Computational Solvent Mapping

Computational solvent mapping is a tool for the identification and characterization of binding sites on proteins. A very important result is that using at least six different solvent as probes, the consensus sites found by the mapping are always in the major subsites of the functional site, and as a result, the amino acid residues that interact with the probes also bind the specific substrates of the enzyme. Thus, computational mapping provides detailed and reliable information on the functional sites of proteins.

Here we describe the computational mapping of thermolysin, for which experimental mapping results are available, and the application of the algorithm to six other enzymes that have no experimental mapping data, but whose binding sites are well characterized.

Thermolysin (2tlx)

Thermolysin was mapped both experimentally and computationally, using isopropanol, acetone, acetonitrile, and phenol as probes.

Table 1 shows the ranking of probe clusters within the consensus sites for thermolysin, obtained by superimposing the five lowest free energy clusters for each probe.

2tlx_exp.jpg - thermolysin structure co-crystallized with the V-K dipeptide (2tlx), and superimposed the results of experimental mapping, i.e., the ligand positions in structures solved in 10% isopropanol, 50% acetone, 50% acetonitrile, and 50 mM phenol. The color scheme used for the ligands is ochre, V-K dipeptide; red, isopropanol (IPA); blue, acetone (ACN); black, acetonitrile (CCN); and purple, phenol (IPH). For the protein side chains we use the standard atomic colors, i.e., carbon, grey; oxygen, red; nitrogen, blue; and hydrogen, white. All solvents bind in the S1’ pocket (IPA1, ACN1, CCN1, and IPH1), and isopropanol also binds at the S1 site (IPA5). The S1’ pocket is found to be the only location that binds all four probes (Site 1 in Table 1), with additional clustering of isopropanol, phenol, and acetone close to the S1 subsite (Site 2 in Table 1).

2tlx_map.jpg - results of computational mapping for thermolysin. The main consensus site is in the S1’ pocket that binds all four solvents (Site 1 in Table 1), and the second consensus site is close to S1, which binds three solvents (Site 2 in Table 1).

2tlx_exp_NB.pdf - nonbonded receptor-ligand interactions in 23 thermolysin structures in the PDB that have been co-crystallized with different ligands The interactions were extracted using the SAS program, available on the webpage http://www.biochem.ucl.ac.uk/bsm/pdbsum/

2tlx_exp_HB.pdf - list of intermolecular hydrogen bonds, also extracted by SAS.

2tlxNB.jpg - distribution of intermolecular nonbonded interactions among thermolysin residues. The interactions were determined from three sources: computational mapping; extracted from 23 complexes of thermolysin with different ligands in the PDB database; and experimental mapping. Computational mapping results are based on the interactions found between various thermolysin residues and the probes in the main consensus site.

2tlxHB.jpg - the same comparison for the distribution of hydrogen bonds.

Six More Enzymes

Acetone, urea, DMSO, isopropanol, t-butanol, and phenol were used as probes. The five lowest free energy clusters of each probe were superimposed to find the consensus sites shown in Table 2. We show that the amino acid residues that interact with the probes are also important for substrate binding and/or catalysis in Table 3. Detailed results for each enzyme are as follows:

Enolase (1ebg)

1ebg_map.jpg - Consensus site 1 (all six solvents), superimposed with the specific ligand of the enzyme, phosphonoacetohydroxamate. The color scheme for the ligands is ochre, phosphonoacetohydroxamate; blue, acetone; yellow, urea; pink, DMSO; red, isopropanol; green, t-butanol; and purple, phenol.

1ebg_exp_NB.pdf - nonbonded receptor-ligand interactions in 16 enolase structures in the PDB that have been co-crystallized with different ligands.

1ebg_exp_HB.pdf - list of intermolecular hydrogen bonds.

1ebgNB.jpg - distribution of intermolecular nonbonded interactions, determined by computational mapping and extracted from the 16 enolase complexes with different ligands in the PDB database.

Ribonuclease T1 (1rnt)

1rnt_map.jpg - Consensus site 1 which contains all six solvents, and the nearby consensus site 6, which contains DMSO and t-butanol (located at the side chain of H92). The probes are superimposed with a specific ligand of the enzyme, 2’-guanylic acid. The color scheme for the ligands is ochre, 2’-guanylic acid; blue, acetone; yellow, urea; pink, DMSO; red, isopropanol; green, t-butanol; and purple, phenol.

