PDBsum entry 2dv0

Oxidoreductase

PDB id: 2dv0

Contents

Protein chain

314 a.a.

Ligands

NAP

ZST

Waters ×223

References listed in PDB file

Key reference

Title

Expect the unexpected or caveat for drug designers: multiple structure determinations using aldose reductase crystals treated under varying soaking and co-Crystallisation conditions.

Authors

H.Steuber, M.Zentgraf, C.Gerlach, C.A.Sotriffer, A.Heine, G.Klebe.

Ref.

J Mol Biol, 2006, 363, 174-187. [DOI no: 10.1016/j.jmb.2006.08.011]

PubMed id

16952371

Abstract

In structure-based drug design, accurate crystal structure determination of protein-ligand complexes is of utmost importance in order to elucidate the binding characteristics of a putative lead to a given target. It is the starting point for further design hypotheses to predict novel leads with improved properties. Often, crystal structure determination is regarded as ultimate proof for ligand binding providing detailed insight into the specific binding mode of the ligand to the protein. This widely accepted practise relies on the assumption that the crystal structure of a given protein-ligand complex is unique and independent of the protocol applied to produce the crystals. We present two examples indicating that this assumption is not generally given, even though the composition of the mother liquid for crystallisation was kept unchanged: Multiple crystal structure determinations of aldose reductase complexes obtained under varying crystallisation protocols concerning soaking and crystallisation exposure times were performed resulting in a total of 17 complete data sets and ten refined crystal structures, eight in complex with zopolrestat and two complexed with tolrestat. In the first example, a flip of a peptide bond is observed, obviously depending on the crystallisation protocol with respect to soaking and co-crystallisation conditions. This peptide flip is accompanied by a rupture of an H-bond formed to the bound ligand zopolrestat. The indicated enhanced local mobility of the complex is in agreement with the results of molecular dynamics simulations. As a second example, the aldose reductase-tolrestat complex is studied. Unexpectedly, two structures could be obtained: one with one, and a second with four inhibitor molecules bound to the protein. They are located in and near the binding pocket facilitated by crystal packing effects. Accommodation of the four ligand molecules is accompanied by pronounced shifts concerning two helices interacting with the additional ligands.

Figure 3.
Figure 3. Selected conformational snapshots obtained from the MD simulation of the ALR2–zopolrestat complex are represented for the residues Cys298, Ala299, and Leu300. These snapshots suggest enhanced mobility in this region: while the conformations shown in green or magenta enable H-bond formation to the ligand's N3, this H-bond is ruptured in the conformations coloured in light blue or yellow. The inhibitor as observed in 10days_cocryst is represented as grey sticks after superimposing this crystal structure with the MD snapshots using a C^α-fit.

Figure 6.
Figure 6. TIM-barrel of ALR2 represented as a tube, emphasizing the local mobility with respect to the refined B-factors. The tube is coloured by B-factor: blue regions correspond to low temperature factors, whereas green, yellow and red colour characterize regions of subsequently increasing B-factor. In particular, dark blue represents B-values in the single-digit range, whereas red depicts regions with average B-factors of 40 Å^2 and higher. Additionally, gain of temperature factor is represented by an increasing diameter of the tube. The inhibitor zopolrestat is shown in magenta: (a) represents the corresponding tube representation for 10days_cocryst, in (b) the one for 6days_soaked_1 is given. Note the remarkable gain of local mobility within the C-terminal loop region lining the ligand binding pocket observed in 6days_soaked_1 ( vert, similar 31 Å^2, shown in yellow) compared to 10days_cocryst ( vert, similar 15 Å^2, represented in light blue). This comparison suggests that extended soaking exposure times provoke increasing mobility with respect to distinct regions represented by higher B-factors.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 363, 174-187) copyright 2006.