Targeted molecular dynamics was used to examine the mechanism of WPD loop closure in PTP1B, which is essential for the activity of the enzyme. Two important regions are identified: the R-loop ...(residues 113–118), which assists in substrate binding, and the S-loop (residues 198–209), which undergoes a conformational change that appears to be vital for the movement of the WPD loop. The S-loop is adjacent to the α3-helix, and its conformational change is coupled with a change of interactions between the α3- and α7-helices. This latter observation is of particular interest in connection with a novel class of allosteric inhibitors of PTP1B Wiesmann et al., Nat. Struc. Mol. Biol. 11 (2004) 730–737. These compounds prevent the closure of the WPD loop, forcing the enzyme to remain in a catalytically inactive conformation, by blocking the rearrangement of the α3-helix relative to the α7-helix.
The structural resolution of a bound ligand–receptor complex is a key asset to efficiently drive lead optimization in drug design. However, structural resolution of many drug targets still remains a ...challenging endeavor. In the absence of structural knowledge, scientists resort to structure–activity relationships (SARs) to promote compound development. In this study, we incorporated ligand-based knowledge to formulate a docking scoring function that evaluates binding poses for their agreement with a known SAR. We showcased this protocol by identifying the binding mode of the pyrazoloquinolinone (PQ) CGS-8216 at the benzodiazepine binding site of the GABAA receptor. Further evaluation of the final pose by molecular dynamics and free energy simulations revealed a close proximity between the pendent phenyl ring of the PQ and γ2D56, congruent with the low potency of carboxyphenyl analogues. Ultimately, we introduced the γ2D56A mutation and in fact observed a 10-fold potency increase in the carboxyphenyl analogue, providing experimental evidence in favor of our binding hypothesis.
One of the key requirements for the accurate calculation of free energy differences is proper sampling of conformational space. Especially in biological applications, molecular dynamics simulations ...are often confronted with rugged energy surfaces and high energy barriers, leading to insufficient sampling and, in turn, poor convergence of the free energy results. In this work, we address this problem by employing enhanced sampling methods. We explore the possibility of using self-guided Langevin dynamics (SGLD) to speed up the exploration process in free energy simulations. To obtain improved free energy differences from such simulations, it is necessary to account for the effects of the bias due to the guiding forces. We demonstrate how this can be accomplished for the Bennett’s acceptance ratio (BAR) and the enveloping distribution sampling (EDS) methods. While BAR is considered among the most efficient methods available for free energy calculations, the EDS method developed by Christ and van Gunsteren is a promising development that reduces the computational costs of free energy calculations by simulating a single reference state. To evaluate the accuracy of both approaches in connection with enhanced sampling, EDS was implemented in CHARMM. For testing, we employ benchmark systems with analytical reference results and the mutation of alanine to serine. We find that SGLD with reweighting can provide accurate results for BAR and EDS where conventional molecular dynamics simulations fail. In addition, we compare the performance of EDS with other free energy methods. We briefly discuss the implications of our results and provide practical guidelines for conducting free energy simulations with SGLD.
A statistical mechanical analysis of the role of bond stretching and angle bending terms, as well as of dummy atoms, in alchemical free energy simulations is presented. It allows one to resolve ...several ambiguities concerning their treatment from first principles. The relative importance of vibrational, Jacobian factor and potential-of-mean-force-like contributions from bonded energy terms to single and dual free energy differences is compared. Additional conclusions can be drawn for dummy atoms. In particular, it is shown that double free energy differences obtained in thermodynamic cycle calculations can always be made independent of the treatment of dummy atoms. Further, the equivalence of single and dual topology calculations in such applications is demonstrated. The theoretical considerations are accompanied and illustrated by simulation results for the free energy difference of solvation between iso-butane and propane.
