| 1. |
- Diehl, Carl, et al.
(författare)
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Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3.
- 2010
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 132, s. 14577-14589
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Tidskriftsartikel (refereegranskat)abstract
- Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.
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| 2. |
- Kadhirvel, Saraboji, et al.
(författare)
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The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:1, s. 296-306
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Tidskriftsartikel (refereegranskat)abstract
- The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.
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| 3. |
- Ciancetta, Antonella, et al.
(författare)
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A QM/MM study of the binding of RAPTA ligands to cathepsin B
- 2011
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Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 1573-4951 .- 0920-654X. ; 25:8, s. 729-742
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Tidskriftsartikel (refereegranskat)abstract
- We have carried out quantum mechanical (QM) and QM/MM (combined QM and molecular mechanics) calculations, as well as molecular dynamics (MD) simulations to study the binding of a series of six RAPTA (Ru(II)-arene-1,3,5-triaza-7-phosphatricyclo-[3.3.1.1] decane) complexes with different arene substituents to cathepsin B. The recently developed QM/MM-PBSA approach (QM/MM combined with Poisson-Boltzmann solvent-accessible surface area solvation) has been used to estimate binding affinities. The QM calculations reproduce the antitumour activities of the complexes with a correlation coefficient (r(2)) of 0.35-0.86 after a conformational search. The QM/MM-PBSA method gave a better correlation (r(2) = 0.59) when the protein was fixed to the crystal structure, but more reasonable ligand structures and absolute binding energies were obtained if the protein was allowed to relax, indicating that the ligands are strained when the protein is kept fixed. In addition, the best correlation (r(2) = 0.80) was obtained when only the QM energies were used, which suggests that the MM and continuum solvation energies are not accurate enough to predict the binding of a charged metal complex to a charged protein. Taking into account the protein flexibility by means of MD simulations slightly improves the correlation (r(2) = 0.91), but the absolute energies are still too large and the results are sensitive to the details in the calculations, illustrating that it is hard to obtain stable predictions when full flexible protein is included in the calculations.
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| 4. |
- Diehl, Carl, et al.
(författare)
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Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3.
- 2009
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 45:1-2, s. 157-169
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Tidskriftsartikel (refereegranskat)abstract
- The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used (15)N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the (15)N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes.
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| 5. |
- Genheden, Samuel, et al.
(författare)
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A comparison of different initialization protocols to obtain statistically independent molecular dynamics simulations.
- 2011
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Ingår i: Journal of Computational Chemistry. - : Wiley. - 1096-987X .- 0192-8651. ; 32:2, s. 187-195
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Tidskriftsartikel (refereegranskat)abstract
- We study how the results of molecular dynamics (MD) simulations are affected by various choices during the setup, e.g., the starting velocities, the solvation, the location of protons, the conformation of His, Asn, and Gln residues, the protonation and titration of His residues, and the treatment of alternative conformations. We estimate the binding affinity of ligands to four proteins calculated with the MM/GBSA method (molecular mechanics combined with a generalized Born and surface area solvation energy). For avidin and T4 lysozyme, all variations gave similar results within 2 kJ/mol. For factor Xa, differences in the solvation or in the selection of alternative conformations gave results that are significantly different from those of the other approaches by 4-6 kJ/mol, whereas for galectin-3, changes in the conformations, rotations, and protonation gave results that differed by 10 kJ/mol, but only if residues close to the binding site were modified. This shows that the results of MM/GBSA calculations are reasonably reproducible even if the MD simulations are set up with different software. Moreover, we show that the sampling of phase space can be enhanced by solvating the systems with different equilibrated water boxes, in addition to the common use of different starting velocities. If different conformations are available in the crystal structure, they should also be employed to enhance the sampling. Protonation, ionization, and conformations of Asn, Gln, and His may also be used to enhance sampling, but great effort should be spent to obtain as reliable predictions as possible close to the active site.
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| 6. |
- Genheden, Samuel, et al.
