Sonja M. Schwarzl

Can the calculation of ligand binding free energies be improved with continuum solvent electrostatics and an ideal‐gas entropy correction?

The prediction of a ligand binding constant requires generating three‐dimensional structures of the complex concerned and reliably scoring these structures. Here, the scoring problem is investigated by examining benzamidine‐like inhibitors of trypsin, a system for which errors in the structures are small. Precise and consistent binding free energies for the inhibitors are determined experimentally for this test system. To examine possible improvement of scoring methods, we test the suitability of continuum electrostatics to account for solvation effects and use an ideal‐gas entropy correction to account for the changes in the degrees of freedom of the ligand. The small observed root‐mean‐square deviation of 0.55 kcal/mol of the calculated relative to the experimental values indicates that the essentials of the binding process have been captured. Even though all six ligands make the same salt bridge and H‐bonds to the protein, the electrostatic contribution varies among the ligands by as much as 2 kcal/mol. Moreover, although the ligands are rigid and similar in size, the entropic terms also significantly affect the relative binding affinities (by up to 2.7 kcal/mol). The present approach to solvation and entropy may allow the ranking of the ligands to be considerably improved at a cost that makes the method applicable to the optimization of lead compounds or to the screening of small collections of ligands.

Role of glucose-6-phosphate dehydrogenase for oxidative stress and apoptosis.

Loss of function of glucose-6-phosphate dehydrogenase (G6PD) represents the most common inborn error of metabolism throughout the world which is known as G6PDdeficiency affecting an estimated 400 million people. Prolonged neonatal jaundice and hemolytic anemia are common clinical manifestations. Infections, ingestion of fava beans, and some drugs can trigger life-threatening hemolytic anemia. G6PD is the first enzyme of the pentose phosphate pathway that converts a-D-glucose-6-phospate into D-glucono-1,5-lactone-6-phosphate and is involved in the generation of NADPH. As erythrocytes lack the citric acid cycle, the pentose phosphate shunt is the only source of NADPH. NADPH is required for the generation of reduced glutathione, which is important for the protection against oxidative damage. As the G6PD gene is located at the X-chromosome at Xq28, the disease is recessively inherited in males. Some 140 missense mutations leading to amino-acid substitutions and in a few cases base pair deletions that do not produce frame shifts are known. Few splicing mutations have been documented. Recently, Fico et al. stated that ‘for the first time a role for G6PD in the protection from redox imbalance-induced apoptosis and necrosis has been clearly assigned’ in a paper published by them. In fact, this has already been reported in 1995 by us. Since glutathione represents a cellular protectant against oxidative damage, which inhibits apoptosis, we hypothesized at that time that nucleated cells of G6PDdeficient patients should be more susceptible to DNA damage and apoptosis induced by oxidative stress. We reported that peripheral mononuclear cells (PBMC) from a G6PD-deficient male showed significantly higher apoptotic rates upon challenge by cytostatic drugs (daunorubicin), gamma-irradiation, or glucocorticoids (dexamethasone) than PBMC from healthy males. The induction of oxidative stress/generation of reactive oxygen species by all three agents has been reported. This implies that diverse ROS-generating agents affect white blood cells of G6PD-deficient patients leading to apoptosis due to insufficient protection from oxidative damage by reduced glutathione. We used PBMC of a German variant of G6PD deficiency, G6PD Aachen. This variant has originally been described by Kahn et al. The mutation in the G6PD Aachen variant has been determined by us previously. A mutation 1089C4G results in a predicted amino-acid change 363Asn4Lys. The 1089C4G point mutation is unique, but produces the identical amino-acid change found in another variant of G6PD deficiency, G6PD Loma Linda. The 363Asn4Lys exchange in G6PD Loma Linda is caused by a 1089C4A mutation. Using the available three-dimensional structure of the human G6PD tetrameric protein complex, the location of the point mutation of amino acid 363 in G6PD Aachen is found at the surface of a monomer in close proximity to NADPþ and more than 20 Å away from the glucose-6-phosphate-binding site (data not shown). It can be speculated that this residue may be involved in NADPþ binding that in turn is required for tetramer stability. Thus, Arg363 may be required to indirectly maintain the structural integrity of the functional unit. Replacing it with a positively charged Lys residue would lead to charge–charge repulsion between Lys363 and NADPþ , thus affecting NADPþ binding and tetramer formation. In the same year as we initially reported on the increased induction of apoptosis in G6PD-deficient cells, another group reported that G6PD is essential for defense against oxidative stress using G6PD knockout mice. Knockout clones of embryonic stem cells were extremely sensitive to hydrogen peroxide and to the sulfydryl group oxidizing agent, diamide. In a subsequent study, we analyzed whether increased induction of apoptosis correlated with increased DNA damage in G6PD-deficient PBMC. In PBMC of three males of the G6PD Aachen variant and one G6PD-deficient male from another family coming from Iran, we found that UV light induced more DNA damage and more apoptosis than in PBMC of healthy male subjects. PBMC of heterozygote females showed intermediate rates of DNA damage and apoptosis. We concluded that increased DNA damage and apoptosis may be a result of increased oxidative stress in G6PD-deficient patients. UV radiation is known to induce DNA damage and apoptosis by the generation of reactive oxygen species. Interestingly, UV radiation induces hemolysis – a major complication in G6PD deficiency – which is reversible by the addition of glutathione. Recently, Mesbah-Namin et al. hypothesized that failure to detoxify hydrogen peroxide in G6PD-deficient leukocytes could induce primary DNA damage. Using the comet assay, these authors found that PBMC of 36 infants suffering from the Mediterranean variant of G6PD deficiency showed a significantly higher level of DNA stand breakage than PBMC of healthy control persons. Cell Death and Differentiation (2006) 13, 527–528 & 2006 Nature Publishing Group All rights reserved 1350-9047/06