Calculations of the free energy difference of solvation are used to study the contributions arising from alchemical changes of bond stretching and angle bending energy terms in the force field. The ...results illustrate the theoretical analysis of such terms given in the companion paper (Boresch, S.; Karplus, M. The Role of Bonded Terms in Free Energy Simulations: 1. Theoretical Analysis. J. Phys. Chem. A 1998, 103, 10310). Three model systems are investigated: (a) two one-dimensional harmonic oscillators interacting with a third particle that represents the solvent, (b) the aqueous solvation of two diatomic molecules, and (c) the aqueous solvation of ethane and methanol. In each case, the computations are carried out with both a single topology and a dual topology methodology. A comparison of free energy components of the single and double free energy differences obtained in the calculations makes it possible to identify the three contributions that the theoretical analysis showed were involved, i.e., vibrational, pmf-type, and Jacobian factor terms.The verification of the theoretical analysis by illustrative examples provides the basis for addressing the question of whether the so-called self-terms can make significant contributions to double free energy differences. This is accomplished by identifying the effect of coupling of the three contributions from bonded energy terms on a double free energy difference. For the model systems studied, coupling and, hence, self-terms are found to be of little importance. The analysis resolves the ambiguities concerning this issue in the literature.
Molecular dynamics simulations were used to compute the frequency-dependent dielectric susceptibility of aqueous solutions of alanine and alanine dipeptide. We studied four alanine solutions, ranging ...in concentration from 0.13-0.55 mol/liter, and two solutions of alanine dipeptide (0.13 and 0.27 mol/liter). In accord with experiment we find a strong dielectric increment for both solutes, whose molecular origin is shown to be the zwitterionic nature of the solutes. The dynamic properties were analyzed based on a dielectric component analysis into solute, a first hydration shell, and all remaining (bulk) waters. The results of this three component decomposition were interpreted directly, as well as by uniting the solute and hydration shell component to a "suprasolute" component. In both approaches three contributions to the frequency-dependent dielectric properties can be discerned. The quantitatively largest and fastest component arises from bulk water i.e., water not influenced by the solute(s). The interaction between waters surrounding the solute(s) (the hydration shell) and bulk water molecules leads to a relaxation process occurring on an intermediate time scale. The slowest relaxation process originates from the solute(s) and the interaction of the solute(s) with the first hydration shell and bulk water. The primary importance of the hydration shell is the exchange of shell and bulk waters; the self-contribution from bound water molecules is comparatively small. While in the alanine solutions the solute-water cross-terms are more important than the solute self-term, the solute contribution is larger in the dipeptide solutions. In the latter systems a much clearer separation of time scales between water and alanine dipeptide related properties is observed. The similarities and differences of the dielectric properties of the amino acid/peptide solutions studied in this work and of solutions of mono- and disaccharides and of the protein ubiquitin are discussed.
This article describes the development, implementation, and use of web-based "lessons" to introduce students and other newcomers to computer simulations of biological macromolecules. These lessons, ...i.e., interactive step-by-step instructions for performing common molecular simulation tasks, are integrated into the collaboratively developed CHARMM INterface and Graphics (CHARMMing) web user interface (http://www.charmming.org). Several lessons have already been developed with new ones easily added via a provided Python script. In addition to CHARMMing's new lessons functionality, web-based graphical capabilities have been overhauled and are fully compatible with modern mobile web browsers (e.g., phones and tablets), allowing easy integration of these advanced simulation techniques into coursework. Finally, one of the primary objections to web-based systems like CHARMMing has been that "point and click" simulation set-up does little to teach the user about the underlying physics, biology, and computational methods being applied. In response to this criticism, we have developed a freely available tutorial to bridge the gap between graphical simulation setup and the technical knowledge necessary to perform simulations without user interface assistance.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Comparative models of the extracellular and transmembrane domains of GABA sub(A) receptors in the agonist-free state were generated based on the recently published structures of the nicotinic ...acetylcholine receptor. The models were validated by computational methods, and their reliability was estimated by analyzing conserved and variable elements of the cys-loop receptor topology. In addition, the methodological limits in the interpretation of such anion channel receptor models are discussed. Alignment ambiguities in the helical domain were resolved for helix 3 by placing two gaps into the linker connecting helices 2 and 3. The resulting models were shown to be consistent with a wide range of pharmacological and mutagenesis data from GABA sub(A) and glycine receptors. The loose packing of the models results in a large amount of solvent-accessible space and offers a natural explanation for the rich pharmacology and the great flexibility of these receptors that are known to exist in numerous drug-induced conformational states. Putative drug binding pockets found within and between subunits are described, and amino acid residues important for the action and subtype selectivity of volatile and intravenous anesthetics, barbiturates, and furosemide are shown to be part of these pockets. The entire helical domain, however, seems to be crucial not only for binding of drugs but also for transduction of binding to gating or of allosteric modulation. These models can now be used to design new experiments for clarification of pharmacological and structural questions as well as for investigating and visualizing drug induced conformational changes.
