Dominant Alcohol–Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water–protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol–protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of <i>h</i> ≈ 0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in a significant negative change in Gibbs free energy due to unmatched enthalpy–entropy compensation. These results show the crucial role of water–protein interplay in alcohol binding

Critical Role of Water in the Binding of Volatile Anesthetics to Proteins

Numerous small molecules exhibit drug-like properties by low-affinity binding to proteins. Such binding is known to be influenced by water, the detailed picture of which, however, remains unclear. One particular example is the controversial role of water in the binding of general anesthetics to proteins as an essential step in general anesthesia. Here we demonstrate that a critical amount of hydration water is a prerequisite for anesthetic–protein binding. Using nuclear magnetic resonance, the concurrent adsorption of hydration water and bound anesthetics on model proteins are simultaneously measured. Halothane binding on proteins can only take place after protein hydration reaches a threshold hydration level of ∼0.31 g of water/g of proteins at the relative water vapor pressure of ∼0.95. Similar dependence on hydration is also observed for several other anesthetics. The ratio of anesthetic partial pressures at which two different anesthetics reach the same fractional load is correlated with the anesthetic potency. The binding of nonimmobilizers, which are structurally similar to known anesthetics but unable to produce anesthesia, does not occur even after the proteins are fully hydrated. Our results provide the first unambiguous experimental evidence that water is absolutely required to enable anesthetic–protein interactions, shedding new light on the general mechanism of molecular recognition and binding

Functionally relevant sites in the EC domain of GLIC.

<p>(<b>a</b>) Residues for the NQN mutation (D91N; E177Q; D178N) and the complementary basic residues (R179 and K148) for salt bridge formation are highlighted in red and blue, respectively. Residues involved in the ketamine binding site (F174, L176, K183; N152, D153, D154) are highlighted in cyan. (<b>b</b>) The C loop region of the crystal structure of the NQN mutant (orange; PDB code: 4IRE), showing an outward movement of the C loop in comparison with the wild type GLIC (yellow and gray; PDB code 4F8H) due to removal of salt bridges in the mutant. R179 and K148 are shown in blue and cyan sticks for GLIC and the NQN mutant respectively. D91N, E177Q, and D178N are shown in red and green sticks, before and after the mutation, respectively. The salt bridge distances in GLIC are highlighted. Note the enlarged gap after the mutation. No hydrogen bonds could be formed for the mutated residues. (<b>c</b>) Two-electrode voltage clamp measurements on <i>Xenopus laevis</i> oocytes expressing the NQN mutant (solid square) and the wild type GLIC (open circle). The half maximal effective concentrations (EC₅₀) for the mutant and GLIC are pH 4.80±0.03 (n = 13) and 5.04±0.02 (n = 10), respectively. The EC₅₀ difference between the wild type GLIC and the NQN mutant is statistically significant (p<0.0001). Error bars represent standard error from the mean. The inserts are the representative traces for GLIC and the NQN mutant.</p

Trajectories of the probability flux over time for each residue upon different initial perturbations.

<p>(<b>a</b>) Initial perturbation at the NQN mutation site; (<b>b</b>) initial perturbation at the ketamine-binding site. The color denotes the normalized intensity of the probability flux (Eq. 1 in the method section). The positive and negative signs describe the net signal flow into and out of the residue, respectively. The time axis is in arbitrary unit. The initially perturbed and immediately affected residues are labeled in blue and red, respectively.</p

Molecular Interactions between Mecamylamine Enantiomers and the Transmembrane Domain of the Human α4β2 Nicotinic Receptor

