density ρ X (R) of any given ligand i to occupy position R in the system (regardless the position of the remaining N −1 ligands). Given our original consideration that the reservoir is a homogeneous volume occupied by ligands with positionindependent density ρ , the probability ρ X (R) simplifies to…”

“…Under these considerations, solution of ligand binding to multiple receptor sites relies fundamentally in determining the equilibrium constant Κ X (n 1, ... , n s ) for the process O X * (0 1 , ... ,0 s )+n L ⇔O X * (n 1 , ... ,n s ) where, O X * (0 1 , ... ,0 s ) is the empty receptor state with all ligands occupying the bulk. As shown in previous work (Stock et al, 2017), Κ X (n 1 ,... ,n s ) can be evaluated from MDbased free energy perturbation (FEP) calculations…”

“…Among all other aspects that might impact channelanesthetic interactions in general, we are specifically interested in determining if sevoflurane binds the wellcharacterized openconductive (O) and restingclosed (C) structures of Kv1.2 (Long et al, 2005;Stock et al, 2013) in a conformationdependent manner to impact protein equilibrium. Very recently, we went through an innovative structurebased study (Stock et al, 2017) of concentrationdependent binding of small ligands to multiple saturable sites in proteins to show that sevoflurane binds the openpore structure of Kv1.2 at the S4S5 linker and the S6Phelix interface a result largely supported by independent photolabeling experiments (Bu et al, 2017;Woll et al, 2017). Here, we aim at extending these previous calculations to investigate sevoflurane interactions with the entire TMdomain of the channel and more importantly, to resolve any conformational dependence for its binding process to channel structures.…”

Location and Character of Volatile General Anesthetics Binding Sites in the Transmembrane Domain of TRPV1

“…This is in agreement with recent work suggesting that small agonists do bind in a concentration-dependent manner in order to enhance their affinity as has been reported in the voltage-gated Kv1.2 ion channel. 71 This can be partly explained due to their size, smaller than capsaicin, and their promiscuous binding poses.…”