Phosphotyrosine (pTyr) is an essential component of biological signaling, often being a determinant of protein−protein interactions. Accordingly, a number of drug discovery efforts targeting signal ...transduction pathways have included phosphotyrosine and analogues as essential components of the lead compounds. Toward the goal of improved biological efficacy, the phosphonate and difluoro phosphonate analogues of pTyr have been employed in inhibitor design because of their stability to hydrolysis and enhanced binding affinity in certain cases. To quantitate the contribution of aqueous solubility of pTyr, phosphonomethyl phenylalanine (Pmp), and difluorophosphonomethyl phenylalanine (F2Pmp) to their relative binding affinities, free energy perturbation calculations were undertaken on the mimetics phenol phosphate (PP), benzyl phosphonate (BP), and difluorobenzyl phosphonate (F2BP), including development of empirical force field parameters compatible with the CHARMM all-atom force fields. Notably, it is shown that the most favorably solvated compound of the series is BP, followed by PP, with F2BP the least favorably solvated for both the mono- and dianionic forms of the compounds. The molecular origin of this ordering is shown to be due to changes in charge distribution, in the comparatively larger size of the fluorine atoms, as well as in differences of local solvation between PP and BP. The implications of the differences in aqueous solubility toward the relative binding potencies of pTyr-, Pmp-, and F2Pmp-containing peptide ligands are discussed. Our results indicate that one general principle explaining the efficacy of selective fluorination to enhance binding affinities may lie in the ability of fluorine atoms to increase the hydrophobicity of a ligand while maintaining its capability to form hydrogen bonds.
We introduce a flexible framework for the correct description of the solvation of biological macromolecules, the
dielectric field equation (DFE). The formalism permits the use of any combination of ...quantum mechanical (QM), molecular mechanical (MM) and continuum electrostatic (CE) based techniques. For the CE region a method that yields the electric field rather than the potential is outlined. The DFE formalism makes clear the need to consider and to calibrate a dielectric boundary region surrounding the simulation system. The details of how to do this are presented for the case of the Ewald summation method; the effects are demonstrated by calculations of the dielectric properties and the spatially resolved Kirkwood
G-factor,
G
K
(
r), of TIP3P water. Computing the dielectric properties of a multi-component system provides a sensitive method to better understand the solvation of biological macromolecules. Towards this goal a rigorous analysis of the dielectric properties of solvated biomolecules based on a decomposition of the frequency-dependent dielectric constant (or susceptibility) of the full system is presented. The meaning of our approach is investigated, and the results of a first application are reported. Using the method of Voronoi polyhedra, the dielectric properties of the first two solvation shells and bulk water are compared by re-analyzing a 12-ns trajectory of a zinc finger peptide in water Löffler et al. J. Mol. Biol. 270 (1997) 520. It is found that the first shell behaves considerably different; in addition, there is a non-negligible contribution to the total susceptibility of the system from coupling between the protein and the bulk water phase, i.e. the water molecules not in the immediate vicinity of the solute.
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