ChargeCalculator:Case studies

We present a few interesting examples of use cases for ACC.

Paracetamol

N-acetyl-p-aminophenol, commonly known as paracetamol, is a widely used analgesic and antipyretic. Its mechanism of action is believed to be the inhibition of the protein cyclooxygenase 2, regulating the production of pro-inflammatory compounds. In order to study the interaction of the drug with its target, it is necessary to look at the electrostatic properties of paracetamol. Further modeling studies will also require atomic charges for paracetamol as parameters of the simulation.

We have computed atomic charges in paracetamol (experimental coordinates from wwPDB CCD). Try out this particular computation setup here, and make sure to use the interactive guides for additional explanations. We have used two EEM parameter sets available in literature, which cover two atomic charge definitions at the B3LYP/6-31G* level of theory. MK charges are more likely to give good electrostatic potentials, whereas NPA charges generally support qualitative chemical concepts like induction and conjugation. In general, it is a good idea to try several charge definitions. One or more of these might prove useful in further modeling studies. If you plan to run molecular dynamics, it is good to use charges which are compatible with the particular force field you plan to use. For instance, if the force field is known to use atomic charges based on electrostatic potential mapping, pick at least one EEM parameter set developed with this charge definition.

The results of the calculation are available here.

The phenolic H appears to be the most positive H in the paracetamol molecule. The H bound to the amide N is the second most positive H. Not surprisingly, these positions represent the reaction sites for all metabolic modifications of paracetamol. While paracetamol is a very small molecule with few polar sites, the same principle can be applied in reasoning out highly reactive sites in more complex molecules.

Different EEM parameter sets produce a range of absolute values for the atomic charges. For instance, in our calculation the phenolic H in paracetamol has an MK charge of +0.31e, and an NPA charge of +0.42e. While the main trends observed within each set of charges hold true, these values cannot be compared between sets, due to the inherently different nature of the charge definitions they are based on. On the other hand, these differences might allow one set of charges to sample conformations not accessible to other sets.

Apoptosis

The protein Bax is important in regulating programmed cell death (apoptosis). Upon activation, Bax oligomerizes and causes a cascade of events which eventually leads to the death of the cell. Bax activation is a transient phenomenon believed to rely on a specific network of charge transfer. The active Bax appears in complex with a charged activator peptide, causing a few conformational changes and a shift in the total charge. The distribution of charges can be studied individually, and further compared between the active and inactive states.

For studying Bax activation we have calculated atomic charges for the active (PDB ID 2k7w) and inactive (PDB ID 1f16) forms of Bax using an EEM parameter set specifically developed for proteins. Try out this particular computation setup here, and make sure to use the interactive guides for additional explanations. Note that the input files were in pqr format, and contained AMBER charges as given by the program pdb2pqr. These are also available for comparison in ACC.

The results of the calculation are available here.

EEM allows inter-residue polarization and charge transfer, and thus the total residue charges given by EEM will deviate from the formal values of +1,0 and -1, respectively. The total residue charges given by AMBER always coincide with the formal values, as they do not account for the influence of the surrounding environment. A number of residues in the inactive Bax deviate from the formal charge, suggesting electrostatically interesting sites. Most are on the surface of Bax, but some are also inside the protein (e.g., ARG 109).

Further, we can compare the total residue charges between active and inactive Bax. For instance, the residue ARG 109 has a charge of +0.07e in inactive Bax, and +0.37e in active Bax. This significant difference may be a clue that this residue is relevant during activation. Indeed, a triple mutant at positions 109-111 shows decreased biological activity.

Note again that the absolute values of the charges are not always relevant. But the trends within a certain set of charges can hold a lot of relevant information. Moreover, not all charge differences may be of interest. For instance, when comparing between the values for highly flexible parts of the protein, the noise (differences caused by entirely different conformation and hence chemical environment) can be higher than the information content. Thus, context is always important.

Proteasome

The 20S subunit is the catalytic machinery of proteasomes, whose main function is to break down unneeded or damaged proteins. The 20S proteasome is a cylindrical nanoparticle with a wide hollow core, where protein degradation takes place at multiple catalytic sites. The proteolysis mechanism is enabled by a threonine-dependent nucleophilic attack, which may depend on associated water molecules for deprotonation of the reactive threonine hydroxyl. It is possible to study the influence of water molecules on the charge distribution of the catalytic threonine before moving on to more complex modeling studies of the catalytic reaction itself.

There are 7 beta catalytic units in the 20s proteasome, each made up of two chains (H-N,V-Z,1,2). The catalytic THR residues occupy the N-terminal position of each chain, so its residue serial number will be 1, and we expect it to be positively charged. In eukariotes, only 4 of these beta subunits contain the expected catalytic THR residues (chains H,I,L,N,V,W,Z,2).

For studying the potential influence of water on the catalytic THR, we have calculated atomic charges for the 20S proteasome of cattle (PDB ID 1IRU) using an EEM parameter set specifically developed for proteins, since the 20S proteasome is actually a large complex of multiple proteins. Try out this particular computation setup here, and make sure to use the interactive guides for additional explanations.

The results of the calculation are available here.

The presence of water molecules generally makes the catalytic THR residues less positive by up to 0.08e. This effect is favorable to the catalytic activity, as the THR hydrogen is partially drawn to water, enabling the reactive hydroxyl to initiate the nucleophilic attack. Maximum effect is seen where water forms an H-bond with the THR hydroxyl (e.g., THR 1 from chain W). A smaller effect is seen where the water is not in optimal position to form an H bond (e.g., THR 1 from chain V). The only exception is the catalytic THR of chain L, where the presence of water actually seems to make the THR more positive by over 0.2e. This is caused by the presence of not one, but three water molecules in close proximity of THR, and is probably related to the different substrate specificity of this site.

Note that the input file also contains Mg ions, which were probably used to stabilize the 20S proteasome in the original experiment that produced the structure with PDB ID 1iru. The influence of Mg ions on the catalytic THR can also be studied in the same straightforward fashion as above. Simply remove the ions from the input file, run ACC with the same setup, and then compare the charge on the catalytic THR residues between the two computations (with and without Mg ions).

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