PDBsum entry 6tli

Hydrolase

PDB id: 6tli

Contents

Protein chain

316 a.a. *

Ligands

DMS

IPA ×3

Metals

_ZN

_CA ×4

Waters ×121

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Locating interaction sites on proteins: the crystal structure of thermolysin soaked in 2% to 100% isopropanol.

Authors

A.C.English, S.H.Done, L.S.Caves, C.R.Groom, R.E.Hubbard.

Ref.

Proteins, 1999, 37, 628-640. [DOI no: 10.1002/(SICI)1097-0134(19991201)37:4<628::AID-PROT13>3.3.CO;2-7]

PubMed id

10651278

Abstract

Multiple-solvent crystal structure determination (MSCS) allows the position and orientation of bound solvent fragments to be identified by determining the structure of protein crystals soaked in organic solvents. We have extended this technique by the determination of high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 2% to 100% isopropanol. The procedure causes only minor changes to the conformation of the protein, and an increasing number of isopropanol interaction sites could be identified as the solvent concentration is increased. Isopropanol occupies all four of the main subsites in the active site, although this was only observed at very high concentrations of isopropanol for three of the four subsites. Analysis of the isopropanol positions shows little correlation with interaction energy computed using a molecular mechanics force field, but the experimentally determined positions of isopropanol are consistent with the structures of known protein-ligand complexes of TLN.

Figure 2.
Figure 2. Stereo drawings to show isopropanol binding reducing the disorder in the interior of TLN. A shows the refined TLN-0 structure. B and C show the refined TLN-10 structure. Red cpk spheres denote bound water molecules in A, B and C. A. 2mF[o(nat)]-DF[c] map (contoured at 1.2 ), at a resolution of 1.65 Å. The side chains of M120 and E143 are disordered. B. 2mF[o(ipa,10)]-DF[c] map (contoured at 1.2 ), at a resolution of 1.95 Å. The side chains of M120 and E143 are in a single conformer, and L144 has changed conformation, highlighting a concerted re-packing .[11] C. mF[o(ipa,10)]-mF[o(nat)] difference density map (contoured at ±5.0 ) (1.95 Å). Positive and negative difference density are shown in green and red, respectively. For clarity, the alternate B conformers of the M120 and E143 side chains, and the original conformation of L144 in the TLN-0 structure have been superimposed (colored yellow). The phases were calculated from the refined TLN-0 model (see Materials and Methods).

Figure 3.
Figure 3. Stereo drawing showing isopropanol (IPA 6) binding at a crystal contact in TLN. The figure shows the mF[o(ipa,90)] map (contoured at 0.3 ), at a resolution of 1.95 Å superimposed with the refined TLN-90 model. Red cpk spheres denote bound water molecules, and yellow bonds indicate the symmetry-related molecule. A nearby molecule of water (B value 28.5 Å^2) was also omitted from the model when calculating the map, to highlight the differences in shape between the electron density of isopropanol and water. For clarity, the mF[o(ipa,90)] map is displayed only over IPA 6 and a nearby water molecule.

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (1999, 37, 628-640) copyright 1999.