Mechanism and energetics of charybdotoxin unbinding from a potassium channel from molecular dynamics simulations.
ABSTRACT: Ion channel-toxin complexes are ideal systems for computational studies of protein-ligand interactions, because, in most cases, the channel axis provides a natural reaction coordinate for unbinding of a ligand and a wealth of physiological data is available to check the computational results. We use a recently determined structure of a potassium channel-charybdotoxin complex in molecular dynamics simulations to investigate the mechanism and energetics of unbinding. Pairs of residues on the channel protein and charybdotoxin that are involved in the binding are identified, and their behavior is traced during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force for the unbinding of charybdotoxin is constructed from the umbrella sampling simulations using the weighted histogram analysis method, and barriers observed are correlated with specific breaking of interactions and influx of water molecules into the binding site. Charybdotoxin is found to undergo conformational changes as a result of the reaction coordinate choice--a nontrivial decision for larger ligands--which we explore in detail, and for which we propose solutions. Agreement between the calculated and the experimental binding energies is obtained once the energetic consequences of these conformational changes are included in the calculations.
Project description:In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.
Project description:Free energy calculations for protein-ligand dissociation have been tested and validated for small ligands (50 atoms or less), but there has been a paucity of studies for larger, peptide-size ligands due to computational limitations. Previously we have studied the energetics of dissociation in a potassium channel-charybdotoxin complex by using umbrella sampling molecular-dynamics simulations, and established the need for carefully chosen coordinates and restraints to maintain the physiological ligand conformation. Here we address the ligand integrity problem further by constructing additional potential of mean forces for dissociation of charybdotoxin using restraints. We show that the large discrepancies in binding free energy arising from simulation artifacts can be avoided by using appropriate restraints on the ligand, which enables determination of the binding free energy within the chemical accuracy. We make several suggestions for optimal choices of harmonic potential parameters and restraints to be used in binding studies of large ligands.
Project description:We use a recently proposed method called Spectral Gap Optimization of Order Parameters (SGOOP) [P. Tiwary and B. J. Berne, Proc. Natl. Acad. Sci. U. S. A. 113, 2839 (2016)], to determine an optimal 1-dimensional reaction coordinate (RC) for the unbinding of a bucky-ball from a pocket in explicit water. This RC is estimated as a linear combination of the multiple available order parameters that collectively can be used to distinguish the various stable states relevant for unbinding. We pay special attention to determining and quantifying the degree to which water molecules should be included in the RC. Using SGOOP with under-sampled biased simulations, we predict that water plays a distinct role in the reaction coordinate for unbinding in the case when the ligand is sterically constrained to move along an axis of symmetry. This prediction is validated through extensive calculations of the unbinding times through metadynamics and by comparison through detailed balance with unbiased molecular dynamics estimate of the binding time. However when the steric constraint is removed, we find that the role of water in the reaction coordinate diminishes. Here instead SGOOP identifies a good one-dimensional RC involving various motional degrees of freedom.
Project description:Unbinding of a spin-labeled dinitrophenyl (DNP) hapten from the monoclonal antibody AN02 F(ab) fragment has been studied by force probe molecular dynamics (FPMD) simulations. In our nanosecond simulations, unbinding was enforced by pulling the hapten molecule out of the binding pocket. Detailed inspection of the FPMD trajectories revealed a large heterogeneity of enforced unbinding pathways and a correspondingly large flexibility of the binding pocket region, which exhibited induced fit motions. Principal component analyses were used to estimate the resulting entropic contribution of approximately 6 kcal/mol to the AN02/DNP-hapten bond. This large contribution may explain the surprisingly large effect on binding kinetics found for mutation sites that are not directly involved in binding. We propose that such "entropic control" optimizes the binding kinetics of antibodies. Additional FPMD simulations of two point mutants in the light chain, Y33F and I96K, provided further support for a large flexibility of the binding pocket. Unbinding forces were found to be unchanged for these two mutants. Structural analysis of the FPMD simulations suggests that, in contrast to free energies of unbinding, the effect of mutations on unbinding forces is generally nonadditive.
Project description:Human microsomal cytochrome P450 2E1 (CYP2E1) can oxidize not only low molecular weight xenobiotic compounds such as ethanol, but also many endogenous fatty acids. The crystal structure of CYP2E1 in complex with indazole reveals that the active site is deeply buried into the protein center. Thus, the unbinding pathways and associated unbinding mechanisms remain elusive. In this study, random acceleration molecular dynamics simulations combined with steered molecular dynamics and potential of mean force calculations were performed to identify the possible unbinding pathways in CYP2E1. The results show that channel 2c and 2a are most likely the unbinding channels of CYP2E1. The former channel is located between helices G and I and the B-C loop, and the latter resides between the region formed by the F-G loop, the B-C loop and the ?1 sheet. Phe298 and Phe478 act as the gate keeper during indazole unbinding along channel 2c and 2a, respectively. Previous site-directed mutagenesis experiments also supported these findings.