To characterize the binding sites of mecamylamine enantiomers on the transmembrane domain (TMD) of human (h) (α4)₃(β2)₂ and (α4)₂(β2)₃ nicotinic acetylcholine receptors (AChRs), we used nuclear magnetic resonance (NMR), molecular docking, and radioligand binding approaches. The interactions of (<i>S</i>)-(+)- and (<i>R</i>)-(−)-mecamylamine with several residues, determined by high-resolution NMR, within the hα4β2-TMD indicate different modes of binding at several luminal (L) and nonluminal (NL) sites. In general, the residues sensitive to each mecamylamine enantiomer are similar at both receptor stoichiometries. However, some differences were observed. The molecular docking experiments were crucial for delineating the location and orientation of each enantiomer in its binding site. In the (α4)₂(β2)₃-TMD, (<i>S</i>)-(+)-mecamylamine interacts with the L1 (i.e., between positions −3′ and −5′) and L2 (i.e., between positions 16′ and 20′) sites, whereas the β2-intersubunit (i.e., cytoplasmic end of two β2-TMDs) and α4/β2-intersubunit (i.e., cytoplasmic end of α4-TM1 and β2-TM3) sites are shared by both enantiomers. In the (α4)₃(β2)₂-TMD, both enantiomers bind with different orientations to the L1′ (closer to ring 2′) and α4-intrasubunit (i.e., at the cytoplasmic ends of α4-TM1 and α4-TM2) sites, but only (<i>R</i>)-(−)-mecamylamine interacts with the L2′ (i.e., closer to ring 20′) and α4-TM3-intrasubunit sites. Our findings are important because they provide, for the first time, a structural understanding of the allosteric modulation elicited by mecamylamine enantiomers at each hα4β2 stoichiometry. This advancement could be beneficial for the development of novel therapies for the treatment of several neurological disorders

Data collection and refinement statistics.

a<p>Values in the parentheses are for highest-resolution shell.</p

Paths with the highest probability to reach the channel gate (I233; 9′) under different initial perturbations in GLIC.

<p>(<b>a</b>) The path within a subunit upon perturbation to D91 of the NQN mutation; (<b>b</b>) the path between D178 of the NQN mutation site and I233 (9′) of the same subunit showing an inter-subunit pathway; (<b>c</b>) the path between D91 of subunit B and I233 (9′) of subunit A; the perturbation to F174 of the ketamine binding site shows both (<b>d</b>) intra- and (<b>e</b>) inter-subunit paths for signal starting and ending in subunit B; (<b>f</b>) the path between F174 of subunit B and I233 (9′) of subunit C. The perturbation starting and ending points are shown in green and red spheres, respectively. The pathways are highlighted in purple spheres. Subunits A, B, and C are colored silver, yellow, and cyan, respectively. All calculations were performed using Yen's algorithm.</p

Enantioselective Synthesis of ABCF Tetracyclic Framework of Daphniphyllum Alkaloid Calyciphylline N

Efforts toward the enantioselective synthesis of Daphniphyllum alkaloid calyciphylline N which leads to efficient preparation of the ABCF tetracyclic framework containing three bridgehead all-carbon quaternary stereocenters are described. This synthetic work features the utilization of an asymmetric conjugate addition to install the C5 all-carbon quaternary center, an efficient successive inter/intramolecular aldol sequence to build the critical bicyclo[2.2.2]­octanone BC core, and a ring closing metathesis reaction followed by stereoselective Nagata conjugate cyanation to deliver the functionalized F ring

Trajectory of the probability flux and the highest probability path in nAChR (PDB code: 2BG9).

<p>(<b>a</b>) Trajectory of the probability flux over time for each residue of the α1 nAChR upon perturbation to the agonist-binding site (Y93, W149, Y190, and Y198). The color denotes the normalized intensity of the flux. Positive and negative signs describe the net signal flow into and out of the residue, respectively. (<b>b</b>) The signaling path with highest probability between Y190 of the C loop and the pore-lining residue L251 (9′) in the α1 nAChR. Perturbation starting and ending points are shown in green and red spheres, respectively. Residues comprising the path are shown in purple spheres. The labeled residues were identified previously in the mutagenesis and functional studies for transferring energy from the extracellular domain to the channel gating <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064326#pone.0064326-Chakrapani1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064326#pone.0064326-Bafna1" target="_blank">[27]</a>, .</p

One-Pot Synthesis of Multisubstituted Butyrolactonimidates: Total Synthesis of (−)-Nephrosteranic Acid

Multisubstituted chiral butyrolactonimidates have been synthesized via a one-pot, three-step cascade reaction in which (<i>R</i>)-<i>N</i>-<i>tert</i>-butanesulfinyl imidates and α,β-unsaturated diesters undergo highly stereoselective Michael addition, anion-oxidative hydroxylation, and cyclization. The synthesized butyrolactonimidates are versatile intermediates for preparation of substituted butyrolactones and furans. The usefulness of this cascade reaction is demonstrated through the concise total synthesis of natural product (−)-nephrosteranic acid