(författare)
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Accurate Predictions of Nonpolar Solvation Free Energies Require Explicit Consideration of Binding-Site Hydration
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:33, s. 13081-13092
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Tidskriftsartikel (refereegranskat)abstract
- Continuum solvation methods are frequently used to increase the efficiency of computational methods to estimate free energies. In this paper, we have evaluated how well such methods estimate the nonpolar solvation free-energy change when a ligand binds to a protein. Three different continuum methods at various levels of approximation were considered, viz., the polarized continuum model (PCM), a method based on cavity and dispersion terms (CD), and a method based on a linear relation to the solvent-accessible surface area (SASA). Formally rigorous double-decoupling thermodynamic integration was used as a benchmark for the continuum methods. We have studied four protein-ligand complexes with binding sites of varying solvent exposure, namely the binding of phenol to ferritin, a biotin analogue to avidin, 2-aminobenzimidazole to trypsin, and a substituted galactoside to galectin-3. For ferritin and avidin, which have relatively hidden binding sites, rather accurate nonpolar solvation free energies could be obtained with the continuum methods if the binding site is prohibited to be filled by continuum water in the unbound state, even though the simulations and experiments show that the ligand replaces several water molecules upon binding. For the more solvent exposed binding sites of trypsin and galectin-3, no accurate continuum estimates could be obtained, even if the binding site was allowed or prohibited to be filled by continuum water. This shows that continuum methods fail to give accurate free energies on a wide range of systems with varying solvent exposure because they lack a microscopic picture of binding-site hydration as well as information about the entropy of water molecules that are in the binding site before the ligand binds. Consequently, binding affinity estimates based upon continuum solvation methods will give absolute binding energies that may differ by up to 200 kJ/mol depending on the method used. Moreover, even relative energies between ligands with the same scaffold may differ by up to 75 kJ/mol. We have tried to improve the continuum solvation methods by adding information about the solvent exposure of the binding site or the hydration of the binding site, and the results are promising at least for this small set of complexes.
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| 7. |
- Genheden, Samuel, et al.
(författare)
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An MM/3D-RISM approach for ligand binding affinities.
- 2010
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Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 114:25, s. 8505-8516
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Tidskriftsartikel (refereegranskat)abstract
- We have modified the popular MM/PBSA or MM/GBSA approaches (molecular mechanics for a biomolecule, combined with a Poisson-Boltzmann or generalized Born electrostatic and surface area nonelectrostatic solvation energy) by employing instead the statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D reference interaction site model, or 3D-RISM-KH) coupled with molecular mechanics or molecular dynamics ( Blinov , N. ; et al. Biophys. J. 2010 ; Luchko , T. ; et al. J. Chem. Theory Comput. 2010 ). Unlike the PBSA or GBSA semiempirical approaches, the 3D-RISM-KH theory yields a full molecular picture of the solvation structure and thermodynamics from the first principles, with proper account of chemical specificities of both solvent and biomolecules, such as hydrogen bonding, hydrophobic interactions, salt bridges, etc. We test the method on the binding of seven biotin analogues to avidin in aqueous solution and show it to work well in predicting the ligand-binding affinities. We have compared the results of 3D-RISM-KH with four different generalized Born and two Poisson-Boltzmann methods. They give absolute binding energies that differ by up to 208 kJ/mol and mean absolute deviations in the relative affinities of 10-43 kJ/mol.
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| 8. |
- Genheden, Samuel, et al.
(författare)
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Binding affinities by alchemical perturbation using QM/MM with a large QM system and polarizable MM model.