Nonuniform charge scaling (NUCS): A practical approximation of solvent electrostatic screening in proteins

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Three-Dimensional Modeling of Glucose-6-phosphate Dehydrogenase-Deficient Variants from German Ancestry

In molecular mechanics calculations, electrostatic interactions between chemical groups are usually represented by a Coulomb potential between the partial atomic charges of the groups. In aqueous solution these interactions are modified by the polarizable solvent. Although the electrostatic effects of the polarized solvent on the protein are well described by the Poisson–Boltzmann equation, its numerical solution is computationally expensive for large molecules such as proteins. The procedure of nonuniform charge scaling (NUCS) is a pragmatic approach to implicit solvation that approximates the solvent screening effect by individually scaling the partial charges on the explicit atoms of the macromolecule so as to reproduce electrostatic interaction energies obtained from an initial Poisson–Boltzmann analysis. Once the screening factors have been determined for a protein the scaled charges can be easily used in any molecular mechanics program that implements a Coulomb term. The approach is particularly suitable for minimization‐based simulations, such as normal mode analysis, certain conformational reaction path or ligand binding techniques for which bulk solvent cannot be included explicitly, and for combined quantum mechanical/molecular mechanical calculations when the interface to more elaborate continuum solvent models is lacking. The method is illustrated using reaction path calculations of the Tyr35 ring flip in the bovine pancreatic trypsin inhibitor.

Time-resolved computational protein biochemistry: Solvent effects on interactions, conformational transitions and equilibrium fluctuations

Background Loss of function of dimeric glucose-6-phosphate dehydrogenase (G6PD) represents the most common inborn error of metabolism throughout the world affecting an estimated 400 million people. In Germany, this enzymopathy is very rare. Methodology/Principal Findings On the basis of G6PD crystal structures, we have analyzed six G6PD variants of German ancestry by three-dimensional modeling. All mutations present in the German population are either close to one of the three G6P or NADP+ units or to the interface of the two monomers. Two of the three mutated amino acids of G6PD Vancouver are closer to the binding site of NADP+. The G6PD Aachen mutation is also closer to the second NADP+ unit. The G6PD Wayne mutation is closer to the G6P binding region. These mutations may affect the binding of G6P and NADP+ units. Three mutations, i.e. G6PD Munich, G6PD Riverside and G6PD Gastonia, lie closer to the interface of the two monomers. These may also affect the interface of two monomers. Conclusion None of these G6PD variants share mutations with the common G6PD variants known from the Mediterranean, Near East, or Africa indicating that they have developed independently. The G6PD variants have been compared with mutants from other populations and the implications for survival of G6PD variants from natural selection have been discussed.

Solvent Electrostatic Screening in Protein Simulations

Solvent plays an important role in modulating internal motions of proteins. Here we present a computational method for including solvent effects on charge-charge interactions and on pathways between functional protein conformations, and examine solvent effects on equilibrium internal fluctuations in proteins. A computationally efficient charge reparametrisation method is presented that satisfactorily reproduces the electrostatic interactions present in a full continuum Poisson-Boltzmann representation. The application of charge reparametrisation in the calculation of a large-scale conformational transition pathway in a protein, annexin V, is illustrated. We also examine solvent effects on fast (picosecond timescale) internal protein dynamics. Nosé-Hoover dual heatbath molecular dynamics simulations are performed. These simulations allow the solvent region to be fixed at one temperature and the protein at another. The results of the Nosé-Hoover simulations on hydrated myoglobin confirm that the solvent temperature strongly influences the protein fluctuations. We consider to what extent the solvent can be considered to determine the high temperature protein dynamics.

Protein/Ligand Binding Free Energies Calculated with Quantum Mechanics/Molecular Mechanics

The treatment of electrostatic interactions is one of the key issues in protein simulations. In particular, solvent effects need to be considered. Several methods have been developed that address this problem, involving a trade-off between accuracy and speed. The current work discusses a method that aims at approximating Poisson—Boltzmann interaction energies with a conventional Coulomb potential by introducing scaling factors that are used to reparametrize the partial atomic charges. Thus, solvent effects can be incorporated into simulations while retaining maximal speed and ease of implementation.