Substrate-enzyme interactions and catalytic mechanism in phospholipase C: a molecular modeling study using the GRID program.

Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics geometry relaxations. The substrate model has been constructed by successively placing phosphate, choline and diacylglycerol moieties in the positions indicated from GRID calculations. On the basis of the resulting orientation of a complete phosphatidylcholine molecule, we propose a mechanism for the hydrolysis of the substrate.     

Conformational behavior of chondroitin and chondroitin sulfate in relation to their physical properties as inferred by molecular modeling

Chondroitin and chondroitin sulfates belong to the family of glycosaminoglycans. They are most widely distributed in animal tissues, where they are involved in structural functions and in cell-cell communication. Their basic structures consist of a disaccharidic repeating unit of beta-D-glucuronic acid (GlcA) and 2-acetamido-2-deoxy-beta-D-galactose (GalNAc), this latter being sulfated at different positions. Molecular mechanics has been applied to calculate the adiabatic energy maps for each of the constituting disaccharides of chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate using the MM3 force field. Based on these maps, higher levels of structural organization have been simulated. On one hand, the disordered state is studied through a Metropolis-based algorithm; the resulting chains present a behavior of semirigid polymers, with an order of stiffness: chondroitin 4-sulfate > chondroitin > chondroitin 6-sulfate. On the other hand, the exploration of the stable ordered forms leads to numerous helical conformations of comparable energies. Several of these conformations correspond to the experimentally observed ones. The ability of coordination with cations has also been explored, resulting in a preferential stereospecificity for calcium ions when compared to sodium ions.     

Substrate binding and catalytic mechanism in phospholipase C from Bacillus Cereus: A molecular mechanics and molecular dynamics study

For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate, phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B. cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme–substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 Å) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme–substrate–water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AM1-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AM1-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure. © 1997 John Wiley and Sons, Inc. Biopoly 42: 319–336, 1997     

New insights into the GABA(A) receptor structure and orthosteric ligand binding: receptor modeling guided by experimental data

GABA(A) receptors (GABA(A)Rs) are ligand gated chloride ion channels that mediate overall inhibitory signaling in the CNS. A detailed understanding of their structure is important to gain insights in, e.g., ligand binding and functional properties of this pharmaceutically important target. Homology modeling is a necessary tool in this regard because experimentally determined structures are lacking. Here we present an exhaustive approach for creating a high quality model of the α(1)β(2)γ(2) subtype of the GABA(A)R ligand binding domain, and we demonstrate its usefulness in understanding details of orthosteric ligand binding. The model was constructed by using multiple templates and by incorporation of knowledge from biochemical/pharmacological experiments. It was validated on the basis of objective energy functions, its ability to account for available residue specific information, and its stability in molecular dynamics (MD) compared with that of the two homologous crystal structures. We then combined the model with extensive structure-activity relationships available from two homologous series of orthosteric GABA(A)R antagonists to create a detailed hypothesis for their binding modes. Excellent agreement with key experimental data was found, including the ability of the model to accommodate and explain a previously developed pharmacophore model. A coupling to agonist binding was thereby established and discussed in relation to activation mechanisms. Our results highlight the importance of critical evaluation and optimization of each step in the homology modeling process. The approach taken here can greatly aid in increasing the understanding of GABA(A)Rs and related receptors where structural insight is limited and reliable models are difficult to obtain.     

Favoured carbonyl binding regions around the QA and QB sites of Rps. viridis

The factors governing quinone protein binding in the photosynthetic bacterium Rps. viridis are investigated via use of empirical intermolecular energy calculations using the GRID method (PJ Goodford (1985) J Med Chem 28: 849–854). A probe atom corresponding to a carbonyl oxygen atom is used to investigate regions exhibiting preference for carbonyl binding in the Q_A and Q_B sites of Rps. viridis. Two distinct regions at the top and bottom of the sites are revealed to be most favourable for carbonyl binding. Overlay of the crystallographically determined positions for the quinone carbonyl groups shows that they lie within the regions predicted theoretically. The method is also shown to be able to give qualitative insight into the affect of amino acid mutations of quinone binding.     

Binding modes of L35 to alpha- and beta-semihemoglobins: structural insights into the inequivalence of alpha- and beta-subunits of hemoglobin

It has been thought for several years that the greatly lowered oxygen affinity, high cooperativity, and heterotropic modulation displayed by tetrameric human hemoglobin (Hb) was an exclusive result of the assembly of high affinity alpha(1)beta(1) dimers into alpha(2)beta(2) tetramers. However, in recent times, it has been shown that alpha- and beta-semihemoglobins, namely alpha(heme)beta(apo) and alpha(apo)beta(heme), which are dimers of Hb characterized by a high affinity for oxygen and lack of cooperativity do respond to effectors such as 2-[4-(3,5-dichlorophenylureido) phenoxy]-2-methylpropionic acid (L35), a bezafibrate (BZF) related compound, by decreasing the ligand affinity to a considerable extent (between 60- and 130-fold). In order to shed some light on the structural basis of this phenomenon, we have developed a binding mode of L35 to semihemoglobins through docking analysis using the program GRID. Molecular modelling studies did identify sites on semihemoglobins where favourable interactions with L35 can occur. We found that the effector binds differently to the two semihemoglobins exhibiting high affinity only for the alpha chain heme pocket. The proposed binding models are consistent with the experimental findings and may be rationalized in terms of different hydrophobic and hydrophilic characteristics between alpha- and beta-heme pockets of Hb.     

D-Serine Potently Drives Ligand-Binding Domain Closure in the Ionotropic Glutamate Receptor GluD2

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Homology modeling and molecular dynamics simulations of Dengue virus NS2B/NS3 protease: insight into molecular interaction

The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor‐complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz‐Nle(P4)‐Lys(P3)‐Arg(P2)‐Arg(P1)‐H) were generated. The first DV protease model (DV‐1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV‐2) was built taking the WNV NS3/NS2B protease in the inhibitor‐complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure‐based design purposes. Copyright © 2009 John Wiley & Sons, Ltd.