- 2015
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Ingår i: Journal of Computational Chemistry. - : Wiley. - 1096-987X .- 0192-8651. ; 36:28, s. 2114-2124
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Tidskriftsartikel (refereegranskat)abstract
- The most general way to improve the accuracy of binding-affinity calculations for protein-ligand systems is to use quantum-mechanical (QM) methods together with rigorous alchemical-perturbation (AP) methods. We explore this approach by calculating the relative binding free energy of two synthetic disaccharides binding to galectin-3 at a reasonably high QM level (dispersion-corrected density functional theory with a triple-zeta basis set) and with a sufficiently large QM system to include all short-range interactions with the ligand (744-748 atoms). The rest of the protein is treated as a collection of atomic multipoles (up to quadrupoles) and polarizabilities. Several methods for evaluating the binding free energy from the 3600 QM calculations are investigated in terms of stability and accuracy. In particular, methods using QM calculations only at the endpoints of the transformation are compared with the recently proposed non-Boltzmann Bennett acceptance ratio (NBB) method that uses QM calculations at several stages of the transformation. Unfortunately, none of the rigorous approaches give sufficient statistical precision. However, a novel approximate method, involving the direct use of QM energies in the Bennett acceptance ratio method, gives similar results as NBB but with better precision, ∼3 kJ/mol. The statistical error can be further reduced by performing a greater number of QM calculations. © 2015 Wiley Periodicals, Inc.
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| 9. |
- Genheden, Samuel, et al.
(författare)
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Binding Affinities of Factor Xa Inhibitors Estimated by Thermodynamic Integration and MM/GBSA.
- 2011
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-960X .- 1549-9596. ; 51:Online March 18, 2011, s. 947-958
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Tidskriftsartikel (refereegranskat)abstract
- We present free energy estimates of nine 3-amidinobenzyl-1H-indole-2-carboxamide inhibitors of factor Xa. Using alchemical thermodynamic integration (TI) calculations, we estimate the difference in binding free energies with high accuracy and precision, except for mutations involving one of the amidinobenzyl rings. Crystal studies show that the inhibitors may bind in two distinct conformations, and using TI, we show that the two conformations give a similar binding affinity. Furthermore, we show that we can reduce the computational demand, while still retaining a high accuracy and precision, by using fewer integration points and shorter protein-ligand simulations. Finally, we have compared the TI results to those obtained with the simpler MM/GBSA method (molecular-mechanics with generalized Born surface-area solvation). MM/GBSA gives better results for the mutations that involve a change of net charge, but if a precision similar to that of the TI method is required, the MM/GBSA method is actually slightly more expensive. Thus, we have shown that TI could be a valuable tool in drug design.
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| 10. |
- Genheden, Samuel, et al.
(författare)
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Comparison of end-point continuum-solvation methods for the calculation of protein-ligand binding free energies.
- 2012
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Ingår i: Proteins. - : Wiley. - 0887-3585. ; 80:5, s. 1326-1342
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Tidskriftsartikel (refereegranskat)abstract
- We have compared the predictions of ligand-binding affinities from several methods based on end-point molecular dynamics simulations and continuum solvation, i.e. methods related to MM/PBSA (molecular mechanics combined with Poisson-Boltzmann and surface area solvation). Two continuum-solvation models were considered, viz. the Poisson-Boltzmann (PB) and generalised Born (GB) approaches. The non-electrostatic energies were also obtained in two different ways, viz. either from the sum of the bonded, van der Waals, non-polar solvation energies, and entropy terms (as in MM/PBSA), or from the scaled protein-ligand van der Waals interaction energy (as in the linear interaction energy approach, LIE). Three different approaches to calculate electrostatic energies were tested, viz. the sum of electrostatic interaction energies and polar solvation energies, obtained either from a single simulation of the complex or from three independent simulations of the complex, the free protein, and the free ligand, or the linear-response approximation (LRA). Moreover, we investigated the effect of scaling the electrostatic interactions by an effective internal dielectric constant of the protein (ε(int) ). All these methods were tested on the binding of seven biotin analogues to avidin and nine 3-amidinobenzyl-1H-indole-2-carboxamide inhibitors to factor Xa. For avidin, the best results were obtained with a combination of the LIE non-electrostatic energies with the MM+GB electrostatic energies from a single simulation, using ε(int) = 4. For fXa, standard MM/GBSA, based on one simulation and using ε(int) = 4-10 gave the best result. The optimum internal dielectric constant seems to be slightly higher with PB than with GB solvation. Proteins 2012. © 2012 Wiley-Liss, Inc.
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