Project description:An 1-ns unbinding trajectory of retinol from the bovine serum retinol-binding protein has been obtained from molecular dynamics simulations. The behavior of water during ligand unbinding has never been studied in detail. I described a new method for defining a binding site, located the water molecules involved in the binding site, and examined their movements during unbinding. I found that there were only small changes in the binding site. During unbinding, the number of water molecules inside the binding site decreased, with some water molecules exhibiting movements similar in magnitude to bulk water, and there were rearrangements of the hydrogen bonds. This work represents the first detailed study of the behavior of water during an unbinding process.
Project description:The binding/unbinding of the human thrombin and its 15-mer single stranded DNA aptamer, under the application of external stimulus in the form of electrostatic potential/electric field, is investigated by a combination of continuum analysis and atomistic molecular dynamics simulation. In agreement with the experiments that demonstrate the influence of electrostatic potential on the thrombin/aptamer complex, our computations show that the application of positive electric field successfully unbinds the thrombin from the aptamer. Results from umbrella sampling simulations reveal that there is a decrease in the free energy of binding between the thrombin and aptamer in presence of positive electric fields. Hydrogen bonding and non-bonded interaction energies, and hence the free energy of binding, between the thrombin and its aptamer reduce as the applied electric field is shifted from negative to positive values. Our analyses demonstrate that application of electrical stimulus modifies the molecular interactions within the complex and consequently, electrical field can be used to modulate the association between the thrombin and its aptamer.
Project description:One of the unaddressed challenges in drug discovery is that drug potency determined in vitro is not a reliable indicator of drug activity in vivo. Accumulated evidence suggests that in vivo activity is more strongly correlated with the binding/unbinding kinetics than the equilibrium thermodynamics of protein-ligand interactions (PLIs). However, existing experimental and computational techniques are insufficient in studying the molecular details of kinetics processes of PLIs on a large scale. Consequently, we not only have limited mechanistic understanding of the kinetic processes but also lack a practical platform for high-throughput screening and optimization of drug leads on the basis of their kinetic properties. For the first time, we address this unmet need by integrating coarse-grained normal mode analysis with multitarget machine learning (MTML). To test our method, HIV-1 protease is used as a model system. We find that computational models based on the residue normal mode directionality displacement of PLIs can not only recapitulate the results from all-atom molecular dynamics simulations but also predict protein-ligand binding/unbinding kinetics accurately. When this is combined with energetic features, the accuracy of combined kon and koff prediction reaches 74.35%. Furthermore, our integrated model provides us with new insights into the molecular determinants of the kinetics of PLIs. We propose that the coherent coupling of conformational dynamics and thermodynamic interactions between the receptor and the ligand may play a critical role in determining the kinetic rate constants of PLIs. In conclusion, we demonstrate that residue normal mode directionality displacement can serve as a kinetic fingerprint to capture long-time-scale conformational dynamics of the binding/unbinding kinetics. When this is coupled with MTML, it is possible to screen and optimize compounds on the basis of their binding/unbinding kinetics in a high-throughput fashion. The further development of such computational tools will bridge one of the critical missing links between in vitro compound screening and in vivo drug activity.
Project description:Potential of mean force (PMF) calculations are used to characterize the free energy landscape of protein-lipid and protein-protein association within membranes. Coarse-grained simulations allow binding free energies to be determined with reasonable statistical error. This accuracy relies on defining a good collective variable to describe the binding and unbinding transitions, and upon criteria for assessing the convergence of the simulation toward representative equilibrium sampling. As examples, we calculate protein-lipid binding PMFs for ANT/cardiolipin and Kir2.2/PIP2, using umbrella sampling on a distance coordinate. These highlight the importance of replica exchange between windows for convergence. The use of two independent sets of simulations, initiated from bound and unbound states, provide strong evidence for simulation convergence. For a model protein-protein interaction within a membrane, center-of-mass distance is shown to be a poor collective variable for describing transmembrane helix-helix dimerization. Instead, we employ an alternative intermolecular distance matrix RMS (DRMS) coordinate to obtain converged PMFs for the association of the glycophorin transmembrane domain. While the coarse-grained force field gives a reasonable Kd for dimerization, the majority of the bound population is revealed to be in a near-native conformation. Thus, the combination of a refined reaction coordinate with improved sampling reveals previously unnoticed complexities of the dimerization free energy landscape. We propose the use of replica-exchange umbrella sampling starting from different initial conditions as a robust approach for calculation of the binding energies in membrane simulations.
Project description:How does a type II inhibitor bind to/unbind from a kinase target is still a confusing question because the small molecule occupies both the ATP pocket and the allosteric pocket of the kinase binding site. Here, by using enhanced sampling simulations (umbrella sampling, US) and two-end-state free energy calculations (MM/GSBA), we systemically studied the dissociation processes of two distinct small molecules escaping from the binding pocket of p38 MAP kinase through the allosteric channel and the ATP channel. The results show that the unbinding pathways along the allosteric channel have much lower PMF depths than those along the ATP channel, suggesting that the allosteric channel is more favorable for the dissociations of the two inhibitors and thereby supporting the general understanding that the largest channel of a target is usually the entry/exit pathway for the binding/dissociation of small molecules. Interestingly, the MM/GBSA approach yielded similar PMF profiles compared with those based on US, a much time consuming approach, indicating that for a general study, such as detecting the important transition state of a ligand binding/unbinding process, MM/GBSA may be a feasible choice.