1rnt_exp_NB.pdf - nonbonded receptor-ligand interactions in 69 ribonuclease T1 structures in the PDB that have been co-crystallized with different ligands.

1rnt_exp_HB.pdf – list of intermolecular hydrogen bonds.

1rntNB.jpg - distribution of intermolecular nonbonded interactions, determined by computational mapping and extracted from the 69 ribonuclease T1 complexes with different ligands in the PDB database

Triosephosphate isomerase (2ypi)

2ypi_map.jpg - Consensus site 1 (all six solvents), superimposed with the specific ligand, 2-phosphoglycolate. The color scheme for the ligands is ochre, 2-phosphoglycolate; blue, acetone; yellow, urea; pink, DMSO; red, isopropanol; green, t-butanol; and purple, phenol.

2ypi_exp_NB.pdf - nonbonded receptor-ligand interactions in 33 triosephosphate isomerase structures in the PDB that have been co-crystallized with different ligands.

2ypi_exp_HB.pdf - list of intermolecular hydrogen bonds.

2ypiNB.jpg - distribution of intermolecular nonbonded interactions, determined by computational mapping and extracted from the 33 triosephosphate isomerase complexes with different ligands in the PDB database

Trypsin (1tng)

1tng_map.jpg – Consensus site 1 (all six solvents), superimposed with a specific ligand, aminomethylcyclohexane. The color scheme for the ligands is ochre, aminomethylcyclohexane.; blue, acetone; yellow, urea; pink, DMSO; red, isopropanol; green, t-butanol; and purple, phenol.

1tng_exp_NB.pdf - nonbonded receptor-ligand interactions in 69 trypsin structures in the PDB that have been co-crystallized with different ligands.

1tng_exp_HB.pdf – list of intermolecular hydrogen bonds.

1tngNB.jpg - distribution of intermolecular nonbonded interactions, determined by computational mapping and extracted from the 69 trypsin complexes with different ligands in the PDB database

Fructose-1,6-bisphosphatase (1fbc)

1fbc_map.jpg Consensus sites 1 and 2 are shown overlapping the two ends of the ligand 2,5-anhydroglucitol-1,6-biphosphate. The color scheme for the ligands is ochre, 2,5-anhydroglucitol-1,6-biphosphate; blue, acetone; yellow, urea; pink, DMSO; red, isopropanol; green, t-butanol; and purple, phenol.

1fbc_exp_NB.pdf - nonbonded receptor-ligand interactions in 64 fructose-1,6-bisphosphatase structures in the PDB that have been co-crystallized with different ligands.

1fbc_exp_HB.pdf – list of intermolecular hydrogen bonds.

1fbcNB.jpg - distribution of intermolecular nonbonded interactions, determined by computational mapping and extracted from 64 fructose-1,6-bisphosphatase complexes with different ligands in the PDB database

Haloalkane dehalogenase (2dhc)

2dhc_map.jpg – There are two consensus sites (Sites 2 and 3), both with all six different solvents, and located at either end of a long and narrow channel through the protein leading to the active site. Consensus Site 1, which includes the lowest free energy clusters for acetone, urea, and isopropanol, is exactly at the active site. An additional consensus site with three solvents (Site 4) is adjacent to Site 2 at the entrance of the channel. The existence of a collision complex formed during halide import is supported by both crystallographic and kinetic evidence, the latter based on the mutations of T197 and F294, which are both at consensus site 4. The free energy values at the consensus site suggest the existence of a pathway from Site 4 to the nearby Site 2, and from there to the catalytic Site 1. The role of Site 3 is not clear. The color scheme for the ligands is ochre, 1,2-dichloroethane; blue, acetone; yellow, urea; pink, DMSO; red, isopropanol; green, t-butanol; and purple, phenol.

2dhc_exp_NB.pdf - nonbonded receptor-ligand interactions in 4 haloalkane dehalogenase structures in the PDB that have been co-crystallized with different ligands.

2dhc_exp_HB.pdf – list of intermolecular hydrogen bonds.

2dhcNB.jpg - distribution of intermolecular nonbonded interactions, determined by computational mapping and extracted from the 4 haloalkane dehalogenase complexes with different ligands in the PDB database