Journal of Molecular Biology
Volume 363, Issue 1, 13 October 2006, Pages 174-187
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Expect the Unexpected or Caveat for Drug Designers: Multiple Structure Determinations Using Aldose Reductase Crystals Treated under Varying Soaking and Co-crystallisation Conditions

https://doi.org/10.1016/j.jmb.2006.08.011Get rights and content

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.

Introduction

During the last four decades protein crystallography emerged as an essential tool to elucidate the binding geometry and interaction patterns of protein–protein and protein–ligand complexes. Especially as prerequisite for drug design accurate crystal structure determination is of utmost importance.1,2 Novel leads are either discovered experimentally by screening existing compound libraries (high-throughput screening) or computationally by enumerating virtual libraries against a given target.3 For the latter strategy, X-ray crystallography is essential as it provides the structure of the target protein allowing detailed insights into the binding characteristics of a bound ligand. Prerequisite for such a structure-based approach are correct model building and error-free interpretation of electron-density maps.4 However, even in the case of successful crystal structure determination a unique and definite answer with respect to the binding mode resembling the in vivo situation is not necessarily provided. One explanation for the observed complexity most likely originates from protein flexibility resulting in different geometries in deviating environments. Protein adaptability is an important prerequisite for biological function.2,3,5 Nevertheless, it also provides a special challenge to inhibitor design as flexibility complicates the prediction of the binding mode of a small molecule ligand.6 Crystallographic B-factors provide evidence for the local atomic displacements, and, in case they are refined anisotropically, even information about the directionality is available. It has often been discussed whether B-factors provide information about local dynamic mobility or whether crystal structures only represent a static frozen-in picture, thereby underestimating the dynamic properties of the crystallised molecules.1,7

In the first part of this study we analyse the dynamic properties of a protein–ligand complex using a molecular dynamics (MD) simulation based on the binding geometry observed in a crystal structure determined in our laboratory. Characteristic changes of the binding geometry observed during the MD simulation prompted us to collect multiple data sets of crystals obtained by different soaking or co-crystallisation conditions in order to investigate whether the computationally indicated flexibility is also reflected by different states in the crystalline phase. Actually, distinct protein conformers are observed, which underline agreement between experimentally observed and computationally predicted adaptability of the protein binding pocket. In a second example, the intricate interplay between flexibility experienced by some amino acid side-chains and cooperative packing effects caused by symmetry-equivalent molecules enable binding of more than one ordered ligand within and close-by the binding pocket.

The studied target protein is human aldose reductase (ALR2; EC 1.1.1.21), a 36 kDa sized enzyme that exhibits pronounced flexibility and adaptability with respect to its active site. The enzyme adopts a TIM-barrel fold and is probably involved in severe diabetic complications such as retinopathy and angiopathy. It is therefore believed to be a promising drug target.8 It converts various aldehydes (including glucose under diabetic conditions) to their corresponding alcohols using NADPH as reducing cofactor. Even though the exact mechanism is currently under discussion, NADPH donates a hydride ion to the carbonyl carbon of the aldehyde resulting in a negatively charged intermediate. Most likely this step is followed by a subsequent transfer of a proton from one of the neighbouring acidic active site residues. The binding site consists of two sub-pockets, one comprising the residues probably involved in catalysis (Tyr48, Lys77, and His110) along with the nicotinamide moiety of the cofactor, while the second so-called specificity pocket is formed by Trp111, Ala299, Leu300 and Phe122 (Figure 1). These latter residues can adopt several conformations depending on the size and properties of the accommodated ligand. Thereby, they form differently shaped sub-pockets. However, upon binding of, e.g. a small-molecule ligand to a protein, the residual dynamic properties of the resulting complex are difficult to predict. The binding event imposes additional restraints onto the formed complex that can either increase or decrease the mobility of one or both partners.5,9 Thus, deeper understanding of dynamic properties with respect to protein–ligand interactions is of utmost importance. Here, we address these questions by means of multiple crystal structure determinations and MD simulations.

Section snippets

Results and Discussion

Human ALR2 is studied in complex with zopolrestat (Figure 2, 1), a potent carboxylic-acid type inhibitor formerly investigated in clinical trials. A crystal structure was obtained from preformed crystals soaked for one day with zopolrestat (1day_soaked, Figure 1). It could be refined to a resolution of 1.48 Å and shows the following binding mode†

Conclusion

Several conclusions can be drawn from our study. It is well known that modified crystallisation conditions with respect to the composition of the mother liquid can result in different crystal forms and, in consequence, in deviating ligand binding modes. However, it is widely assumed that the same binding mode will be produced for a protein–ligand complex independent of the applied protocol concerning the soaking exposure time or the co-crystallisation procedure. To our best knowledge, this is

Materials and Methods

Cloning, expression, purification and crystallisation of aldose reductase have been described elsewhere.11,17,18 Prior to crystallisation, ALR2 solutions were concentrated to 20 mg/ml in 50 mM di-ammonium hydrogen citrate (pH 5) and mixed with a solution of the cofactor in oxidized state to achieve a molar ratio of ALR2/NADP+ of 1:3. After an equilibration period of one week, a microseeding was performed. Crystals were grown at 293 K using the hanging drop vapour diffusion method. For the

Acknowledgement

We thank Pfizer, Inc., Connecticut for providing us with samples of zopolrestat. Dr Alberto Podjarny is gratefully acknowledged for making a clone of human aldose reductase and appropriate seeds available to us. The bilateral financial support of CNRS and DFG under the CERC3 program (KL1204/3, KL1204/4) as well as the Graduiertenkolleg “Protein function at the atomic level” is gratefully acknowledged.

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